CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/540,097, filed Jul. 2, 2012, which is a divisional of U.S. application Ser. No. 13/359,271, filed Jan. 26, 2012, now U.S. Pat. No. 8,546,350, which is a continuation of U.S. application Ser. No. 12/346,940, filed Dec. 31, 2008, now U.S. Pat. No. 8,106,025, which is a continuation of U.S. application Ser. No. 10/909,125, filed Jul. 30, 2004, now U.S. Pat. No. 7,683,036, which claims priority to U.S. Provisional Application No. 60/492,056, filed Jul. 31, 2003; 60/516,303, filed Oct. 31, 2003; 60/531,596, filed Dec. 19, 2003; and 60/562,417, filed Apr. 14, 2004; each which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulation of small non-coding RNAs. In particular, this invention relates to compounds, particularly oligomeric compounds, which, in some embodiments, hybridize with or sterically interfere with nucleic acid molecules comprising or encoding small non-coding RNA targets. Such compounds are shown herein to modulate the levels of small non-coding RNAs. The oligomeric compounds of the invention may include one or more modifications thereon resulting in differences in physical or chemical properties compared to unmodified nucleic acids. These modified oligomeric compounds are used as single compounds or in compositions to modulate or mimic the targeted nucleic acid comprising or encoding the small non-coding RNA. In some embodiments of the invention, modifications include chemical modifications that improve activity of the oligomeric compound. In some embodiments, the modifications include moieties that modify or enhance the pharmacokinetic or pharmacodynamic properties, stability or nuclease resistance of the oligomeric compound. In some embodiments, the modifications render the oligomeric compounds capable of sterically interfering with the natural processing of the nucleic acids comprising or encoding the small non-coding RNA targets.

BACKGROUND OF THE INVENTION

RNA genes were once considered relics of a primordial â€œRNA worldâ€ that was largely replaced by more efficient proteins. More recently, however, it has become clear that non-coding RNA genes produce functional RNA molecules with important roles in regulation of gene expression, developmental timing, viral surveillance, and immunity. Not only the classic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), tiny non-coding RNAs (tncRNAs), repeat-associated small interfering RNAs (rasiRNAs) and microRNAs (miRNAs) are now believed to act in diverse cellular processes such as chromosome maintenance, gene imprinting, pre-mRNA splicing, guiding RNA modifications, transcriptional regulation, and the control of mRNA translation (Eddy, Nat. Rev. Genet., 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003, 423, 838-842; Aravin, et al., Dev. Cell, 2003, 5, 337-350). RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, and DNA elimination (Cerutti, Trends Genet., 2003, 19, 39-46; Couzin, Science, 2002, 298, 2296-2297).

The recently described phenomenon known as RNA interference (RNAi) is involves the processing of double stranded RNA into siRNAs by an RNase III-like dsRNA-specific enzyme known as Dicer (also known as helicase-moi) which are then incorporated into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC). RISC is believed to use the siRNA molecules as a guide to identify complementary RNAs, and an endoribonuclease (to date unidentified) cleaves these target RNAs, resulting in their degradation (Cerutti, Trends Genet., 2003, 19, 39-46; Grishok et al., Cell, 2001, 106, 23-34). In addition to the siRNAs, a large class of small non-coding RNAs known as microRNAs (miRNAs, originally termed stRNA for â€œshort temporal RNAsâ€ ) is believed to play a role in regulation of gene expression employing some of the same players involved in the RNAi pathway (Novina and Sharp, Nature, 2004, 430, 161-164).

Like siRNAs, miRNAs are believed to be processed endogenously by the Dicer enzyme, and are approximately the same length, and possess the characteristic 5â€²-phosphate and 3â€²-hydroxyl termini. The miRNAs are also incorporated into a ribonucleoprotein complex, the miRNP, which is similar, and may be identical to the RISC (Bartel and Bartel, Plant Physiol., 2003, 132, 709-717). More than 200 different miRNAs have been identified in plants and animals (Ambros et al., Curr. Biol., 2003, 13, 807-818).

In spite of their biochemical and mechanistic similarities, there are also some differences between siRNAs and miRNAs, based on unique aspects of their biogenesis. siRNAs are generated from the cleavage of long exogenous or possibly endogenous dsRNA molecules, such as very long hairpins or bimolecular duplexed dsRNA, and numerous siRNAs accumulate from both strands of dsRNA precursors. In contrast, mature miRNAs appear to originate from long endogenous primary miRNA transcripts (also known as pri-miRNAs, pri-mirs or pri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee, et al., EMBO J., 2002, 21(17), 4663-4670).

The current model of miRNA processing involves primary miRNA transcripts being processed by a nuclear enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs or foldback miRNA precursors) which are subsequently processed by the Dicer RNase into mature miRNAs, approximately 21-25 nucleotides in length. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3â€² overhang (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, Plant Physiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12; Lee, et al., EMBO J., 2002, 21(17), 4663-4670; Lee, et al., Nature, 2003, 425, 415-419). The 3â€² two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9). Both the primary RNA transcripts (pri-miRNAs) and foldback miRNA precursors (pre-miRNAs) are believed to be single-stranded RNA molecules with at least partial double-stranded character, often containing smaller, local internal hairpin structures. Primary miRNA transcripts may be processed such that one single-stranded mature miRNA molecule is generated from one arm of the hairpin-like structure of the pri-miRNA. Alternatively, a polycistronic pri-miRNA may contain multiple pre-miRNAs, each processed into a different, single-stranded mature miRNA.

Naturally occurring miRNAs are characterized by imperfect complementarity to their target sequences. Artificially modified miRNAs with sequences completely complementary to their target RNAs have been designed and found to function as double stranded siRNAs that inhibit gene expression by reducing RNA transcript levels. Synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules were demonstrated to target genes for silencing by degradation and not translational repression (McManus et al., RNA, 2002, 8, 842-850).

Tiny non-coding RNA (tncRNA), one class of small non-coding RNAs (Ambros et al., Curr. Biol., 2003, 13, 807-818) produce transcripts similar in length (20-21 nucleotides) to miRNAs, and are also thought to be developmentally regulated but, unlike miRNAs, tncRNAs are reportedly not processed from short hairpin precursors and are not phylogenetically conserved. Although none of these tncRNAs are reported to originate from miRNA hairpin precursors, some are predicted to form potential foldback structures reminiscent of pre-miRNAs; these putative tncRNA precursor structures deviate significantly from those of pre-miRNAs in key characteristics, i.e., they exhibit excessive numbers of bulged nucleotides in the stem or have fewer than 16 base pairs involving the small RNA (Ambros et al., Curr. Biol., 2003, 13, 807-818).

Recently, another class of small non-coding RNAs, the repeat-associated small interfering RNAs (rasiRNAs) has been isolated from Drosophila melanogaster. The rasiRNAs are associated with repeated sequences, transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs may participate in defining chromatin structure (Aravin, et al., Dev. Cell, 2003, 5, 337-350).

A total of 201 different expressed RNA sequences potentially encoding novel small non-messenger species (smnRNAs) has been identified from mouse brain cDNA libraries. Based on sequence and structural motifs, several of these have been assigned to the snoRNA class of nucleolar localized molecules known to act as guide RNAs for rRNA modification, whereas others are predicted to direct modification within the U2, U4, or U6 small nuclear RNAs (snRNAs). Some of these newly identified smnRNAs remained unclassified and have no identified RNA targets. It was suggested that some of these RNA species may have novel functions previously unknown for snoRNAs, namely the regulation of gene expression by binding to and/or modifying mRNAs or their precursors via their antisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953).

To date, the binding and regulatory sites within nucleic acid targets of the small non-coding RNAs are largely unknown, although a few putative motifs have been suggested to exist in the 3â€²UTR of certain genes (Lai and Posakony, Development, 1997, 124, 4847-4856; Lai, et al., Development, 2000, 127, 291-306; Lai, Nat Genet. 2002, 30(4), 363-364).

One miRNA is also believed to act as a cell death regulator, implicating it in mechanisms of human disease such as cancer. Recently, the Drosophila mir-14 miRNA was identified as a suppressor of apoptotic cell death and is required for normal fat metabolism. (Xu et al., Curr. Biol., 2003, 13, 790-795).

Downregulation or deletion of other miRNAs has been associated with B-cell chronic lymphocytic leukemia (B-CLL) (Calin et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15524-15529), and human homologues of the murine mir-143 and mir-145 mature miRNAs were recently reported to be expressed and processed at reduced steady-state levels at the adenomatous and cancerous stages of colorectal neoplasia (Michael, et al., Mol. Cancer Res., 2003, 1, 882-891).

Expression of the human mir-30 miRNA specifically blocked the translation in human cells of an mRNA containing artificial mir-30 target sites. In these studies, putative miRNAs were excised from transcripts encompassing artificial miRNA precursors and could inhibit the expression of mRNAs containing a complementary target site. These data indicate that novel miRNAs can be readily produced in vivo and can be designed to specifically inactivate the expression of selected target genes in human cells (Zeng et al., Mol. Cell, 2002, 9, 1327-1333).

Disclosed and claimed in PCT Publication WO 03/029459 are miRNAs from several species, or a precursor thereof; a nucleotide sequence which is the complement of said nucleotide sequence which has an identity of at least 80% to said sequence; and a nucleotide sequence which hybridizes under stringent conditions to said sequence. Also claimed is a pharmaceutical composition containing as an active agent at least one of said nucleic acid and optionally a pharmaceutically acceptable carrier, and a method of identifying microRNA molecules or precursor molecules thereof comprising ligating 5â€²- and 3â€²-adapter molecules to the ends of a size-fractionated RNA population, reverse transcribing said adapter containing RNA population and characterizing the reverse transcription products (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

Small non-coding RNA-mediated regulation of gene expression is an attractive approach to the treatment of diseases as well as infection by pathogens such as bacteria, viruses and prions and other disorders associated with RNA expression or processing.

Consequently, there remains a long-felt need for agents that regulate gene expression via the mechanisms mediated by small non-coding RNAs. Identification of modified miRNAs or miRNA mimics that can increase or decrease gene expression or activity is therefore desirable.

The present invention therefore provides oligomeric compounds and methods useful for modulating gene levels, expression, function or pathways, including those relying on mechanisms of action such as RNA interference and dsRNA enzymes, as well as antisense and non-antisense mechanisms. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify compounds, compositions and methods for these uses.

SUMMARY OF THE INVENTION

The present invention provides oligomeric compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to or mimic nucleic acids comprising or encoding small non-coding RNAs, and which act to modulate the levels of small non-coding RNAs, or interfere with their function.

The present invention also provides oligomeric compounds comprising a first strand and a second strand wherein at least one strand contains a modification and wherein a portion of one of the oligomeric compound strands is capable of hybridizing to a small non-coding RNA target nucleic acid.

The present invention also provides oligomeric compounds comprising a first region and a second region and optionally a third region wherein at least one region contains a modification and wherein a portion of the oligomeric compound is capable of hybridizing to a small non-coding RNA target nucleic acid.

The present invention also provides oligomeric compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding human Dicer, and which act to modulate the levels of the human Dicer RNase III enzyme and interfere with its function, as well as modulating the levels of small non-coding RNAs.

Pharmaceutical and other compositions comprising the compounds of the invention are also provided.

Also provided are methods of screening for modulators of small non-coding RNAs and methods of modulating the levels of small non-coding RNAs in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention.

Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of small non-coding RNAs are also set forth herein. Such methods comprise optionally identifying such an animal, and administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the animal or person.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the interaction of the mir-143 miRNA with three novel binding sites in the ERK5 mRNA coding sequence (GenBank Accession NM_â€”139032.1) identified herein, along with their bimolecular hybridization free energies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides oligomeric compounds useful in, for example, the modulation of expression, endogenous levels or the function of small non-coding RNAs. As used herein, the term â€œsmall non-coding RNAâ€ is used to encompass, without limitation, a polynucleotide molecule ranging from about 17 to about 450 nucleotides in length, which can be endogenously transcribed or produced exogenously (chemically or synthetically), but is not translated into a protein. Small non-coding RNAs may include isolated single-, double-, or multiple-stranded molecules, any of which may include regions of intrastrand nucleobase complementarity, said regions capable of folding and forming a molecule with fully or partially double-stranded or multiple-stranded character based on regions of perfect or imperfect complementarity. Examples of small non-coding RNAs include, but are not limited to, primary miRNA transcripts (also known as pri-pre-miRNAs, pri-mirs and pri-miRNAs, which range from around 70 nucleotides to about 450 nucleotides in length and often taking the form of a hairpin structure); pre-miRNAs (also known as pre-mirs and foldback miRNA precursors, which range from around 50 nucleotides to around 110 nucleotides in length); miRNAs (also known as microRNAs, Mirs, miRs, mirs, and mature miRNAs, and generally refer either to double-stranded intermediate molecules around 17 to about 25 nucleotides in length, or to single-stranded miRNAs, which may comprise a bulged structure upon hybridization with a partially complementary target nucleic acid molecule); or mimics of pri-miRNAs, pre-miRNAs or miRNAs. Small non-coding RNAs can be endogenously transcribed in cells, or can be synthetic oligonucleotides, in vitro transcribed polynucleotides or nucleic acid oligomeric compounds expressed from vectors. Pri-miRNAs and pre-miRNAs, or mimics thereof, may be processed into smaller molecules.

As used herein, the term â€œmiRNA precursorâ€ is used to encompass, without limitation, primary RNA transcripts, pri-miRNAs and pre-miRNAs.

In some embodiments, pri-miRNAs, or mimics thereof, are 70 to 450 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 or 450 nucleobases in length, or any range therewithin.

In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 430 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 nucleobases in length, or any range therewithin.

In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 280 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280 nucleobases in length, or any range therewithin.

In some embodiments, pre-miRNAs, or mimics thereof, are 50 to 110 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleobases in length, or any range therewithin. In some embodiments, pre-miRNAs, or mimics thereof, are 60 to 80 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length, or any range therewithin.

In some embodiments, miRNAs, or mimics thereof, are 15 to 49 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleobases in length, or any range therewithin. In some embodiments, miRNAs, or mimics thereof, are 17 to 25 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleobases in length, or any range therewithin.

Oligomeric compounds of the invention modulate the levels, expression or function of small non-coding RNAs by hybridizing to a nucleic acid comprising or encoding a small non-coding RNA nucleic acid target resulting in alteration of normal function by, for example, facilitating destruction of the small non-coding RNA through cleavage, by sequestration, or by sterically occluding the function of the small non-coding RNA. Further, modified synthetic oligomeric compounds of the present invention may be designed to mimic endogenous small non-coding RNAs. These modifications include, but are not limited to, improved pharmacokinetic or pharmacodynamic properties, binding affinity, stability, charge, localization or uptake. Synthetic mimics can therefore act as replacements for small non-coding RNAs, as competitive inhibitors of naturally occurring small non-coding RNAs or as delivery systems wherein the mimic construct contains one or more functional components.

As used herein, the terms â€œtarget nucleic acid,â€ â€œtarget RNA,â€ â€œtarget RNA transcriptâ€ or â€œnucleic acid targetâ€ are used to encompass any nucleic acid capable of being targeted including, without limitation, RNA (including microRNAs, stRNAs, small nuclear RNAs, small nucleolar RNAs, small ribosomal RNAs, small hairpin RNAs, endogenous antisense RNAs, guide RNAs, tiny noncoding RNAs, small single or double stranded RNAs that are encoded by heterochromatic repeats at centromeres or other chromosomal origin, and any precursors thereof). These nucleic acid targets can be coding or non-coding sequences; pre-mRNAs or mRNAs; single- or double-stranded, or single-stranded with partial double-stranded character; may occur naturally within introns or exons of messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), or transfer RNAs (tRNAs); and can be endogenously transcribed or exogenously produced.

In some embodiments of this invention, modulation of small non-coding RNA levels, expression or function is achieved via oligomeric compounds which target a further RNA associated with the particular small non-coding RNA. This association can be a physical association between that RNA and the particular small non-coding RNA such as, but not limited to, in an RNA or ribonucleoprotein complex. This association can also be within the context of a biological pathway, such as but not limited to, the regulation of levels, expression or function of a protein-encoding mRNA or its precursor by a small non-coding RNA. As such, the invention provides for modulation of the levels, expression or function of a target nucleic acid where the target nucleic acid is a messenger RNA whose expression levels and/or function are associated with one or more small non-coding RNAs. The messenger RNA function or processing may be disrupted by degradation through an antisense mechanism, including but not limited to, RNA interference, or RNase H, as well as other mechanisms wherein double stranded nucleic acid structures are recognized and degraded, cleaved, sterically occluded, sequestered or otherwise rendered inoperable.

The compounds or compositions of the present invention may also interfere with the function of endogenous RNA molecules. The functions of RNA to be interfered with can include, for example, nuclear events such as replication or transcription as the compounds of the present invention could target or mimic small non-coding RNAs in these cellular processes. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include cytoplasmic events such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, RNA signaling and regulatory activities, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA as the compounds of the present invention could target or mimic small non-coding RNAs in these cellular processes.

In the context of the present invention, â€œmodulationâ€ and â€œmodulation of expressionâ€ mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a small non-coding RNA, nucleic acid target, an RNA or protein associated with a small non-coding RNA, or a downstream target of the small non-coding RNA (e.g., a mRNA representing a protein-coding nucleic acid that is regulated by a small non-coding RNA). Inhibition is a suitable form of modulation and small non-coding RNA is a suitable target nucleic acid.

In the context of the present invention, â€œmodulation of functionâ€ means an alteration in the function of the small non-coding RNA or an alteration in the function of any cellular component with which the small non-coding RNA has an association or downstream effect.

The present invention provides, inter alia, oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications that render the compound capable of supporting modulation of the levels, expression or function of the small non-coding RNA by a degradation or cleavage mechanism.

The present invention also provides methods of maintaining a pluripotent stem cell comprising contacting the cell with an effective amount of an oligomeric compound targeting human Dicer. The pluripotent stem cell can be present in a sample of cord blood or bone marrow, or may be present as part of a cell line. In addition, the pluripotent stem cell can be an embryonic stem cell.

The present invention also provides oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications that render the compound capable of blocking or interfering with the levels, expression or function of one or more small non-coding RNAs by steric occlusion.

The present invention also provides oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications or structural elements or motifs that render the compound capable of mimicking or replacing one or more small non-coding RNAs.

Oligomeric Compounds

In the context of the present invention, the term â€œoligomeric compound(s)â€ refers to polymeric structures which are capable of hybridizing to at least a region of a small non-coding RNA molecule or a target of small non-coding RNAs, or polymeric structures which are capable of mimicking small non-coding RNAs. The term â€œoligomeric compoundâ€ includes, but is not limited to, compounds comprising oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and combinations of these. Oligomeric compounds also include, but are not limited to, antisense oligomeric compounds, antisense oligonucleotides, siRNAs, alternate splicers, primers, probes and other compounds that hybridize to at least a portion of the target nucleic acid. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and may also include branching. Separate oligomeric compounds can hybridize to form double stranded compounds that can be blunt-ended or may include overhangs on one or both termini. In general, an oligomeric compound comprises a backbone of linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. The linkages joining the monomeric subunits, the sugar moieties or sugar surrogates and the heterocyclic base moieties can be independently modified giving rise to a plurality of motifs for the resulting oligomeric compounds including hemimers, gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base moiety. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2â€², 3â€² or 5â€² hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of this linear polymeric structure can be joined to form a circular structure by hybridization or by formation of a covalent bond. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide. The normal internucleoside linkage of RNA and DNA is a 3â€² to 5â€² phosphodiester linkage.

In the context of this invention, the term â€œoligonucleotideâ€ refers generally to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside linkages. The term â€œoligonucleotide analogâ€ refers to oligonucleotides that have one or more non-naturally occurring portions which function in a similar manner to oligonucleotides. Such non-naturally occurring oligonucleotides are often selected over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases.

In the context of this invention, the term â€œoligonucleosideâ€ refers to nucleosides that are joined by internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic. These internucleoside linkages include but are not limited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH₂component parts. In addition to the modifications described above, the nucleosides of the oligomeric compounds of the invention can have a variety of other modifications. Additional nucleosides amenable to the present invention having altered base moieties and or altered sugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323.

For nucleotides that are incorporated into oligonucleotides of the invention, these nucleotides can have sugar portions that correspond to naturally occurring sugars or modified sugars. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2â€², 3â€² or 4â€² positions and sugars having substituents in place of one or more hydrogen atoms of the sugar.

Altered base moieties or altered sugar moieties also include other modifications consistent with the spirit of this invention. Such oligomeric compounds are best described as being structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified oligonucleotides. All such oligomeric compounds are comprehended by this invention so long as they function effectively to mimic the structure or function of a desired RNA or DNA oligonucleotide strand.

A class of representative base modifications include tricyclic cytosine analog, termed â€œG clampâ€ (Lin, et al., J. Am. Chem. Soc. 1998, 120, 8531). This analog can form four hydrogen bonds with a complementary guanine (G) by simultaneously recognizing the Watson-Crick and Hoogsteen faces of the targeted G. This G clamp modification when incorporated into phosphorothioate oligomeric compounds, dramatically enhances potencies as measured by target reduction in cell culture. The oligomeric compounds of the invention also can include phenoxazine-substituted bases of the type disclosed by Flanagan, et al., Nat. Biotechnol. 1999, 17(1), 48-52.

The oligomeric compounds in accordance with this invention comprise from about 8 to about 80 monomeric subunits (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 12 to 50 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 13 to 80 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 15 to 30 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 70 to 450 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 or 450 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 110 to 430 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 110 to 280 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 50 to 110 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 60 to 80 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 15 to 49 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 17 to 25 subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 17, 18, 19, 20, 21, 22, 23, 24 or 25 subunits in length, or any range therewithin.

In accordance with the present invention, oligomeric compounds designed to mimic pri-miRNAs are from about 70 to about 450 monomeric subunits in length, or from about 110 to 430 subunits in length. Oligomeric compounds of the invention designed to mimic pre-miRNAs are from about 50 to about 110 monomeric subunits in length, or from about 60 to about 80 subunits in length. Oligomeric compounds of the invention designed to mimic mature miRNAs are from about 17 to about 25 monomeric subunits in length, and can be single- or double-stranded with either or both strands comprising from about 17 to about 25 subunits.

As used herein, the term â€œaboutâ€ meansÂ±5% of the variable thereafter.

The size or length of any oligomeric compound of the present invention, within any range cited herein, can be determined as follows:

Let R(n, n+mâˆ’1) be a region from a target nucleobase sequence, where â€œnâ€ is the 5â€²-most nucleobase position of the region, where â€œn+mâˆ’1â€ is the 3â€²-most nucleobase position of the region and where â€œmâ€ is the length of the region. A set â€œS(m)â€ , of regions of length â€œmâ€ is defined as the regions where n ranges from 1 to Lâˆ’m+1, where L is the length of the target nucleic acid sequence and L>m. A set, â€œAâ€ , of all regions can be constructed as a union of the sets of regions for each length from where m is greater than or equal to a lower limit of any recited range (8 in this example) and is less than or equal to the upper limit of any recited range (80 in this example).

This set of regions can be represented using the following mathematical notation:

$A = \underset{m}{â‹ƒ} î¢ž S î¢ (m)$ $where$ $m âˆˆ N | 8 â‰¤ m â‰¤ 80$ $and$ $S î¢ (m) = {R_{n, n + m - 1} î¡œ n âˆˆ {1, 2, 3, â€¦ î¢ž, L - m + 1}}$

where the mathematical operator | indicates â€œsuch thatâ€ ,

where the mathematical operator Îµ indicates â€œa member of a setâ€ (e.g. yÎµZ indicates that element y is a member of set Z),

where x is a variable,

where N indicates all natural numbers, defined as positive integers,

and where the mathematical operator âˆª indicates â€œthe union of setsâ€ . For example, the set of regions for m equal to 8, 20 and 80 can be constructed in the following manner. The set of regions, each 8 monomeric subunits in length, S(m=8), in a target nucleic acid sequence 100 subunits in length (L=100), beginning at position 1 (n=1) of the target nucleic acid sequence, can be created using the following expression:

S(8)={R_1,8|nÎµ{1,2,3, . . . ,93}}

and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91, 85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 monomeric subunits in length, in a target sequence 100 subunits in length, beginning at position 1 of the target nucleic acid sequence, can be described using the following expression:

S(20)={R_1,20|nÎµ{1,2,3, . . . ,81}}

and describes the set of regions comprising nucleobases 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 monomeric subunits in length, in a target sequence 100 subunits in length, beginning at position 1 of the target nucleic acid sequence, can be described using the following expression:

S(80)={R_1,80|nÎµ{1,2,3, . . . ,21}}

and describes the set of regions comprising nucleobases 1-80, 2-81, 3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

The union of these aforementioned example sets and other sets for lengths from 10 to 19 and 21 to 79 can be described using the mathematical expression

$A = \underset{m}{â‹ƒ} î¢ž S î¢ (m)$

where âˆª represents the union of the sets obtained by combining all members of all sets.

Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . . 93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The mathematical expressions described herein define all possible target regions in a target nucleic acid sequence of any length L, where the region is of length m, and where m is greater than or equal to the lower limit and less than or equal to the upper limit of monomeric units, and where m is less than L, and where n is less than Lâˆ’m+1.

In the context of this invention, â€œhybridizationâ€ means the pairing of complementary strands of oligomeric compounds. In the present invention, the mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

An oligomeric compound of the invention is â€œspecifically hybridizableâ€ when association of the compound with the target nucleic acid interferes with the normal function of the target nucleic acid to alter the activity, disrupt the function, or modulate the level of the target nucleic acid, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences under conditions in which specific hybridization is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under standard assay conditions in the case of in vitro assays.

In the present invention the phrase â€œstringent hybridization conditionsâ€ or â€œstringent conditionsâ€ refers to conditions under which an oligomeric compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary with different circumstances and in the context of this invention; â€œstringent conditionsâ€ under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. One having ordinary skill in the art will understand variability in the experimental protocols and be able to determine when conditions are optimal for stringent hybridization with minimal non-specific hybridization events.

â€œComplementary,â€ as used herein, refers to the capacity for precise pairing of two monomeric subunits regardless of where in the oligomeric compound or target nucleic acid the two are located. For example, if a monomeric subunit at a certain position of an oligomeric compound is capable of hydrogen bonding with a monomeric subunit at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligomeric compound and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the target nucleic acid are â€œsubstantially complementaryâ€ to each other when a sufficient number of complementary positions in each molecule are occupied by monomeric subunits that can hydrogen bond with each other. Thus, the term â€œsubstantially complementaryâ€ is used to indicate a sufficient degree of precise pairing over a sufficient number of monomeric subunits such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

Generally, an oligomeric compound is â€œantisenseâ€ to a target nucleic acid when, written in the 5â€² to 3â€² direction, it comprises the reverse complement of the corresponding region of the target nucleic acid. â€œAntisense compoundsâ€ are also often defined in the art to comprise the further limitation of, once hybridized to a target, being able to induce or trigger a reduction in target gene expression.

It is understood in the art that the sequence of the oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligomeric compound may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization (e.g., a bulge, a loop structure or a hairpin structure).

In some embodiments of the invention, the oligomeric compounds comprise at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid. In other embodiments of the invention, the oligomeric compounds comprise at least 90% sequence complementarity to a target region within the target nucleic acid. In other embodiments of the invention, the oligomeric compounds comprise at least 95% or at least 99% sequence complementarity to a target region within the target nucleic acid. For example, an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target sequence would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

In some embodiments of the invention, the oligomeric compounds act as mimics or replacements for small non-coding RNAs. In this case, the oligomeric compounds of the invention can comprise at least 70% sequence identity to a small non-coding RNA or a region thereof. In some embodiments the oligomeric compounds of the invention can comprise at least 90% sequence identity and in some embodiments can comprise at least 95% sequence identity to a small non-coding RNA or a region thereof

â€œTargetingâ€ an oligomeric compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose levels, expression or function is to be modulated. This target nucleic acid may be, for example, a mRNA transcribed from a cellular gene whose expression is associated with a particular disorder or disease state, a small non-coding RNA or its precursor, or a nucleic acid molecule from an infectious agent.

The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the interaction to occur such that the desired effect, e.g., modulation of levels, expression or function, will result. Within the context of the present invention, the term â€œregionâ€ is defined as a portion of the target nucleic acid having at least one identifiable sequence, structure, function, or characteristic. Within regions of target nucleic acids are segments. â€œSegmentsâ€ are defined as smaller or sub-portions of regions within a target nucleic acid. â€œSites,â€ as used in the present invention, are defined as specific positions within a target nucleic acid. The terms region, segment, and site can also be used to describe an oligomeric compound of the invention such as for example a gapped oligomeric compound having three separate segments.

Targets of the present invention include both coding and non-coding nucleic acid sequences. For coding nucleic acid sequences, the translation initiation codon is typically 5â€²-AUG (in transcribed mRNA molecules; 5â€²-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the â€œAUG codon,â€ the â€œstart codonâ€ or the â€œAUG start codon.â€ A minority of genes have a translation initiation codon having the RNA sequence 5â€²-GUG, 5â€²-UUG or 5â€²-CUG, and 5â€²-AUA, 5â€²-ACG and 5â€²-CUG have been shown to function in vivo. Thus, the terms â€œtranslation initiation codonâ€ and â€œstart codonâ€ can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, â€œstart codonâ€ and â€œtranslation initiation codonâ€ refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding a nucleic acid target, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or â€œstop codonâ€ ) of a gene may have one of three sequences, i.e., 5â€²-UAA, 5â€²-UAG and 5â€²-UGA (the corresponding DNA sequences are 5â€²-TAA, 5â€²-TAG and 5â€²-TGA, respectively).

The terms â€œstart codon regionâ€ and â€œtranslation initiation codon regionâ€ refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5â€² or 3â€²) from a translation initiation codon. Similarly, the terms â€œstop codon regionâ€ and â€œtranslation termination codon regionâ€ refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5â€² or 3â€²) from a translation termination codon. Consequently, the â€œstart codon regionâ€ (or â€œtranslation initiation codon regionâ€ ) and the â€œstop codon regionâ€ (or â€œtranslation termination codon regionâ€ ) are all regions which may be targeted effectively with the oligomeric compounds of the present invention.

The open reading frame (ORF) or â€œcoding region,â€ which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a further suitable region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

Other target regions include the 5â€² untranslated region (5â€²UTR), known in the art to refer to the portion of an mRNA in the 5â€² direction from the translation initiation codon, and thus including nucleotides between the 5â€² cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3â€² untranslated region (3â€²UTR), known in the art to refer to the portion of an mRNA in the 3â€² direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3â€² end of an mRNA (or corresponding nucleotides on the gene). The 5â€² cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5â€²-most residue of the mRNA via a 5â€²-5â€² triphosphate linkage. The 5â€² cap region of an mRNA is considered to include the 5â€² cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also suitable to target the 5â€² cap region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as â€œintrons,â€ which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as â€œexonsâ€ and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as â€œfusion transcripts.â€ It is also known that introns can be effectively targeted using oligomeric compounds targeted to, precursor molecules for example, pre-mRNA.

It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as â€œvariants.â€ More specifically, â€œpre-mRNA variantsâ€ are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequences.

Upon excision of one or more exon or intron regions, or portions thereof, during splicing, pre-mRNA variants produce smaller â€œmRNA variants.â€ Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as â€œalternative splice variants.â€ If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as â€œalternative start variantsâ€ of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as â€œalternative stop variantsâ€ of that pre-mRNA or mRNA. One specific type of alternative stop variant is the â€œpolyA variantâ€ in which the multiple transcripts produced result from the alternative selection of one of the â€œpolyA stop signalsâ€ by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also target nucleic acids.

Certain non-coding RNA genes are known to produce functional RNA molecules with important roles in diverse cellular processes. Such non-translated, non-coding RNA molecules can include ribosomal RNAs, tRNAs, snRNAs, snoRNAs, tncRNAs, rasiRNAs, short hairpin RNAs (shRNAs), short temporal RNAs (stRNAs), short hairpin RNAs (shRNAs), siRNAs, miRNAs and smnRNAs. These non-coding RNA genes and their products are also suitable targets of the compounds of the invention. Such cellular processes include transcriptional regulation, translational regulation, developmental timing, viral surveillance, immunity, chromosome maintenance, ribosomal structure and function, gene imprinting, subcellular compartmentalization, pre-mRNA splicing, and guidance of RNA modifications. RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, cellular differentiation and DNA elimination.

The locations on the target nucleic acid to which compounds and compositions of the invention hybridize are herein referred to as â€œsuitable target segments.â€ As used herein the term â€œsuitable target segmentâ€ is defined as at least an 8-nucleobase portion of a target region to which oligomeric compound is targeted.

Once one or more targets, target regions, segments or sites have been identified, oligomeric compounds are designed to be sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. The desired effect may include, but is not limited to modulation of the levels, expression or function of the target.

In accordance with the present invention, a series of single stranded oligomeric compounds can be designed to target or mimic one or more specific small non-coding RNAs. These oligomeric compounds can be of a specified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 70 to 450, 110 to 430, 110 to 280, 50 to 110, 60 to 80, 15 to 49, 17 to 25 or 19 to 23 nucleotides long and have one or more modifications.

In accordance with one embodiment of the invention, a series of double-stranded oligomeric compounds (duplexes) comprising, as the antisense strand, the single-stranded oligomeric compounds of the present invention, and the fully or partially complementary sense strand, can be designed to modulate the levels, expression or function of one or more small non-coding RNAs or small non-coding RNA targets. One or both termini of the duplex strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the duplex may be designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the duplex would be complementary over the central region of the duplex, each having overhangs at one or both termini.

For the purposes of this invention, the combination of an antisense strand and a sense strand, each of which can be of a specified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 15 to 49, 17 to 25 or 19 to 23 subunits long, is identified as a complementary pair of oligomeric compounds. This complementary pair of oligonucleotides can include additional nucleotides on either of their 5â€² or 3â€² ends. They can include other molecules or molecular structures on their 3â€² or 5â€² ends, such as a phosphate group on the 5â€² end, or non-nucleic acid moieties conjugated to either terminus of either strand or both strands. One group of compounds of the invention includes a phosphate group on the 5â€² end of the antisense strand compound. Other compounds also include a phosphate group on the 5â€² end of the sense strand compound. Some compounds include additional nucleotides such as a two base overhang on the 3â€² end as well as those lacking overhangs.

For example, a complementary pair of oligomeric compounds may comprise an antisense strand oligomeric compound having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181), having a two-nucleobase overhang of deoxythymidine (dT) and its complement sense strand. This complementary pair of oligomeric compounds would have the following structure:

[Image Omitted]

In some embodiments, a single-stranded oligomeric compound may be designed comprising the antisense portion as a first region and the sense portion as a second region. The first and second regions can be linked together by either a nucleotide linker (a string of one or more nucleotides that are linked together in a sequence) or by a non-nucleotide linker region or by a combination of both a nucleotide and non-nucleotide structure. In any of these structures, the oligomeric compound, when folded back on itself, would form at least a partially complementary structure at least between a portion of the first region, the antisense portion, and a portion of the second region, the sense portion.

In one embodiment, the invention includes an oligomeric compound/protein composition. This composition has both an oligomeric compound component and a protein component. The oligomeric compound component comprises at least one oligomeric compound, either the antisense or the sense oligomeric compound but preferably the antisense oligomeric compound (the oligomeric compound that is antisense to the target nucleic acid). The protein component of the composition comprises at least one protein that forms a portion of the RNA-induced silencing complex, i.e., the RISC complex. The oligomeric compound component can also comprise both antisense and sense strand oligomeric compounds.

RISC is a ribonucleoprotein complex that contains proteins of the Argonaute family of proteins. While not wishing to be bound by theory, it is believed that the Argonaute proteins are a class of proteins, some of which have been shown to contain a PAZ and/or a Piwi domain and that have been implicated in processes previously linked to posttranscriptional silencing. The Argonaute family of proteins includes, but depending on species, is not necessary limited to elF2C1 and elF2C2. It is also believed that at least the antisense strand of double-stranded compounds shown to act as siRNAs is bound to one of the protein components that form the RISC complex, and that the RISC complex interacts with the ribosomes or polyribosome complexes which may contain small non-coding RNA molecules amenable to targeting with the oligomeric compounds of the present invention. Consequently, one embodiment of the invention includes oligomeric compounds that mimic RNA components of the RISC complex.

In one embodiment, the oligomeric compounds of the invention are designed to exert their modulatory effects via mimicking or targeting small non-coding RNAs associated with cellular factors such as transporters or chaperones. These cellular factors can be protein, lipid or carbohydrate based and can have structural or enzymatic functions that may or may not require the complexation of one or more metal ions.

Furthermore, the oligomeric compounds of the invention can have one or more moieties bound or conjugated, which facilitates the active or passive transport, localization, or compartmentalization of the oligomeric compound. Cellular localization includes, but is not limited to, localization to within the nucleus, the nucleolus, or the cytoplasm. Compartmentalization includes, but is not limited to, any directed movement of the oligonucleotides of the invention to a cellular compartment including the nucleus, nucleolus, mitochondrion, or imbedding into a cellular membrane.

In some embodiments of the invention, the oligomeric compounds are designed to exert their modulatory effects via mimicking or targeting small non-coding RNAs associated with cellular factors that affect gene expression, more specifically those involved in RNA or DNA modifications. These modifications include, but are not limited to, posttranscriptional or chromosomal modifications such as methylation, acetylation, pseudouridylation or amination.

Furthermore, the oligomeric compounds of the invention comprise one or more conjugate moieties which facilitate posttranscriptional modification.

The oligomeric compounds of the invention may be in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the oligomeric compounds of the invention may elicit the action of one or more enzymes or proteins to effect modulation of the levels, expression or function of the target nucleic acid.

One non-limiting example of such a protein is the Drosha RNase III enzyme. Drosha is a nuclear enzyme that processes long primary RNA transcripts (pri-miRNAs) from approximately 70 to 450 nucleotides in length into pre-miRNAs (from about 50 to about 80 nucleotides in length) which are exported from the nucleus to encounter the human Dicer enzyme which then processes pre-miRNAs into miRNAs. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3â€² overhang (Lee, et al., Nature, 2003, 425, 415-419). The 3â€² two-nucleotide overhang structure, a signature of RNaseIII enzymatic cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9).

A further non-limiting example involves the enzymes of the RISC complex. Use of the RISC complex to effect cleavage of RNA targets thereby greatly enhances the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

Oligomeric compounds or compositions of the invention are used to induce potent and specific modulation of gene function through interactions with or mimicry of small non-coding RNAs that are processed by the RISC complex. These compounds include single-stranded oligomeric compounds that bind in a RISC complex, double-stranded antisense/sense pairs of oligomeric compounds, or single-stranded oligomeric compounds that include both an antisense portion and a sense portion.

General Oligomer Synthesis:

Oligomerization of modified and unmodified nucleosides is performed according to literature procedures for DNA like compounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. In addition, specific protocols for the synthesis of oligomeric compounds of the invention are illustrated in the examples below.

RNA oligomers can be synthesized by methods disclosed herein or purchased from various RNA synthesis companies such as for example Dharmacon Research Inc., (Lafayette, Colo.).

Irrespective of the particular protocol used, the oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.

Synthesis of Nucleoside Phosphoramidites:

The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5â€²-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5â€²-O-Dimethoxytrityl-2â€²-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5â€²-O-Dimethoxytrityl-2â€²-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, (5â€²-O-(4,4â€²-Dimethoxytriphenylmethyl)-2â€²-deoxy-N⁴-benzoyl-5-methylcytidin-3â€²-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2â€²-Fluorodeoxyadenosine, 2â€²-Fluorodeoxyguanosine, 2â€²-Fluorouridine, 2â€²-Fluorodeoxycytidine, 2â€²-O-(2-Methoxyethyl) modified amidites, 2â€²-O-(2-methoxyethyl)-5-methyluridine intermediate, 5â€²-O-DMT-2â€²-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, (5â€²-O-(4,4â€²-Dimethoxytriphenylmethyl)-2â€²-O-(2-methoxyethyl)-5-methyluridin-3â€²-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5â€²-O-Dimethoxytrityl-2â€²-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5â€²-O-dimethoxytrityl-2â€²-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine penultimate intermediate, (5â€²-O-(4,4â€²-Dimethoxytriphenylmethyl)-2â€²-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3â€²-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), (5â€²-O-(4,4â€²-Dimethoxytriphenylmethyl)-2â€²-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3â€²-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), (5â€²-O-(4,4â€²-Dimethoxytriphenylmethyl)-2â€²-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3â€²-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2â€²-O-(Aminooxyethyl) nucleoside amidites and 2â€²-O-(dimethylaminooxyethyl) nucleoside amidites, 2â€²-(Dimethylaminooxyethoxy) nucleoside amidites, 5â€²-O-tert-Butyldiphenylsilyl-O²-2â€²-anhydro-5-methyluridine, 5â€²-O-tert-Butyldiphenylsilyl-2â€²-O-(2-hydroxyethyl)-5-methyluridine, 2â€²-O-((2-phthalimidoxy)ethyl)-5â€²-t-butyldiphenylsilyl-5-methyluridine, 5â€²-O-tert-butyldiphenylsilyl-2â€²-O-((2-formadoximinooxy)ethyl)-5-methyluridine, 5â€²-O-tert-Butyldiphenylsilyl-2â€²-Oâ€”(N,N dimethylaminooxyethyl)-5-methyluridine, 2â€²-O-(dimethylaminooxyethyl)-5-methyluridine, 5â€²-O-DMT-2â€²-O-(dimethylaminooxyethyl)-5-methyluridine, 5â€²-O-DMT-2â€²-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3â€²-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2â€²-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2â€²-O-(2-ethylacetyl)-5â€²-O-(4,4â€²-dimethoxytrityl)guanosine-3â€²-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2â€²-dimethylaminoethoxyethoxy (2â€²-DMAEOE) nucleoside amidites, 2â€²-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine, 5â€²-O-dimethoxytrityl-2â€²-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine and 5â€²-O-Dimethoxytrityl-2â€²-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine-3â€²-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Oligonucleotide and Oligonucleoside Synthesis:

Oligonucleotides: Unsubstituted and substituted phosphodiester (Pâ• O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (Pâ• S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55Â° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

3â€²-Deoxy-3â€²-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

3â€²-Deoxy-3â€²-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for instance, alternating MMI and Pâ• O or Pâ• S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

RNA Synthesis:

In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5â€²-hydroxyl in combination with an acid-labile orthoester protecting group on the 2â€²-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2â€² hydroxyl.

Following this procedure for the sequential protection of the 5â€²-hydroxyl in combination with protection of the 2â€²-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3â€²- to 5â€²-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3â€²-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5â€²-end of the first nucleoside. The support is washed and any unreacted 5â€²-hydroxyl groups are capped with acetic anhydride to yield 5â€²-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5â€²-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55Â° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2â€²-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

The 2â€²-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

The present invention is also useful for the preparation of oligomeric compounds incorporating at least one 2â€²-O-protected nucleoside. After incorporation and appropriate deprotection the 2â€²-O-protected nucleoside will be converted to a ribonucleoside at the position of incorporation. The number and position of the 2-ribonucleoside units in the final oligomeric compound can vary from one at any site or the strategy can be used to prepare up to a full 2â€²-OH modified oligomeric compound. All 2â€²-O-protecting groups amenable to the synthesis of oligomeric compounds are included in the present invention.

In general a protected nucleoside is attached to a solid support by for example a succinate linker. Then the oligonucleotide is elongated by repeated cycles of deprotecting the 5â€²-terminal hydroxyl group, coupling of a further nucleoside unit, capping and oxidation (alternatively sulfurization). In a more frequently used method of synthesis the completed oligonucleotide is cleaved from the solid support with the removal of phosphate protecting groups and exocyclic amino protecting groups by treatment with an ammonia solution. Then a further deprotection step is normally required for the more specialized protecting groups used for the protection of 2â€²-hydroxyl groups which will give the fully deprotected oligonucleotide.

A large number of 2â€²-O-protecting groups have been used for the synthesis of oligoribonucleotides but over the years more effective groups have been discovered. The key to an effective 2â€²-O-protecting group is that it is capable of selectively being introduced at the 2â€²-O-position and that it can be removed easily after synthesis without the formation of unwanted side products. The protecting group also needs to be inert to the normal deprotecting, coupling, and capping steps required for oligoribonucleotide synthesis. Some of the protecting groups used initially for oligoribonucleotide synthesis included tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groups are not compatible with all 5â€²-O-protecting groups so modified versions were used with 5â€²-DMT groups such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identified a number of piperidine derivatives (like Fpmp) that are useful in the synthesis of oligoribonucleotides including 1-((chloro-4-methyl)phenyl)-4â€²-methoxypiperidin-4-yl (Reese et al., Tetrahedron Lett., 1986, (27), 2291). Another approach was to replace the standard 5â€²-DMT (dimethoxytrityl) group with protecting groups that were removed under non-acidic conditions such as levulinyl and 9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile 2â€²-protecting groups for oligoribonucleotide synthesis. Another more widely used protecting group initially used for the synthesis of oligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al., Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett., 1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The 2â€²-O-protecting groups can require special reagents for their removal such as for example the t-butyldimethylsilyl group is normally removed after all other cleaving/deprotecting steps by treatment of the oligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2â€²-O-protecting groups (Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoride labile and photolabile protecting groups that are removed using moderate conditions. One photolabile group that was examined was the (2-(nitrobenzyl)oxy)methyl (nbm) protecting group (Schwartz et al., Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examined included a number structurally related formaldehyde acetal-derived, 2â€²-O-protecting groups. Also prepared were a number of related protecting groups for preparing 2â€²-O-alkylated nucleoside phosphoramidites including 2â€²-O-((triisopropylsilyl)oxy)methyl (2â€²-Oâ€”CH₂â€”Oâ€”Si(iPr)₃, TOM). One 2â€²-O-protecting group that was prepared to be used orthogonally to the TOM group was 2â€²-O-((R)-1-(2-nitrophenyl)ethyloxy)methyl) ((R)-mnbm).

Another strategy using a fluoride labile 5â€²-O-protecting group (non-acid labile) and an acid labile 2â€²-O-protecting group has been reported (Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number of possible silyl ethers were examined for 5â€²-O-protection and a number of acetals and orthoesters were examined for 2â€²-O-protection. The protection scheme that gave the best results was 5â€²-O-silyl ether-2â€²-ACE (5â€²-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2â€²-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses a modified phosphoramidite synthesis approach in that some different reagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis of oligoribonucleotides the main RNA synthesis strategies that are presently being used commercially include 5â€²-O-DMT-2â€²-O-t-butyldimethylsilyl (TBDMS), 5â€²-O-DMT-2â€²-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP), 2â€²-O-((triisopropylsilyl)oxy)methyl (2â€²-Oâ€”CH₂â€”Oâ€”Si(iPr)₃(TOM), and the 5â€²-O-silyl ether-2â€²-ACE (5â€²-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2â€²-O-bis(2-acetoxyethoxy)methyl (ACE). A current list of some of the major companies currently offering RNA products include Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri Biotechnologies Inc., and Integrated DNA Technologies, Inc. One company, Princeton Separations, is marketing an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the present invention.

The structures corresponding to these protecting groups are shown below.

TBDMS=5â€²-O-DMT-2â€²-O-t-butyldimethylsilyl;

TOM=2â€²-O-((triisopropylsilyl)oxy)methyl;

DOD/ACE=(5â€²-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2â€²-O-bis(2-acetoxyethoxy)methyl

FPMP=5â€²-O-DMT-2â€²-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl)

[Image Omitted]

All of the aforementioned RNA synthesis strategies are amenable to the present invention. Strategies that would be a hybrid of the above e.g. using a 5â€²-protecting group from one strategy with a 2â€²-O-protecting from another strategy is also amenable to the present invention.

The preparation of ribonucleotides and oligomeric compounds having at least one ribonucleoside incorporated and all the possible configurations falling in between these two extremes are encompassed by the present invention. The corresponding oligomeric compounds can be hybridized to further oligomeric compounds including oligoribonucleotides having regions of complementarity to form double-stranded (duplexed) oligomeric compounds.

The methods of preparing oligomeric compounds of the present invention can also be applied in the areas of drug discovery and target validation.

Oligonucleotide Isolation:

After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55Â° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the âˆ’16 amu product (+/âˆ’32+/âˆ’48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Oligonucleotide Synthesisâ€”96 Well Plate Format:

Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60Â° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Oligonucleotide Analysisâ€”96-Well Plate Format:

The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACEâ„¢ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACEâ„¢ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the oligomeric compounds on the plate were at least 85% full length.

For double-stranded compounds of the invention, once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 Once diluted, 30 Î¼L of each strand is combined with 15 Î¼L of a 5Ã— solution of annealing buffer. The final concentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 Î¼L. This solution is incubated for 1 minute at 90Â° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37Â° C. at which time the double-stranded compounds are used in experimentation. The final concentration of the duplexed compound is 20 Î¼M. This solution can be stored frozen (âˆ’20Â° C.) and freeze-thawed up to 5 times.

Once prepared, the double-stranded compounds are evaluated for their ability to modulate target levels, expression or function. When cells reach 80% confluency, they are treated with synthetic double-stranded compounds comprising at least one oligomeric compound of the invention. For cells grown in 96-well plates, wells are washed once with 200 Î¼L OPTI-MEMâ„¢-1 reduced-serum medium (Gibco BRL) and then treated with 130 Î¼L of OPTI-MEMâ„¢-1 containing 12 Î¼g/mL LIPOFECTINâ„¢ (Invitrogen Corporation, Carlsbad, Calif.) and the desired double stranded compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by real-time RT-PCR.

Specific examples of oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage (phosphorothioate) did not significantly interfere with RNAi activity. Based on this observation, it is suggested that certain oligomeric compounds of the invention can also have one or more modified internucleoside linkages. A suitable phosphorus-containing modified internucleoside linkage is the phosphorothioate internucleoside linkage.

Modified oligonucleotide backbones (internucleoside linkages) containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3â€²-alkylene phosphonates, 5â€²-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3â€²-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3â€²-5â€² linkages, 2â€²-5â€² linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3â€² to 3â€², 5â€² to 5â€² or 2â€² to 2â€² linkage. Oligonucleotides having inverted polarity comprise a single 3â€² to 3â€² linkage at the 3â€²-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

In other embodiments of the invention, oligomeric compounds have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular â€”CH₂â€”NHâ€”Oâ€”CH₂â€”, â€”CH₂â€”N(CH₃)â€”Oâ€”CH₂â€” (known as a methylene (methylimino) or MMI backbone), â€”CH₂â€”Oâ€”N(CH₃)â€”CH₂â€”, â€”CH₂â€”N(CH₃)â€”N(CH₃)â€”CH₂â€” and â€”Oâ€”N(CH₃)â€”CH₂â€”CH₂â€” (wherein the native phosphodiester internucleotide linkage is represented as â€”Oâ€”P(â• O)(OH)â€”Oâ€”CH₂â€”). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.

Modified oligonucleotide backbones (internucleoside linkages) that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Another group of oligomeric compounds amenable to the present invention includes oligonucleotide mimetics. The term mimetic as it is applied to oligonucleotides is intended to include oligomeric compounds wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Teaching of PNA oligomeric compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

PNA has been modified to incorporate numerous modifications since the basic PNA structure was first prepared. The basic structure is shown below:

[Image Omitted]

wherein

Bx is a heterocyclic base moiety;

T₄is hydrogen, an amino protecting group, â€”C(O)R₅, substituted or unsubstituted C₁-C₁₀alkyl, substituted or unsubstituted C₂-C₁₀alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group, a reporter group, a conjugate group, a D or L Î±-amino acid linked via the Î±-carboxyl group or optionally through the Ï‰-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

T₅is â€”OH, â€”N(Z₁)Z₂, R₅, D or L Î±-amino acid linked via the Î±-amino group or optionally through the Ï‰-amino group when the amino acid is lysine or ornithine or a peptide derived from D, L or mixed D and L amino acids linked through an amino group, a chemical functional group, a reporter group or a conjugate group;

Z₁is hydrogen, C₁-C₆alkyl, or an amino protecting group;

Z₂is hydrogen, C₁-C₆alkyl, an amino protecting group, â€”C(â• O)â€”(CH₂)_n-J-Z₃, a D or L Î±-amino acid linked via the Î±-carboxyl group or optionally through the Ï‰-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group;

Z₃is hydrogen, an amino protecting group, â€”C₁-C₆alkyl, â€”C(â• O)â€”CH₃, benzyl, benzoyl, or â€”(CH₂)_nâ€”N(H)Z₁;

each J is O, S or NH;

R₅is a carbonyl protecting group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. A suitable class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits.

Morpholino nucleic acids have been prepared having a variety of different linking groups (L₂) joining the monomeric subunits. The basic formula is shown below:

[Image Omitted]

wherein

T₁is hydroxyl or a protected hydroxyl;

T₅is hydrogen or a phosphate or phosphate derivative;

L₂is a linking group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in an DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. coli RNase resulting in cleavage of the target RNA strand.

The general formula of CeNA is shown below:

[Image Omitted]

wherein

each Bx is a heterocyclic base moiety;

T₁is hydroxyl or a protected hydroxyl;

T₂is hydroxyl or a protected hydroxyl;

L₃is a linking group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid) can be prepared from one or more anhydrohexitol nucleosides (see, Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) and would have the general formula:

[Image Omitted]

Another group of modifications includes nucleosides having sugar moieties that are bicyclic thereby locking the sugar conformational geometry. The most studied of these nucleosides is a bicyclic sugar moiety having a 4â€²-CH₂â€”O-2â€² bridge. As can be seen in the structure below the 2â€²-Oâ€” has been linked via a methylene group to the 4â€² carbon. This bridge attaches under the sugar as shown forcing the sugar ring into a locked 3â€²-endo conformation geometry. The V-L nucleoside has also been reported wherein the linkage is above the ring and the heterocyclic base is in the V rather than the âˆƒ-conformation (see U.S. Patent Application Publication No.: Application 2003/0087230). The xylo analog has also been prepared (see U.S. Patent Application Publication No.: 2003/0082807). The preferred bridge for a locked nucleic acid (LNA) is 4â€²-(â€”CH₂â€”)_nâ€”O-2â€² wherein n is 1 or 2. The literature is confusing when the term locked nucleic acid is used but in general locked nucleic acids refers to n=1, ENAâ„¢ refers to n=2 (Kaneko et al., U.S. Patent Application Publication No.: US 2002/0147332, Singh et al., Chem. Commun, 1998, 4, 455-456, also see U.S. Pat. Nos. 6,268,490 and 6,670,461 and U.S. Patent Application Publication No. US 2003/0207841). However the term locked nucleic acids can also be used in a more general sense to describe any bicyclic sugar moiety that has a locked conformation.

ENAâ„¢ along with LNA (n=1) have been studied more than the myriad of other analogs. Oligomeric compounds incorporating LNA and ENA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3â€²-exonucleolytic degradation and good solubility properties.

The basic structure of LNA showing the bicyclic ring system is shown below:

[Image Omitted]

wherein

each Bx is a heterocyclic base moiety;

each L₁is an internucleoside linking group;

T₁is hydroxyl or a protected hydroxyl;

T₂is hydroxyl or a protected hydroxyl, and

n is from 1 to about 80.

The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA: DNA duplex confirmed the 3â€²-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are useful in a wide range of diagnostic and therapeutic applications. Among these are antisense applications, PCR applications, strand-displacement oligomers, substrates for nucleic acid polymerases and generally as nucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that LNAs confer several desired properties to antisense agents. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. LIPOFECTINâ„¢-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2â€²-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., PCT International Application WO 98-DK393 19980914). Furthermore, synthesis of 2â€²-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog with a handle has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2â€²-Amino- and 2â€²-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

Some oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs having the formulas (amidite monomers shown):

[Image Omitted]

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acid and incorporates a phosphorus group in the backbone. This class of olignucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by reference in their entirety) is shown below.

[Image Omitted]

Another oligonucleotide mimetic has been reported wherein the furanosyl ring has been replaced by a cyclobutyl moiety.

Modified Sugars

Oligomeric compounds of the invention may also contain one or more substituted sugar moieties. These oligomeric compounds comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly suitable are O((CH₂)_nO)_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON((CH₂)_nCH₃)₂, where n and m are from 1 to about 10. Some oligonucleotides comprise a sugar substituent group selected from: C₁to C₁₀lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2â€²-methoxyethoxy (2â€²-Oâ€”CH₂CH₂OCH₃, also known as 2â€²-O-(2-methoxyethyl) or 2â€²-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. One modification includes 2â€²-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2â€²-DMAOE, as described in examples hereinbelow, and 2â€²-dimethylaminoethoxyethoxy (also known in the art as 2â€²-O-dimethyl-amino-ethoxy-ethyl or 2â€²-DMAEOE), i.e., 2â€²-Oâ€”CH₂â€”Oâ€”CH₂â€”N(CH₃)₂.

Other sugar substituent groups include methoxy (â€”Oâ€”CH₃), aminopropoxy (â€”OCH₂CH₂CH₂NH₂), allyl (â€”CH₂â€”CHâ• CH₂), â€”O-allyl (â€”Oâ€”CH₂â€”CHâ• CH₂) and fluoro (F). 2â€²-Sugar substituent groups may be in the arabino (up) position or ribo (down) position. One 2â€²-arabino modification is 2â€²-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3â€² position of the sugar on the 3â€² terminal nucleoside or in 2â€²-5â€² linked oligonucleotides and the 5â€² position of 5â€² terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

Representative sugar substituent groups include groups of formula I_aor II_a:

[Image Omitted]

wherein:

R_bis O, S or NH;

R_dis a single bond, O, S or C(â• O);

R_eis C₁-C₁₀alkyl, N(R_k)(R_m), N(R_k)(R_n), Nâ• C(R_p)(R_q), Nâ• C(R_p)(R_r) or has formula

[Image Omitted]

R_pand R_qare each independently hydrogen or C₁-C₁₀alkyl;

R_ris â€”R_x-R_y;

each R_s, R_t, R_uand R_vis, independently, hydrogen, C(O)R_w, substituted or unsubstituted C₁-C₁₀alkyl, substituted or unsubstituted C₂-C₁₀alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_uand R_v, together form a phthalimido moiety with the nitrogen atom to which they are attached;

each R_wis, independently, substituted or unsubstituted C₁-C₁₀alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R_kis hydrogen, a nitrogen protecting group or â€”R_x-R_y;

R_pis hydrogen, a nitrogen protecting group or â€”R_x-R_y;

R_xis a bond or a linking moiety;

R_yis a chemical functional group, a conjugate group or a solid support medium;

each R_mand R_nis, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀alkyl, substituted or unsubstituted C₂-C₁₀alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃⁺, N(R_u)(R_v), guanidino and acyl where said acyl is an acid amide or an ester;

or R_mand R_n, together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group;

R_iis OR_z, SR_z, or N(R_z)₂;

each R_zis, independently, H, C₁-C₈alkyl, C₁-C₈haloalkyl, C(â• NH)N(H)R_u, C(â• O)N(H)R_uor OC(â• O)N(H)R_u;

R_f, R_gand R_hcomprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;

R_jis alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R_k)(R_m)OR_k, halo, SR_kor CN;

m_ais 1 to about 10;

each mb is, independently, 0 or 1;

mc is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups are disclosed in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled â€œCapped 2â€²-Oxyethoxy Oligonucleotides,â€ hereby incorporated by reference in its entirety.

Representative cyclic substituent groups are disclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998, entitled â€œRNA Targeted 2â€²-Oligomeric compounds that are Conformationally Preorganized,â€ hereby incorporated by reference in its entirety.

Particular sugar substituent groups include O((CH₂)_nO)_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON((CH₂)_nCH₃))₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups are disclosed in U.S. patent application Ser. No. 09/349,040, entitled â€œFunctionalized Oligomers,â€ filed Jul. 7, 1999, hereby incorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat. No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups are disclosed in International Patent Application PCT/US99/17895, entitled â€œ2â€²-O-Dimethylaminoethyloxyethyl-Oligomeric compoundsâ€ , filed Aug. 6, 1999, hereby incorporated by reference in its entirety.

Synthesis of Chimeric Oligonucleotides:

Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the â€œgapâ€ segment of linked nucleosides is positioned between 5â€² and 3â€² â€œwingâ€ segments of linked nucleosides and a second â€œopen endâ€ type wherein the â€œgapâ€ segment is located at either the 3â€² or the 5â€² terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as â€œgapmersâ€ or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as â€œhemimersâ€ or â€œwingmers.â€

(2â€²-O-Me)-(2â€²-deoxy)-(2â€²-O-Me) Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2â€²-O-alkyl phosphorothioate and 2â€²-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2â€²-deoxy-5â€²-dimethoxytrityl-3â€²-O-phosphoramidite for the DNA portion and 5â€²-dimethoxytrityl-2â€²-O-methyl-3â€²-O-phosphoramidite for 5â€² and 3â€² wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5â€²-dimethoxytrityl-2â€²-O-methyl-3â€²-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55Â° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

(2â€²-O-(2-Methoxyethyl))-(2â€²-deoxy)-(2â€²-O-(Methoxyethyl)) Chimeric Phosphorothioate Oligonucleotides

(2â€²-O-(2-methoxyethyl))-(2â€²-deoxy)-(-2â€²-O-(methoxyethyl)) chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2â€²-O-methyl chimeric oligonucleotide, with the substitution of 2â€²-O-(methoxyethyl) amidites for the 2â€²-O-methyl amidites.

(2â€²-O-(2-Methoxyethyl)Phosphodiester)-(2â€²-deoxy Phosphorothioate)-(2â€²-O-(2-Methoxyethyl) Phosphodiester) Chimeric Oligonucleotides

(2â€²-O-(2-methoxyethyl phosphodiester)-(2â€²-deoxy phosphorothioate)-(2â€²-O-(methoxyethyl) phosphodiester) chimeric oligonucleotides are prepared as per the above procedure for the 2â€²-O-methyl chimeric oligonucleotide with the substitution of 2â€²-O-(methoxyethyl) amidites for the 2â€²-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

The terms used to describe the conformational geometry of homoduplex nucleic acids are â€œA Formâ€ for RNA and â€œB Formâ€ for DNA. The respective conformational geometry for RNA and DNA duplexes was determined from X-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2â€² hydroxyl in RNA biases the sugar toward a C3â€² endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2â€² hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2â€² endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). As used herein, B-form geometry is inclusive of both C2â€²-endo pucker and O4â€²-endo pucker. This is consistent with Berger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, who pointed out that in considering the furanose conformations which give rise to B-form duplexes consideration should also be given to a O4â€²-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of the duplex formed between a target RNA and a synthetic sequence is central to therapies such as, but not limited to, antisense mechanisms, including RNase H-mediated and RNA interference mechanisms, as these mechanisms involved the hybridization of a synthetic sequence strand to an RNA target strand. In the case of RNase H, effective inhibition of the mRNA requires that the antisense sequence achieve at least a threshold of hybridization.

One routinely used method of modifying the sugar puckering is the substitution of the sugar at the 2â€²-position with a substituent group that influences the sugar geometry. The influence on ring conformation is dependent on the nature of the substituent at the 2â€²-position. A number of different substituents have been studied to determine their sugar puckering effect. For example, 2â€²-halogens have been studied showing that the 2â€²-fluoro derivative exhibits the largest population (65%) of the C3â€²-endo form, and the 2â€²-iodo exhibits the lowest population (7%). The populations of adenosine (2â€²-OH) versus deoxyadenosine (2â€²-H) are 36% and 19%, respectively. Furthermore, the effect of the 2â€²-fluoro group of adenosine dimers (2â€²-deoxy-2â€²-fluoroadenosine-2â€²-deoxy-2â€²-fluoro-adenosine) is also correlated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced by replacement of 2â€²-OH groups with 2â€²-F groups thereby increasing the C3â€²-endo population. It is assumed that the highly polar nature of the 2â€²-F bond and the extreme preference for C3â€²-endo puckering may stabilize the stacked conformation in an A-form duplex. Data from UV hypochromicity, circular dichroism, and ¹H NMR also indicate that the degree of stacking decreases as the electronegativity of the halo substituent decreases. Furthermore, steric bulk at the 2â€²-position of the sugar moiety is better accommodated in an A-form duplex than a B-form duplex. Thus, a 2â€²-substituent on the 3â€²-terminus of a dinucleoside monophosphate is thought to exert a number of effects on the stacking conformation: steric repulsion, furanose puckering preference, electrostatic repulsion, hydrophobic attraction, and hydrogen bonding capabilities. These substituent effects are thought to be determined by the molecular size, electronegativity, and hydrophobicity of the substituent. Melting temperatures of complementary strands is also increased with the 2â€²-substituted adenosine diphosphates. It is not clear whether the 3â€²-endo preference of the conformation or the presence of the substituent is responsible for the increased binding. However, greater overlap of adjacent bases (stacking) can be achieved with the 3â€²-endo conformation.

Nucleoside conformation is influenced by various factors including substitution at the 2â€², 3â€² or 4â€²-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2â€² position to favor the 3â€²-endo conformation can be achieved while maintaining the 2â€²-OH as a recognition element, as illustrated in FIG. 2, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3â€²-endo conformation can be achieved by deletion of the 2â€²-OH as exemplified by 2â€² deoxy-2â€²F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3â€²-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4â€²-position to give 4â€²-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3â€²-endo conformation.

In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3â€²-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3â€²-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3â€²-endo conformational geometry (see Scheme 1). There is an apparent preference for an RNA type duplex (A form helix, predominantly 3â€²-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2â€²-deoxy-2â€²-F-nucleosides appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3â€²-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric compounds designed to act as triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3â€²-endo type conformation.

[Image Omitted]

Along similar lines, oligomeric triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3â€²-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modified nucleosides amenable to the present invention are shown below. These examples are meant to be representative and not exhaustive.

[Image Omitted]

Oligomeric compounds may also include nucleobase (often referred to in the art simply as â€œbaseâ€ or â€œheterocyclic base moietyâ€ ) modifications or substitutions. As used herein, â€œunmodifiedâ€ or â€œnaturalâ€ nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (â€”Câ‰¡Câ€”CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Some nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2Â° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2â€²-O-methoxyethyl sugar modifications.

In one aspect of the present invention oligomeric compounds are prepared having polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Many of these polycyclic heterocyclic compounds have the general formula:

[Image Omitted]

Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀â• O, R₁₁-R₁₄â• H) (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one (R₁₀â• S, R₁₁-R₁₄â• H), (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀â• O, R₁₁-R₁₄â• F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). When incorporated into oligonucleotides, these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Patent Application Publication 20030207804 and U.S. Patent Application Publication 20030175906, both of which are incorporated herein by reference in their entirety).

Helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (R₁₀â• O, R₁₁â• â€”Oâ€”(CH₂)₂â€”NH₂, R_12-14â• H) (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a Î”T_mof up to 18Â° relative to 5-methyl cytosine (dC5^me), which is the highest known affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides. The T_mdata indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5^me. It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the O6, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.

Tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Pat. No. 6,028,183, and U.S. Pat. No. 6,007,992, the contents of both are incorporated herein in their entirety.

The enhanced binding affinity of the phenoxazine derivatives together with their sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions can activate RNaseH, enhance cellular uptake and exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20mer 2â€²-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).

Modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Patent Application Publication 20030158403, each of which is incorporated herein by reference in its entirety.

One substitution that can be appended to the oligomeric compounds of the invention involves the linkage of one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligomeric compounds. In one embodiment such modified oligomeric compounds are prepared by covalently attaching conjugate groups to functional groups such as hydroxyl or amino groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, carbohydrates, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

The oligomeric compounds of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

Oligomeric compounds used in the compositions of the present invention can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of oligomeric compounds to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By â€œcap structure or terminal cap moietyâ€ is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5â€²-terminus (5â€²-cap) or at the 3â€²-terminus (3â€²-cap) or can be present on both termini. For double-stranded oligomeric compounds, the cap may be present at either or both termini of either strand. In non-limiting examples, the 5â€²-cap includes inverted abasic residue (moiety), 4â€²,5â€²-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4â€²-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3â€²,4â€²-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3â€²-3â€²-inverted nucleotide moiety; 3â€²-3â€²-inverted abasic moiety; 3â€²-2â€²-inverted nucleotide moiety; 3â€²-2â€²-inverted abasic moiety; 1,4-butanediol phosphate; 3â€²-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3â€²-phosphate; 3â€²-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).

Particularly preferred 3â€²-cap structures of the present invention include, for example 4â€²,5â€²-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4â€²-thio nucleotide, carbocyclic nucleotide; 5â€²-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3â€²,4â€²-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5â€²-5â€²-inverted nucleotide moiety; 5â€²-5â€²-inverted abasic moiety; 5â€²-phosphoramidate; 5â€²-phosphorothioate; 1,4-butanediol phosphate; 5â€²-amino; bridging and/or non-bridging 5â€²-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5â€²-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

Further 3â€² and 5â€²-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

It is not necessary for all positions in an oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligomeric compound or even at a single monomeric subunit such as a nucleoside within a oligomeric compound. The present invention also includes oligomeric compounds which are chimeric oligomeric compounds. â€œChimericâ€ oligomeric compounds or â€œchimeras,â€ in the context of this invention, are oligomeric compounds that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, an oligomeric compound may be designed to comprise a region that serves as a substrate for RNase H. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H by an oligomeric compound having a cleavage region, therefore, results in cleavage of the RNA target, thereby enhancing the efficiency of the oligomeric compound. Consequently, comparable results can often be obtained with shorter oligomeric compounds having substrate regions when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric oligomeric compounds of the invention may be formed as composite structures of two or more oligonucleotides, oligonucleotide mimics, oligonucleotide analogs, oligonucleosides and/or oligonucleotide mimetics as described above. Such oligomeric compounds have also been referred to in the art as hybrids, hemimers, gapmers or inverted gapmers. Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

The conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA-like conformations (A-form duplex geometry in an oligomeric context), are useful in the oligomeric compounds of the present invention. The synthesis of modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum Press.)

In one aspect, the present invention is directed to oligomeric compounds that are designed to have enhanced properties compared to native RNA. One method to design optimized or enhanced oligomeric compounds involves each nucleoside of the selected sequence being scrutinized for possible enhancing modifications. One modification would be the replacement of one or more RNA nucleosides with nucleosides that have the same 3â€²-endo conformational geometry. Such modifications can enhance chemical and nuclease stability relative to native RNA while at the same time being much cheaper and easier to synthesize and/or incorporate into an oligonucleotide. The sequence can be further divided into regions and the nucleosides of each region evaluated for enhancing modifications that can be the result of a chimeric configuration. Consideration is also given to the 5â€² and 3â€²-termini as there are often advantageous modifications that can be made to one or more of the terminal nucleosides. The oligomeric compounds of the present invention may include at least one 5â€²-modified phosphate group on a single strand or on at least one 5â€²-position of a double-stranded sequence or sequences. Other modifications considered are internucleoside linkages, conjugate groups, substitute sugars or bases, substitution of one or more nucleosides with nucleoside mimetics and any other modification that can enhance the desired property of the oligomeric compound.

One synthetic 2â€²-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2-methoxyethoxy (2â€²-MOE, 2â€²-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages of the 2â€²-MOE substitution is the improvement in binding affinity, which is greater than many similar 2â€² modifications such as O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2â€²-O-methoxyethyl substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotides having the 2â€²-MOE modification displayed improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides having 2â€²-MOE substituents in the wing nucleosides and an internal region of deoxyphosphorothioate nucleotides (also termed a gapped oligonucleotide or gapmer) have shown effective reduction in the growth of tumors in animal models at low doses. 2â€²-MOE substituted oligonucleotides have also shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.

Unless otherwise defined herein, alkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbyl containing at least one, or about 1 to about 3 hetero atoms in the chain, including the terminal portion of the chain. Suitable heteroatoms include N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, C₃-C₈, or C₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkenyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkynyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moiety containing at least three ring members, at least one of which is carbon, and of which 1, 2 or three ring members are other than carbon. The number of carbon atoms can vary from 1 to about 12, from 1 to about 6, and the total number of ring members varies from three to about 15, or from about 3 to about 8. Suitable ring heteroatoms are N, O and S. Suitable heterocycloalkyl groups include, but are not limited to, morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ring structure containing at least one aryl ring. Suitable aryl rings have about 6 to about 20 ring carbons. Especially suitable aryl rings include phenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containing at least one fully unsaturated ring, the ring consisting of carbon and non-carbon atoms. The ring system can contain about 1 to about 4 rings. The number of carbon atoms can vary from 1 to about 12, from 1 to about 6, and the total number of ring members varies from three to about 15, or from about 3 to about 8. Suitable ring heteroatoms are N, O and S. Suitable hetaryl moieties include, but are not limited to, pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl, etc.

Unless otherwise defined herein, where a moiety is defined as a compound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl and alkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is a group, such as the cyano or isocyanato group that draws electronic charge away from the carbon to which it is attached. Other electron withdrawing groups of note include those whose electronegativities exceed that of carbon, for example halogen, nitro, or phenyl substituted in the ortho- or para-position with one or more cyano, isothiocyanato, nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have their ordinary meanings. Suitable halo (halogen) substituents are Cl, Br, and I.

The aforementioned optional substituents are, unless otherwise herein defined, suitable substituents depending upon desired properties. Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties, NO₂, NH₃(substituted and unsubstituted), acid moieties (e.g. â€”CO₂H, â€”OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, aryl moieties, etc. In all the preceding formulae, the squiggle (Ëœ) indicates a bond to an oxygen or sulfur of the 5â€²-phosphate.

Phosphate protecting groups include those described in U.S. Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expressly incorporated herein by reference in its entirety.

Screening methods for the identification of effective modulators of small non-coding RNAs are also comprehended by the instant invention and comprise the steps of contacting a small non-coding RNA, or portion thereof, with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the levels, expression or alter the function of the small non-coding RNA. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the levels, expression or altering the function of the small non-coding RNA, the modulator may then be employed in further investigative studies, or for use as a target validation, research, diagnostic, or therapeutic agent in accordance with the present invention.

Screening methods for the identification of small non-coding RNA mimics are also within the scope of the invention. Screening for small non-coding RNA modulators or mimics can also be performed in vitro, ex vivo, or in vivo by contacting samples, tissues, cells or organisms with candidate modulators or mimics and selecting for one or more candidate modulators which show modulatory effects.

Design and Screening of Duplexed Oligomeric Compounds:

In screening and target validation studies, oligomeric compounds of the invention can be used in combination with their respective complementary strand oligomeric compound to form stabilized double-stranded (duplexed) oligonucleotides. In accordance with the present invention, a series of duplexes comprising the oligomeric compounds of the present invention and their complements can be designed to target a small non-coding RNA. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in some embodiments, both strands of the duplex would be complementary over the central nucleobases, each having overhangs at one or both termini, as described supra.

In some embodiments, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181) may be prepared with blunt ends (no single stranded overhang) as shown:

[Image Omitted]

In other embodiments, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG, having a two-nucleobase overhang of deoxythymidine (dT) and its complement sense strand may be prepared with overhangs as shown:

[Image Omitted]

RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).

For use in drug discovery, oligomeric compounds of the present invention are used to elucidate relationships that exist between small non-coding RNAs, genes or proteins and a disease state, phenotype, or condition. These methods include detecting or modulating a target comprising contacting a sample, tissue, cell, or organism with the oligomeric compounds and compositions of the present invention, measuring the levels of the target and/or the levels of downstream gene products including mRNA or proteins encoded thereby, a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to an untreated sample, a positive control or a negative control. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a disease.

The oligomeric compounds and compositions of the present invention can additionally be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Such uses allows for those of ordinary skill to elucidate the function of particular non-coding or coding nucleic acids or to distinguish between functions of various members of a biological pathway.

For use in kits and diagnostics, the oligomeric compounds and compositions of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of non-coding or coding nucleic acids expressed within cells and tissues.

As one non-limiting example, expression patterns within cells or tissues treated with one or more oligomeric compounds or compositions of the invention are compared to control cells or tissues not treated with the compounds or compositions and the patterns produced are analyzed for differential levels of nucleic acid expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.

Cell Culture and Oligonucleotide Treatment:

The effects of oligomeric compounds on target nucleic acid expression or function can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or real-time RT-PCR. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is present in the cell type chosen.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells harvested when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in real-time RT-PCR analysis.

A549 Cells:

The human lung carcinoma cell line A549 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

HMECs:

Normal human mammary epithelial cells (HMECs) are obtained from American Type Culture Collection (Manassas, Va.). HMECs are routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. HMECs are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of 50,000-60,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. HMECs are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 10,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.

MCF7 Cells:

The breast carcinoma cell line MCF7 is obtained from American Type Culture Collection (Manassas, Va.). MCF7 cells are routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. MCF7 cells are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of approximately 140,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. MCF7 cells are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 20,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.

T47D Cells:

The breast carcinoma cell line T47D is obtained from American Type Culture Collection (Manassas, Va.). T47D cells are deficient in expression of the tumor suppressor gene p53. T47D cells are cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. T47D cells are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of approximately 170,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. T47D cells are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 20,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.

BJ Cells:

The normal human foreskin fibroblast BJ cell line was obtained from American Type Culture Collection (Manassas, Va.). BJ cells were routinely cultured in MEM high glucose with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate (all media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were plated on collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well, and allowed to attach to wells overnight.

B16-F10 Cells:

The mouse melanoma cell line B16-F10 was obtained from American Type Culture Collection (Manassas, Va.). B16-F10 cells were routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were seeded into collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well and allowed to attach overnight.

HUVECs:

Human vascular endothelial cells (HUVECs) are obtained from American Type Culture Collection (Manassas, Va.). HUVECs are routinely cultured in EBM (Clonetics Corporation, Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence and are maintained for up to 15 passages. HUVECs are plated at approximately 3000 cells/well in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) and treated with oligomeric compounds one day later.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) cells are obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) are obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

293T Cells:

The human 293T cell line is obtained from American Type Culture Collection (Manassas, Va.). 293T cells are a highly transfectable cell line constitutively expressing the simian virus 40 (SV40) large T antigen. 293T cells were maintained in Dulbeccos' Modified Medium (DMEM) (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum and antibiotics (Life Technologies).

HepG2 Cells:

The human hepatoblastoma cell line HepG2 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mM non-essential amino acids, and 1 mM sodium pyruvate (medium and all supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. For treatment with oligomeric compounds, cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well prior to treatment with oligomeric compounds. For the caspase assay, cells are seeded into collagen coated 96-well plates (BIOCOAT cellware, Collagen type I, B-D #354407/356407, Becton Dickinson, Bedford, Mass.) at a density of 7500 cells/well.

Preadipocytes:

Human preadipocytes are obtained from Zen-Bio, Inc. (Research Triangle Park, N.C.). Preadipocytes were routinely maintained in Preadipocyte Medium (ZenBio, Inc., Research Triangle Park, N.C.) supplemented with antibiotics as recommended by the supplier. Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were routinely maintained for up to 5 passages as recommended by the supplier. To induce differentiation of preadipocytes, cells are then incubated with differentiation media consisting of Preadipocyte Medium further supplemented with 2% more fetal bovine serum (final total of 12%), amino acids, 100 nM insulin, 0.5 mM IBMX, 1 Î¼M dexamethasone and 1 Î¼M BRL49653. Cells are left in differentiation media for 3-5 days and then re-fed with adipocyte media consisting of Preadipocyte Medium supplemented with 33 Î¼M biotin, 17 Î¼M pantothenate, 100 nM insulin and 1 Î¼M dexamethasone. Cells differentiate within one week. At this point cells are ready for treatment with the oligomeric compounds of the invention. One day prior to transfection, 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) are seeded with approximately 3000 cells/well prior to treatment with oligomeric compounds.

Differentiated Adipocytes:

Human adipocytes are obtained from Zen-Bio, Inc. (Research Triangle Park, N.C.). Adipocytes were routinely maintained in Adipocyte Medium (ZenBio, Inc., Research Triangle Park, N.C.) supplemented with antibiotics as recommended by the supplier. Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were routinely maintained for up to 5 passages as recommended by the supplier.

NT2 Cells:

The NT2 cell line is obtained from the American Type Culture Collection (ATCC; Manassas, Va.). The NT2 cell line, which has the ATCC designation NTERA-2 cl.D1, is a pluripotent human testicular embryonal carcinoma cell line derived by cloning the NTERA-2 cell line. The parental NTERA-2 line was established in 1980 from a nude mouse xenograft of the Tera-2 cell line (ATCC HTB-106). NT2 cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells harvested when they reached 90% confluence.

HeLa Cells:

The human epitheloid carcinoma cell line HeLa is obtained from the American Tissue Type Culture Collection (Manassas, Va.). HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells were harvested when they reached 90% confluence.

For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

Treatment with Antisense Oligomeric Compounds:

In general, when cells reach approximately 80% confluency, they are treated with oligomeric compounds of the invention. Oligomeric compounds are introduced into cells using the cationic lipid transfection reagent LIPOFECTINâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.). Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired final concentration of oligomeric compound and LIPOFECTINâ„¢. Before adding to cells, the oligomeric compound, LIPOFECTINâ„¢ and OPTI-MEMâ„¢ are mixed thoroughly and incubated for approximately 0.5 hrs. The medium is removed from the plates and the plates are tapped on sterile gauze. Each well of a 96-well plate is washed with 150 Î¼l of phosphate-buffered saline or Hank's balanced salt solution. Each well of a 24-well plate is washed with 250 Î¼L of phosphate-buffered saline or Hank's balanced salt solution. The wash buffer in each well is replaced with 100 Î¼L or 250 Î¼L of the oligomeric compound/OPTI-MEMâ„¢/LIPOFECTINâ„¢ cocktail for 96-well or 24-well plates, respectively. Untreated control cells receive LIPOFECTINâ„¢ only. The plates are incubated for approximately 4 to 7 hours at 37Â° C., after which the medium is removed and the plates are tapped on sterile gauze. 100 Î¼l or 1 mL of full growth medium is added to each well of a 96-well plate or a 24-well plate, respectively. Cells are harvested 16-24 hours after oligonucleotide treatment, at which time RNA can be isolated and target reduction measured by real-time RT-PCR, or other phenotypic assays performed. In general, data from treated cells are obtained in triplicate, and results presented as an average of the three trials.

In some embodiments, cells are transiently transfected with oligomeric compounds of the instant invention. In some embodiments, cells are transfected and selected for stable expression of an oligomeric compound of the instant invention.

The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide may be selected from ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2) or another suitable positive control. Controls are 2â€²-O-methoxyethyl gapmers (2â€²-O-methoxyethyls shown in bold) with a phosphorothioate backbone or having chemical modifications similar to the oligonucleotides being tested. For mouse or rat cells the positive control oligonucleotide may be ISIS 15770 (ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3), a 2â€²-O-methoxyethyl gapmer (2â€²-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) or other suitable control target RNA may then be utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of target expression or function is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. The concentrations of oligonucleotides used herein can range from 10 nM to 300 nM.

Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904), mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41) and real-time quantitative RT-PCR (Heid, et al., Genome Res., 1996, 6(10), 986-94).

Analysis of Oligonucleotide Inhibition of a Target Levels or Expression:

Modulation of target levels or expression can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time quantitative RT-PCR (also known as RT-PCR). Real-time quantitative RT-PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative RT-PCR can be conveniently accomplished using the commercially available ABI PRISMâ„¢ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

RNA Isolation:

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 cold phosphate-buffered saline (PBS). 60 Î¼L lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 Î¼L of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 Î¼L of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 Î¼L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70Â° C., was added to each well, the plate was incubated on a 90Â° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96â„¢ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 Î¼L cold PBS. 150 Î¼L Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 Î¼L of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96â„¢ well plate attached to a QIAVACâ„¢ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 Î¼L of Buffer RW1 was added to each well of the RNEASY96â„¢ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 Î¼L of Buffer RW1 was added to each well of the RNEASY96â„¢ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96â„¢ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVACâ„¢ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACâ„¢ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 Î¼L of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Real-Time Quantitative PCR Analysis of a Target RNA Levels:

Quantitation of a target RNA levels was accomplished by real-time quantitative PCR using the ABI PRISMâ„¢ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5â€² end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3â€² end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3â€² quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5â€²-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMâ„¢ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of RNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer/probe sets specific to the target gene (or RNA) being measured are evaluated for their ability to be â€œmultiplexedâ€ with a GAPDH amplification reaction. In multiplexing, both the target gene (or RNA) and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, RNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer/probe sets specific for GAPDH only, target gene (or RNA) only (â€œsingle-plexingâ€ ), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target RNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer/probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 Î¼L PCR cocktail (2.5Ã—PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 Î¼M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUMÂ® Taq, 5 Units MuLV reverse transcriptase, and 2.5Ã—ROX dye) to 96-well plates containing 30 Î¼L total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48Â° C. Following a 10 minute incubation at 95Â° C. to activate the PLATINUMÂ® Taq, 40 cycles of a two-step PCR protocol were carried out: 95Â° C. for 15 seconds (denaturation) followed by 60Â° C. for 1.5 minutes (annealing/extension).

Gene (or RNA) target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenâ„¢ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenâ„¢ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreenâ„¢ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 Î¼L of RiboGreenâ„¢ working reagent (RiboGreenâ„¢ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 Î¼L purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

Probes and primers are designed to hybridize to the target sequence.

Northern Blot Analysis of Target RNA Levels:

Eighteen hours after treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLâ„¢ (TEL-TEST â€œBâ€ Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBONDâ„¢-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST â€œBâ€ Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKERâ„¢ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYBâ„¢ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

To detect a target, a target specific primer/probe set is prepared for analysis by PCR. To normalize for variations in loading and transfer efficiency, membranes can be stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERâ„¢ and IMAGEQUANTâ„¢ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data can be normalized to GAPDH levels in untreated controls.

The compounds and compositions of the invention are useful for research and diagnostics, because these compounds and compositions hybridize to nucleic acids or interfere with the normal function of these nucleic acids. Hybridization of the compounds and compositions of the invention with a nucleic acid can be detected by means known in the art. Such means may include conjugation of an enzyme to the compound or composition, radiolabeling or any other suitable detection means. Kits using such detection means for detecting the level of selected proteins in a sample may also be prepared.

The specificity and sensitivity of compounds and compositions can also be harnessed by those of skill in the art for therapeutic uses. Antisense oligomeric compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligomeric compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having a disease or disorder presenting conditions that can be treated, ameliorated, or improved by modulating the expression of a selected small non-coding target nucleic acid is treated by administering the compounds and compositions. For example, in one non-limiting embodiment, the methods comprise the step of administering to or contacting the animal, an effective amount of a modulator or mimic to treat, ameliorate or improve the conditions associated with the disease or disorder. The compounds of the present invention effectively modulate the activity or function of the small non-coding RNA target or inhibit the expression or levels of the small non-coding RNA target. In one embodiment, the activity or expression of the target in an animal is inhibited by about 10%. In another embodiment the activity or expression of a target in an animal is inhibited by about 30%. Further, the activity or expression of a target in an animal is inhibited by 50% or more, by 60% or more, by 70% or more, by 80% or more, by 90% or more, or by 95% or more. In another embodiment, the present invention provides for the use of a compound of the invention in the manufacture of a medicament for the treatment of any and all conditions disclosed herein.

The reduction of target levels may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal known to contain the small non-coding RNA or its precursor. Further, the cells contained within the fluids, tissues or organs being analyzed contain a nucleic acid molecule of a downstream target regulated or modulated by the small non-coding RNA target itself.

The oligomeric compounds and compositions of the invention can be utilized in pharmaceutical compositions by adding an effective amount of the compound or composition to a suitable pharmaceutically acceptable diluent or carrier. Use of the oligomeric compounds and methods of the invention may also be useful prophylactically.

The oligomeric compounds and compositions of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative U.S. patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

The oligomeric compounds and compositions of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the oligomeric compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term â€œprodrugâ€ indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligomeric compounds of the invention can be prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al. Larger oligomeric compounds that are processed to supply, as cleavage products, compounds capable of modulating the function or expression of small non-coding RNAs or their downstream targets are also considered prodrugs.

The term â€œpharmaceutically acceptable saltsâ€ refers to physiologically and pharmaceutically acceptable salts of the compounds and compositions of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Suitable examples include, but are not limited to, sodium and potassium salts. For oligonucleotides, examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions and formulations that include the oligomeric compounds and compositions of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Oligomeric compounds may be formulated for delivery in vivo in an acceptable dosage form, e.g. as parenteral or non-parenteral formulations. Parenteral formulations include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intravitreal and intramuscular (IM) formulations, as well as formulations for delivery via pulmonary inhalation, intranasal administration, topical administration, etc. Non-parenteral formulations include formulations for delivery via the alimentary canal, e.g. oral administration, rectal administration, intrajejunal instillation, etc. Rectal administration includes administration as an enema or a suppository. Oral administration includes administration as a capsule, a gel capsule, a pill, an elixir, etc.

In some embodiments, an oligomeric compound can be administered to a subject via an oral route of administration. The subject may be an animal or a human (man). An animal subject may be a mammal, such as a mouse, a rat, a dog, a guinea pig, a monkey, a non-human primate, a cat or a pig. Non-human primates include monkeys and chimpanzees. A suitable animal subject may be an experimental animal, such as a mouse, rat, mouse, a rat, a dog, a monkey, a non-human primate, a cat or a pig.

In some embodiments, the subject may be a human. In certain embodiments, the subject may be a human patient. In certain embodiments, the subject may be in need of modulation of expression of one or more genes as discussed in more detail herein. In some particular embodiments, the subject may be in need of inhibition of expression of one or more genes as discussed in more detail herein. In particular embodiments, the subject may be in need of modulation, i.e. inhibition or enhancement, of a nucleic acid target in order to obtain therapeutic indications discussed in more detail herein.

In some embodiments, non-parenteral (e.g. oral) oligomeric compound formulations according to the present invention result in enhanced bioavailability of the compound. In this context, the term â€œbioavailabilityâ€ refers to a measurement of that portion of an administered drug which reaches the circulatory system (e.g. blood, especially blood plasma) when a particular mode of administration is used to deliver the drug. Enhanced bioavailability refers to a particular mode of administration's ability to deliver oligonucleotide to the peripheral blood plasma of a subject relative to another mode of administration. For example, when a non-parenteral mode of administration (e.g. an oral mode) is used to introduce the drug into a subject, the bioavailability for that mode of administration may be compared to a different mode of administration, e.g. an IV mode of administration. In some embodiments, the area under a compound's blood plasma concentration curve (AUC₀) after non-parenteral (e.g. oral, rectal, intrajejunal) administration may be divided by the area under the drug's plasma concentration curve after intravenous (i.v.) administration (AUC_iv) to provide a dimensionless quotient (relative bioavailability, RB) that represents the fraction of compound absorbed via the non-parenteral route as compared to the IV route. A composition's bioavailability is said to be enhanced in comparison to another composition's bioavailability when the first composition's relative bioavailability (RB₁) is greater than the second composition's relative bioavailability (RB₂).

In general, bioavailability correlates with therapeutic efficacy when a compound's therapeutic efficacy is related to the blood concentration achieved, even if the drug's ultimate site of action is intracellular (van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300). Bioavailability studies have been used to determine the degree of intestinal absorption of a drug by measuring the change in peripheral blood levels of the drug after an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1451-1458).

In general, an oral composition's bioavailability is said to be â€œenhancedâ€ when its relative bioavailability is greater than the bioavailability of a composition substantially consisting of pure oligonucleotide, i.e. oligonucleotide in the absence of a penetration enhancer.

Organ bioavailability refers to the concentration of compound in an organ. Organ bioavailability may be measured in test subjects by a number of means, such as by whole-body radiography. Organ bioavailability may be modified, e.g. enhanced, by one or more modifications to the oligomeric compound, by use of one or more carrier compounds or excipients. In general, an increase in bioavailability will result in an increase in organ bioavailability.

Oral oligomeric compound compositions according to the present invention may comprise one or more â€œmucosal penetration enhancers,â€ also known as â€œabsorption enhancersâ€ or simply as â€œpenetration enhancers.â€ Accordingly, some embodiments of the invention comprise at least one oligomeric compound in combination with at least one penetration enhancer. In general, a penetration enhancer is a substance that facilitates the transport of a drug across mucous membrane(s) associated with the desired mode of administration, e.g. intestinal epithelial membranes. Accordingly it is desirable to select one or more penetration enhancers that facilitate the uptake of one or more oligomeric compounds, without interfering with the activity of the compounds, and in such a manner the compounds can be introduced into the body of an animal without unacceptable side-effects such as toxicity, irritation or allergic response.

Embodiments of the present invention provide compositions comprising one or more pharmaceutically acceptable penetration enhancers, and methods of using such compositions, which result in the improved bioavailability of oligomeric compounds administered via non-parenteral modes of administration. Heretofore, certain penetration enhancers have been used to improve the bioavailability of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that the uptake and delivery of oligonucleotides can be greatly improved even when administered by non-parenteral means through the use of a number of different classes of penetration enhancers.

In some embodiments, compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property. Modifications may be made to the base, the linker, or the sugar, in general, as discussed in more detail herein with regards to oligonucleotide chemistry. In some embodiments of the invention, compositions for administration to a subject, and in particular oral compositions for administration to an animal or human subject, will comprise modified oligonucleotides having one or more modifications for enhancing affinity, stability, tissue distribution, or other biological property.

Suitable modified linkers include phosphorothioate linkers. In some embodiments according to the invention, the oligomeric compound has at least one phosphorothioate linker. Phosphorothioate linkers provide nuclease stability as well as plasma protein binding characteristics to the compound. Nuclease stability is useful for increasing the in vivo lifetime of oligomeric compounds, while plasma protein binding decreases the rate of first pass clearance of oligomeric compound via renal excretion. In some embodiments according to the present invention, the oligomeric compound has at least two phosphorothioate linkers. In some embodiments, wherein the oligomeric compound has exactly n nucleosides, the oligomeric compound has from one to nâˆ’1 phosphorothioate linkages. In some embodiments, wherein the oligomeric compound has exactly n nucleosides, the oligomeric compound has nâˆ’1 phosphorothioate linkages. In other embodiments wherein the oligomeric compound has exactly n nucleoside, and n is even, the oligomeric compound has from 1 to n/2 phosphorothioate linkages, or, when n is odd, from 1 to (nâˆ’1)/2 phosphorothioate linkages. In some embodiments, the oligomeric compound has alternating phosphodiester (PO) and phosphorothioate (PS) linkages. In other embodiments, the oligomeric compound has at least one stretch of two or more consecutive PO linkages and at least one stretch of two or more PS linkages. In other embodiments, the oligomeric compound has at least two stretches of PO linkages interrupted by at least one PS linkage.

In some embodiments, at least one of the nucleosides is modified on the ribosyl sugar unit by a modification that imparts nuclease stability, binding affinity or some other beneficial biological property to the sugar. In some cases, the sugar modification includes a 2â€²-modification, e.g. the 2â€²-OH of the ribosyl sugar is replaced or substituted. Suitable replacements for 2â€²-OH include 2â€²-F and 2â€²-arabino-F. Suitable substitutions for OH include 2â€²-O-alkyl, e.g. 2â€²-O-methyl, and 2â€²-O-substituted alkyl, e.g. 2â€²-O-methoxyethyl, 2â€²-O-aminopropyl, etc. In some embodiments, the oligomeric compound contains at least one 2â€²-modification. In some embodiments, the oligomeric compound contains at least 2 2â€²-modifications. In some embodiments, the oligomeric compound has at least one 2â€²-modification at each of the termini (i.e. the 3â€²- and 5â€²-terminal nucleosides each have the same or different 2â€²-modifications). In some embodiments, the oligomeric compound has at least two sequential 2â€²-modifications at each end of the compound. In some embodiments, oligomeric compounds further comprise at least one deoxynucleoside. In particular embodiments, oligomeric compounds comprise a stretch of deoxynucleosides such that the stretch is capable of activating RNase (e.g. RNase H) cleavage of an RNA to which the oligomeric compound is capable of hybridizing. In some embodiments, a stretch of deoxynucleosides capable of activating RNase-mediated cleavage of RNA comprises about 8 to about 16, e.g. about 8 to about 16 consecutive deoxynucleosides. In further embodiments, oligomeric compounds are capable of eliciting cleaveage by dsRNAse enzymes.

Oral compositions for administration of non-parenteral oligomeric compounds and compositions of the present invention may be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The term â€œalimentary deliveryâ€ encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration. A common requirement for these modes of administration is absorption over some portion or all of the alimentary tract and a need for efficient mucosal penetration of the nucleic acid(s) so administered.

Delivery of a drug via the oral mucosa, as in the case of buccal and sublingual administration, has several desirable features, including, in many instances, a more rapid rise in plasma concentration of the drug than via oral delivery (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).

Endoscopy may be used for delivery directly to an interior portion of the alimentary tract. For example, endoscopic retrograde cystopancreatography (ERCP) takes advantage of extended gastroscopy and permits selective access to the biliary tract and the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl), 1591). Pharmaceutical compositions, including liposomal formulations, can be delivered directly into portions of the alimentary canal, such as, e.g., the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington et al., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct alimentary delivery of pharmaceutical compositions.

In some embodiments, oligomeric compound formulations may be administered through the anus into the rectum or lower intestine. Rectal suppositories, retention enemas or rectal catheters can be used for this purpose and may be preferred when patient compliance might otherwise be difficult to achieve (e.g., in pediatric and geriatric applications, or when the patient is vomiting or unconscious). Rectal administration can result in more prompt and higher blood levels than the oral route. (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of the drug that is absorbed from the rectum will bypass the liver, administration by this route significantly reduces the potential for first-pass metabolism (Benet et al., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).

Some embodiments of the present invention employ various penetration enhancers in order to effect transport of oligomeric compounds and compositions across mucosal and epithelial membranes. Penetration enhancers may be classified as belonging to one of five broad categories-surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Penetration enhancers and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Accordingly, some embodiments comprise oral oligomeric compound compositions comprising at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants. Further embodiments comprise oral oligomeric compound comprising at least one fatty acid, e.g. capric or lauric acid, or combinations or salts thereof. Other embodiments comprise methods of enhancing the oral bioavailability of an oligomeric compound, the method comprising co-administering the oligomeric compound and at least one penetration enhancer.

Other excipients that may be added to oral oligomeric compound compositions include surfactants (or â€œsurface-active agentsâ€ ), which are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligomeric compounds through the alimentary mucosa and other epithelial membranes is enhanced. In addition to bile salts and fatty acids, surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252).

Fatty acids and their derivatives which act as penetration enhancers and may be used in compositions of the present invention include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono- and di-glycerides thereof and/or physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651).

In some embodiments, oligomeric compound compositions for oral delivery comprise at least two discrete phases, which phases may comprise particles, capsules, gel-capsules, microspheres, etc. Each phase may contain one or more oligomeric compounds, penetration enhancers, surfactants, bioadhesives, effervescent agents, or other adjuvant, excipient or diluent. In some embodiments, one phase comprises at least one oligomeric compound and at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and substantially no oligomeric compound. In some embodiments, at least one phase is compounded with at least one degradation retardant, such as a coating or a matrix, which delays release of the contents of that phase. In some embodiments, a first phase comprises at least one oligomeric compound, at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and a release-retardant. In particular embodiments, an oral oligomeric compound comprises a first phase comprising particles containing an oligomeric compound and a penetration enhancer, and a second phase comprising particles coated with a release-retarding agent and containing penetration enhancer.

A variety of bile salts also function as penetration enhancers to facilitate the uptake and bioavailability of drugs. The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term â€œbile saltâ€ includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579).

In some embodiments, penetration enhancers useful in some embodiments of present invention are mixtures of penetration enhancing compounds. One such penetration enhancer is a mixture of UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof e.g. sodium. Such mixtures are useful for enhancing the delivery of biologically active substances across mucosal membranes, in particular intestinal mucosa. Other penetration enhancer mixtures comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid. Particular penetration enhancers are mixtures of the sodium salts of UDCA, capric acid and lauric acid in a ratio of about 1:2:2 respectively. Anther such penetration enhancer is a mixture of capric and lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In particular embodiments capric acid and lauric acid are present in molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular about 0.5:1 to about 1:0.5.

Other excipients include chelating agents, i.e. compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligomeric compounds through the alimentary and other mucosa is enhanced. With regard to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the invention include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. Control Rel., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers may be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligomeric compounds through the alimentary and other mucosal membranes (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of penetration enhancers includes, but is not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligomeric compounds at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), can be used.

Some oral oligomeric compound compositions also incorporate carrier compounds in the formulation. As used herein, â€œcarrier compoundâ€ or â€œcarrierâ€ can refer to a nucleic acid, or analog thereof, which may be inert (i.e., does not possess biological activity per se) or may be necessary for transport, recognition or pathway activation or mediation, or is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of an oligomeric compound having biological activity by, for example, degrading the biologically active oligomeric compound or promoting its removal from circulation. The coadministration of a oligomeric compound and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of oligomeric compound recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the oligomeric compound for a common receptor. For example, the recovery of a partially phosphorothioate oligomeric compound in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4â€² isothiocyano-stilbene-2,2â€²-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).

A â€œpharmaceutical carrierâ€ or â€œexcipientâ€ may be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more oligomeric compounds to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with an oligomeric compound and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Oral oligomeric compound compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipuritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The oligomeric compounds and compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.

Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 Î¼m in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

Formulations of the present invention include liposomal formulations. As used in the present invention, the term â€œliposomeâ€ means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged nucleic acid molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap nucleic acids rather than complex with it. Both cationic and noncationic liposomes have been used to deliver nucleic acids and oligomeric compounds to cells.

Liposomes also include â€œsterically stabilizedâ€ liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

Formulations for topical administration include those in which the oligomeric compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

For topical or other administration, oligomeric compounds and compositions of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, they may be complexed to lipids, in particular to cationic lipids. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Oral formulations are those in which oligomeric compounds of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. A particularly suitable combination is the sodium salt of lauric acid, capric acid and UDCA. Penetration enhancers also include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Compounds and compositions of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Certain oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and U.S. Application Publication 20030027780, each of which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositions containing one or more of the compounds and compositions of the invention and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the oligomeric compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of oligomeric compounds and compositions of the invention and other drugs are also within the scope of this invention. Two or more combined compounds such as two oligomeric compounds or one oligomeric compound combined with further compounds may be used together or sequentially.

In another embodiment, compositions of the invention may contain one or more of the compounds and compositions of the invention targeted to a first nucleic acid target and one or more additional oligomeric compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more oligomeric compounds and compositions targeted to different regions, segments or sites of the same target. Two or more combined compounds may be used together or sequentially.

The formulation of therapeutic compounds and compositions of the invention and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligomeric compounds, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 Î¼g to 100 g per kg of body weight, from 0.1 Î¼g to 10 g per kg of body weight, from 1.0 Î¼g to 1 g per kg of body weight, from 10.0 Î¼g to 100 mg per kg of body weight, from 100 Î¼g to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily determine repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomeric compound is administered in maintenance doses, ranging from 0.01 Î¼g to 100 g per kg of body weight, from 0.1 Î¼g to 10 g per kg of body weight, from 1 Î¼g to 1 g per kg of body weight, from 10 Î¼g to 100 mg per kg of body weight, from 100 Î¼g to 10 mg per kg of body weight, or from 100 Î¼g to 1 mg per kg of body weight, once or more daily, to once every 20 years. The effects of treatments with therapeutic compositions can be assessed following collection of tissues or fluids from a patient or subject receiving said treatments. It is known in the art that a biopsy sample can be procured from certain tissues without resulting in detrimental effects to a patient or subject. In certain embodiments, a tissue and its constituent cells comprise, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺ cells CD4⁺ cells), lymphocytes and other blood lineage cells, bone marrow, breast, cervix, colon, esophagus, lymph node, muscle, peripheral blood, oral mucosa and skin. In other embodiments, a fluid and its constituent cells comprise, but are not limited to, blood, urine, semen, synovial fluid, lymphatic fluid and cerebro-spinal fluid. Tissues or fluids procured from patients can be evaluated for expression levels of a target small non-coding RNA, mRNA or protein. Additionally, the mRNA or protein expression levels of other genes known or suspected to be associated with the specific disease state, condition or phenotype can be assessed. mRNA levels can be measured or evaluated by real-time PCR, Northern blot, in situ hybridization or DNA array analysis. Protein levels of a downstream target modulated or regulated by a small non-coding RNA can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Western Blot Analysis of Protein Levels:

When small non-coding RNAs have effects on expression of downstream genes or proteins encoded by genes, it is advantageous to measure the protein levels of those gene products. To do this, western blot analysis may be employed.

Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligomeric compound treatment, washed once with PBS, suspended in Laemmli buffer (100 Î¼l/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gradient gels (4-20%) may also be used for the separation of proteins, as is known in the art. Gels are typically run for 1.5 hours at 150 V, and transferred to a membrane, such as PVDF, for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGERâ„¢ (Molecular Dynamics, Sunnyvale Calif.).

Furthermore, the effects of treatment can be assessed by measuring biomarkers associated with the disease or condition in the aforementioned tissues and fluids, collected from a patient or subject receiving treatment, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation; testosterone, estrogen and other hormones; tumor markers; vitamins, minerals and electrolytes.

In Vitro and In Vivo Assays:Phenotypic Assays

Once modulators are designed or identified by the methods disclosed herein, the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive or suggestive of efficacy in the treatment, amelioration or improvement of physiologic conditions associated with a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with an oligomeric compound identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the oligomeric compound. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

Cell Proliferation and Survival Assays:

In some embodiments, cell proliferation and survival assays are used. Cell cycle regulation is the basis for many cancer therapeutic agents. Unregulated cell proliferation is a characteristic of cancer cells, thus most current chemotherapy agents target dividing cells, for example, by blocking the synthesis of new DNA required for cell division. However, cells in healthy tissues are often also affected by agents that modulate cell proliferation.

In some cases, a cell cycle inhibitor will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected (Blagosklonny, Bioessays, 1999, 21, 704-709; Chen et al., Cancer Res., 1997, 57, 2013-2019; Evan and Littlewood, Science, 1998, 281, 1317-1322; Lees and Weinberg, Proc. Natl. Acad. Sci. USA, 1999, 96, 4221-4223). An example of sensitization to anti-cancer agents is observed in cells that have reduced or absent expression of the tumor suppressor genes p53 (Bunz et al., Science, 1998, 282, 1497-1501; Bunz et al., J. Clin. Invest., 1999, 104, 263-269; Stewart et al., Cancer Res., 1999, 59, 3831-3837; Wahl et al., Nat. Med., 1996, 2, 72-79). However, cancer cells often escape apoptosis (Lowe and Lin, Carcinogenesis, 2000, 21, 485-495; Reed, Cancer J Sci. Am., 1998, 4 Suppl 1, S8-14). Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected.

Cell Cycle Assay:

A cell cycle assay is employed to identify genes whose modulation affects cell cycle progression. In addition to normal cells, cells lacking functional p53 are utilized to identify genes whose modulation will sensitize p53-deficient cells to anti-cancer agents. Oligomeric compounds of the invention are tested for their effects on the cell cycle in normal human mammary epithelial cells (HMECs) as well as the breast carcinoma cell lines MCF7 and T47D. The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of cell cycle progression. An oligomeric compound targeting kinesin-like 1 is known to inhibit cell cycle progression and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 200 nM of oligomeric compound and 6 Î¼g/mL LIPOFECTINâ„¢. Compounds of the invention and the positive control are tested in triplicate. The negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTINâ„¢ only. Approximately 24, 48 or 72 hours following transfection, routine procedures are used to prepare cells for flow cytometry analysis and cells are stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile is analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces an increase in cells with a hypodiploid DNA content, which are categorized as â€œsubG1.â€ An increase in cells in G1 phase is indicative of a cell cycle arrest prior to entry into S phase; an increase in cells in S phase is indicative of cell cycle arrest during DNA synthesis; and an increase in cells in the G2/M phase is indicative of cell cycle arrest just prior to or during mitosis. Cell cycle profiles of cells treated with oligomeric compounds can be normalized to those of untreated control cells, and values above or below 100% are considered to indicate an increase or decrease, respectively, in the proportion of cells in a particular phase of the cell cycle.

Oligomeric compounds that prevent cell cycle progression are candidate therapeutic agents for the treatment of hyperproliferative disorders, such as cancer or inflammation.

Caspase Assay:

Programmed cell death, or apoptosis, is an important aspect of various biological processes, including normal cell turnover, immune system development and embryonic development. Apoptosis involves the activation of caspases, a family of intracellular proteases through which a cascade of events leads to the cleavage of a select set of proteins. The caspase family can be divided into two groups: the initiator caspases, such as caspase-8 and -9, and the executioner caspases, such as caspase-3, -6 and -7, which are activated by the initiator caspases. The caspase family contains at least 14 members, with differing substrate preferences (Thornberry and Lazebnik, Science, 1998, 281, 1312-1316). A caspase assay is utilized to identify genes whose modulation causes apoptosis. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.

In a further embodiment, a caspase assay is employed to identify genes or targets whose modulation affects apoptosis. In addition to normal cells, cells lacking functional p53 are utilized to identify genes or targets whose modulation will sensitize p53-deficient cells to agents that induce apoptosis. Oligomeric compounds of the invention are assayed for their affects on apoptosis in normal HMECs as well as the breast carcinoma cell lines MCF7 and T47D. HMECs and MCF7 cells express p53, whereas T47D cells do not express this tumor suppressor gene. Cells are cultured in 96-well plates with black sides and flat, transparent bottoms (Corning Incorporated, Corning, N.Y.). DMEM medium, with and without phenol red, is obtained from Invitrogen Life Technologies (Carlsbad, Calif.). MEGM medium, with and without phenol red, is obtained from Cambrex Bioscience (Walkersville, Md.). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of caspase activity. An oligomeric compound targeted to human Jagged2 or human Notch1, both of which are known to induce caspase activity, may be used as a positive control for caspase activation.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 200 nM of oligomeric compound and 6 Î¼g/mL LIPOFECTINâ„¢. Compounds of the invention and the positive controls are tested in triplicate, and the negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTINâ„¢ only.

Caspase-3 activity is evaluated with a fluorometric HTS Caspase-3 assay (Catalog #HTS02; EMD Biosciences, San Diego, Calif.) that detects cleavage after aspartate residues in the peptide sequence DEVD. The DEVD substrate is labeled with a fluorescent molecule, which exhibits a blue to green shift in fluorescence upon cleavage by caspase-3. Active caspase-3 in the oligomeric compound-treated cells is measured by this assay according to the manufacturer's instructions. Approximately 48 hours following treatment, 50 Î¼L of assay buffer containing 10 Î¼M dithiothreitol is added to each well, followed by addition 20 Î¼L of the caspase-3 fluorescent substrate conjugate. Fluorescence in wells is immediately detected (excitation/emission 400/505 nm) using a fluorescent plate reader (SpectraMAX GeminiXS, Molecular Devices, Sunnyvale, Calif.). The plate is covered and incubated at 3Â° C. for an additional three hours, after which the fluorescence is again measured (excitation/emission 400/505 nm). The value at time zero is subtracted from the measurement obtained at 3 hours. The measurement obtained from the untreated control cells is designated as 100% activity. Caspase-3 activity in cells treated with oligomeric compounds is normalized to that in untreated control cells. Values for caspase activity above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit caspase activity, respectively.

Oligomeric compounds that cause a significant induction in apoptosis are candidate therapeutic agents with applications in the treatment of conditions in which the induction of apoptosis is desirable, for example, in hyperproliferative disorders. Oligomeric compounds that inhibit apoptosis are candidate therapeutic agents with applications in the treatment of conditions where the reduction of apoptosis is useful, for example, in neurodegenerative disorders.

Angiogenesis Assays:

In some embodiments, angiogenesis assays are used. Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell. Cardiol., 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol. Histopathol., 1999, 14, 1287-1294).

Expression of Angiogenic Genes as a Measure of Angiogenesis:

During the process of angiogenesis, endothelial cells perform several distinct functions, including the degradation of the extracellular matrix (ECM), migration, proliferation and the formation of tube-like structures (Liekens et al., Biochem. Pharmacol., 2001, 61, 253-270). Endothelial cells must regulate the expression of many genes in order to perform the functions necessary for angiogenesis. This gene regulation has been the subject of intense scrutiny, and many genes have been identified as being important for the angiogenic phenotype. Genes highly expressed in angiogenic endothelial cells include integrin Î²3, endoglin/CD105, TEM5 and MMP-14/MT-MMP1.

Integrin Î²3 is part of a family of heterodimeric transmembrane receptors that consist of alpha and beta subunits (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822). Each subunit recognizes a unique set of ECM ligands, thereby allowing cells to transmit angiogenic signals from the extracellular matrix. Integrin Î²3 is prominently expressed on proliferating vascular endothelial cells, and it plays roles in allowing new blood vessels to form at tumor sites as well as allowing the epithelial cells of breast tumors to spread (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Drake et al., J. Cell Sci., 1995, 108 (Pt 7), 2655-2661). Blockage of integrin Î²3 with monoclonal antibodies or low molecular weight antagonists inhibits blood vessel formation in a variety of in-vivo models, including tumor angiogenesis and neovascularization during oxygen-induced retinopathy (Brooks et al., Science, 1994, 264, 569-571; Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Hammes et al., Nat. Med., 1996, 2, 529-533).

Endoglin is a transforming growth factor receptor-associated protein highly expressed on endothelial cells, and present on some leukemia cells and minor subsets of bone marrow cells (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634; Haruta and Seon, Proc. Natl. Acad. Sci. US A, 1986, 83, 7898-7902). Its expression is upregulated in endothelial cells of angiogenic tissues and is therefore used as a prognostic indicator in various tumors (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634). Endoglin functions as an ancillary receptor influencing binding of the transforming growth factor beta (TGF-beta) family of ligands to signaling receptors, thus mediating cell survival (Massague and Chen, Genes Dev., 2000, 14, 627-644).

Tumor endothelial marker 5 (TEM5) is a putative 7-pass transmembrane protein (GPCR) (Carson-Walter et al., Cancer Res., 2001, 61, 6649-6655). The mRNA transcript, designated KIAA1531, encodes one of many tumor endothelium markers (TEMs) that display elevated expression (greater than 10-fold) during tumor angiogenesis (St Croix et al., Science, 2000, 289, 1197-1202). TEM5 is coordinately expressed with other TEMs on tumor endothelium in humans and mice.

Matrix metalloproteinase 14 (MMP-14), a membrane-type MMP covalently linked to the cell membrane, is involved in matrix detachment and migration. MMP-14 is thought to promote tumor angiogenesis; antibodies directed against the catalytic domain of MMP-14 block endothelial-cell migration, invasion and capillary tube formation in vitro (Galvez et al., J. Biol. Chem., 2001, 276, 37491-37500). MMP-14 can degrade the fibrin matrix that surrounds newly formed vessels potentially allowing the endothelial cells to invade further into the tumor tissue (Hotary et al., J. Exp. Med., 2002, 195, 295-308). MMP-14 null mice have impaired angiogenesis during development, further demonstrating the role of MMP-14 in angiogenesis (Vu and Werb, Genes Dev., 2000, 14, 2123-2133; Zhou et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 4052-4057).

In some embodiments, HUVECs are used to measure the effects of oligomeric compounds of the invention on the activity of endothelial cells stimulated with human vascular endothelial growth factor (VEGF). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of HUVEC activity.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 Î¼g/mL LIPOFECTINâ„¢. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTINâ„¢ only.

Approximately twenty hours after transfection, cells are induced to express angiogenic genes with recombinant VEGF. Total RNA is harvested approximately 52 hours following transfection, and the amount of total RNA from each sample is determined using a Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.). Real-time RT-PCR is performed on the total RNA using primer/probe sets for four angiogenic hallmark genes described herein: integrin Î²3, endoglin, TEM5 and MMP14. Expression levels for each gene are normalized to total RNA. Gene expression in cells treated with oligomeric compounds is normalized to that in untreated control cells. A value above or below 100% is considered to indicated an increase or decrease in gene expression, respectively.

Oligomeric compounds resulting in a decrease in the expression of angiogenic hallmark genes are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that increase the expression of angiogenic hallmark genes are candidate therapeutic agents with applications where the stimulation of angiogenesis is desired, for example, in wound healing.

Endothelial Tube Formation Assay as a Measure of Angiogenesis:

Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluated the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. In this assay, HUVECs are plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HUVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.

In some embodiments, HUVECs are used to measure the effects of oligomeric compounds of the invention on endothelial tube formation activity. The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of endothelial tube formation.

Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 Î¼g/mL LIPOFECTINâ„¢. Untreated control cells receive LIPOFECTINâ„¢ only. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicates.

Approximately fifty hours after transfection, cells are transferred to 96-well plates coated with ECMatrixâ„¢ (Chemicon International). Under these conditions, untreated HUVECs form tube-like structures. After an overnight incubation at 37Â° C., treated and untreated cells are inspected by light microscopy. Tube formation in cells treated with oligomeric compounds is compared to that in untreated control cells. Individual wells are assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network.

Oligomeric compounds resulting in a decrease in tube formation are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that promote endothelial tube formation are candidate therapeutic agents with applications where the stimulation of angiogenesis is desired, for example, in wound healing.

Matrix Metalloproteinase Activity:

During angiogenesis, endothelial cells must degrade the extracellular matrix (ECM) and thus secrete matrix metalloproteinases (MMPs) in order to accomplish this degradation. MMPs are a family of zinc-dependent endopeptidases that fall into eight distinct classes: five are secreted and three are membrane-type MMPs (MT-MMPs) (Egeblad and Werb, J. Cell Science, 2002, 2, 161-174). MMPs exert their effects by cleaving a diverse group of substrates, which include not only structural components of the extracellular matrix, but also growth-factor-binding proteins, growth-factor precursors, receptor tyrosine-kinases, cell-adhesion molecules and other proteinases (Xu et al., J. Cell Biol., 2002, 154, 1069-1080).

In some embodiments, oligomeric compounds of the invention are evaluated for their effects on MMP activity in the medium above cultured HUVECs. MMP activity is measured using the EnzChek Gelatinase/Collagenase Assay Kit (Molecular Probes, Eugene, Oreg.). In this assay, HUVECs are plated at approximately 4000 cells per well in 96-well plates and transfected one day later. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of MMP activity. An oligomeric compound targeted to integrin Î²3 is known to inhibit MMP activity and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 Î¼g/mL LIPOFECTINâ„¢. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Untreated control cells receive LIPOFECTINâ„¢ only.

Approximately 50 hours after transfection, a p-aminophenylmercuric acetate (APMA, Sigma-Aldrich, St. Louis, Mo.) solution is added to each well of a Corning-Costar 96-well clear bottom plate (VWR International, Brisbane, Calif.). The APMA solution is used to promote cleavage of inactive MMP precursor proteins. Medium above the HUVECs is then transferred to the wells in the 96-well plate. After approximately 30 minutes, the quenched, fluorogenic MMP cleavage substrate is added, and baseline fluorescence is read immediately at 485 nm excitation/530 nm emission. Following an overnight incubation at 37Â° C. in the dark, plates are read again to determine the amount of fluorescence, which corresponds to MMP activity. Total protein from HUVEC lysates is used to normalize the readings, and MMP activity from cells treated with oligomeric compounds is normalized to that of untreated control cells. MMP activities above or below 100% are considered to indicate a stimulation or inhibition, respectively, of MMP activity.

Oligomeric compounds resulting in a decrease in MMP activity are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that increase the expression of angiogenic hallmark genes are candidate therapeutic agents with applications in conditions requiring angiogenesis, for example, in wound healing.

Adipocyte Assays:

In some embodiments, adipocytes assays are used. Insulin is an essential signaling molecule throughout the body, but its major target organs are the liver, skeletal muscle and adipose tissue. Insulin is the primary modulator of glucose homeostasis and helps maintain a balance of peripheral glucose utilization and hepatic glucose production. The reduced ability of normal circulating concentrations of insulin to maintain glucose homeostasis manifests in insulin resistance which is often associated with diabetes, central obesity, hypertension, polycystic ovarian syndrome, dyslipidemia and atherosclerosis (Saltiel, Cell, 2001, 104, 517-529; Saltiel and Kahn, Nature, 2001, 414, 799-806).

Response of Undifferentiated Adipocytes to Insulin:

Insulin promotes the differentiation of preadipocytes into adipocytes. The condition of obesity, which results in increases in fat cell number, occurs even in insulin-resistant states in which glucose transport is impaired due to the antilipolytic effect of insulin. Inhibition of triglyceride breakdown requires much lower insulin concentrations than stimulation of glucose transport, resulting in maintenance or expansion of adipose stores (Kitamura et al., Mol. Cell. Biol., 1999, 19, 6286-6296; Kitamura et al., Mol. Cell. Biol., 1998, 18, 3708-3717).

One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and peroxisome proliferator-activated receptor gamma (PPAR-Î³). Insulin signaling is improved by compounds that bind and inactivate PPAR-Î³, a key regulator of adipocyte differentiation (Olefsky, J. Clin. Invest., 2000, 106, 467-472). Insulin induces the translocation of GLUT4 to the adipocyte cell surface, where it transports glucose into the cell, an activity necessary for triglyceride synthesis. In all forms of obesity and diabetes, a major factor contributing to the impaired insulin-stimulated glucose transport in adipocytes is the downregulation of GLUT4. Insulin also induces hormone sensitive lipase (HSL), which is the predominant lipase in adipocytes that functions to promote fatty acid synthesis and lipogenesis (Fredrikson et al., J. Biol. Chem., 1981, 256, 6311-6320). Adipocyte fatty acid binding protein (aP2) belongs to a multi-gene family of fatty acid and retinoid transport proteins. aP2 is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization (Fu et al., J. Lipid Res., 2000, 41, 2017-2023; Pelton et al., Biochem. Biophys. Res. Commun., 1999, 261, 456-458). Together, these genes play important roles in the uptake of glucose and the metabolism and utilization of fats.

Leptin secretion and an increase in triglyceride content are also well-established markers of adipocyte differentiation. In addition to its role in adipocytes differentiation, leptin also regulates glucose homeostasis through mechanisms (autocrine, paracrine, endocrine and neural) independent of the adipocyte's role in energy storage and release. As adipocytes differentiate, insulin increases triglyceride accumulation by both promoting triglyceride synthesis and inhibiting triglyceride breakdown (Spiegelman and Flier, Cell, 2001, 104, 531-543). As triglyceride accumulation correlates tightly with cell size and cell number, it is an excellent indicator of differentiated adipocytes.

Oligomeric compounds of the invention are tested for their effects on preadipocyte differentiation. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of adipocyte differentiation. Tumor necrosis factor alpha (TNF-Î±) is known to inhibit adipocyte differentiation and may be used as a positive control for the inhibition of adipocyte differentiation as evaluated by leptin secretion. For the other adipocyte differentiation markers assayed, an oligomeric compound targeted to PPAR-Î³, also known to inhibit adipocyte differentiation, may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 250 nM of oligomeric compound and 7.5 Î¼g/mL LIPOFECTINâ„¢. Untreated control cells receive LIPOFECTINâ„¢ only. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicate wells.

After the cells have reach confluence (approximately three days), they are exposed for an additional three days to differentiation medium (Zen-Bio, Inc., Research Triangle Park, N.C.) containing a PPAR-Î³ agonist, IBMX, dexamethasone, and insulin. Cells are then fed adipocyte medium (Zen-Bio, Inc.), which is replaced at 2 or 3 day intervals.

Leptin secretion into the medium in which adipocytes are cultured is measured by protein ELISA. On day nine post-transfection, 96-well plates are coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, Minn.) and left at 4Â° C. overnight. The plates are blocked with bovine serum albumin (BSA), and a dilution of the treated adipoctye medium is incubated in the plate at room temperature for approximately 2 hours. After washing to remove unbound components, a second monoclonal antibody to human leptin (conjugated with biotin) is added. The plate is then incubated with strepavidin-conjugated horse radish peroxidase (HRP) and enzyme levels are determined by incubation with 3,3â€²,5,5â€²-tetramethlybenzidine, which turns blue when cleaved by HRP. The OD₄₅₀is read for each well, where the dye absorbance is proportional to the leptin concentration in the cell lysate. Leptin secretion from cells treated with oligomeric compounds is normalized to that from untreated control cells. With respect to leptin secretion, values above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit leptin secretion, respectively.

The triglyceride accumulation assay measures the synthesis of triglyceride by adipocytes. Triglyceride accumulation is measured using the Infinityâ„¢ Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.). On day nine post-transfection, cells are washed and lysed at room temperature, and the triglyceride assay reagent is added. Triglyceride accumulation is measured based on the amount of glycerol liberated from triglycerides by the enzyme lipoprotein lipase. Liberated glycerol is phosphorylated by glycerol kinase, and hydrogen peroxide is generated during the oxidation of glycerol-1-phosphate to dihydroxyacetone phosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP) uses H₂O₂to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye. Dye absorbance, which is proportional to the concentration of glycerol, is measured at 515 nm using an UV spectrophotometer. Glycerol concentration is calculated from a standard curve for each assay, and data are normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Triglyceride accumulation in cells treated with oligomeric compounds is normalized to that in untreated control cells. Values for triglyceride accumulation above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit triglyceride accumulation, respectively.

Expression of the four hallmark genes, HSL, aP2, Glut4, and PPARÎ³, is also measured in adipocytes transfected with oligomeric compounds of the invention. Cells are lysed on day nine post-transfection and total RNA is harvested. The amount of total RNA in each sample is determined using a Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.). Real-time PCR is performed on the total RNA using primer/probe sets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2, and PPAR-Î³. Gene expression in cells treated with oligomeric compounds is normalized to that in untreated control cells. With respect to the four adipocyte differentiation hallmark genes, values above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit adipocyte differentiation, respectively.

Oligomeric compounds that reduce the expression levels of markers of adipocyte differentiation are candidate therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells. Oligomeric compounds of the invention resulting in a significant increase in leptin secretion are potentially useful for the treatment of obesity.

Response of Liver-Derived Cells to Insulin:

Insulin mediates its effects by suppressing the RNA expression levels of enzymes important for gluconeogenesis and glycogenolysis, and also by controlling the activities of some metabolic enzymes through post-translational mechanisms (Hall and Granner, J. Basic Clin. Physiol. Pharmacol., 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In liver cells, genes involved in regulating glucose metabolism can be identified by monitoring changes in the expression of selective insulin-responsive genes in a cell culture model. However, primary human hepatocytes are difficult to obtain and work with in culture. Therefore, the insulin signaling assay described herein is performed in the hepatocellular carcinoma cell line HepG2, the most widely used cell culture model for hepatocytes. The insulin responsive genes evaluate in this assay are phosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factor binding protein 1 (IGFBP-1) and follistatin.

IGFBP-1 is one of a family of six secreted proteins that bind insulin-like growth factor (IGF) with high affinity and thereby modulate IGFs action in vivo (Baxter, Am. J. Physiol. Endocrinol. Metab., 2000, 278, E967-976; Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). IGFBP-1 is characterized by dynamic variability of levels in circulation due to the regulation of its hepatic secretion (Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). The multi-hormonal regulation of PEPCK and IGFBP-1 are similar. Glucocorticoids and cyclic AMP (cAMP) stimulate transcription of the IGFBP-1 gene expression whereas insulin acts in a dominant manner to suppress both basal and cAMP or glucocorticoid-stimulated IGFBP-1 gene transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). PEPCK catalyzes the rate-limiting step in gluconeogenesis, and thereby contributes to hepatic glucose output (Hall and Granner, J. Basic Clin. Physiol. Pharmacol., 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In hepatoma cells, studies have shown that the expression of PEPCK is stimulated by glucocorticoids, glucagon (via cAMP), and retinoic acid. Insulin acts in a dominant manner to suppress these stimulations as well as basal transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). In HepG2 cells, prolonged serum starvation induces the expression of PEPCK and subsequent insulin stimulation significantly reduces the PEPCK mRNA level.

Follistatin is significantly stimulated by insulin in HepG2 cells. Interestingly, follistatin levels have been shown to be higher in women with polycystic ovary syndrome (PCOS) (Norman et al., Hum. Reprod., 2001, 16, 668-672). PCOS is a metabolic as well as a reproductive disorder, and an important cause of type 2 diabetes mellitus in women. It is often associated with profound insulin resistance and hyperinsulinemia as well as with a defect in insulin secretion (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil. Steril., 2002, 77, 209-215). PCOS is the most common cause of female infertility in the U.S. and affects 5%-10% of women of child-bearing age (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil. Steril., 2002, 77, 209-215).

In some embodiments, HepG2 cells are used to measure the effects of compounds of the invention on hepatic gene expression following insulin stimulation. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of hepatic gene expression. Insulin at a concentration of 100 nM may be used as a positive control for the stimulation of hepatic gene expression. An oligomeric compound targeted to human forkhead is known to inhibit hepatic gene expression and may be used as a positive control for the inhibition of gene expression in the presence of insulin.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 100 nM of oligomeric compound and 3 Î¼g/mL LIPOFECTINâ„¢. Untreated control cells receive LIPOFECTINâ„¢ only. Compounds of the invention and the positive controls are tested in triplicate, and the negative control is tested in up to six replicate wells.

Approximately 28 hours after transfection, the cells are subjected to serum starvation for a period of 12 to 16 hours, using serum-free growth medium. Following serum starvation, cells are treated with 1 nM insulin (insulin-treated) or are left untreated (basal conditions) for approximately four hours. At the same time, untreated control cells in both plates are treated with 100 nM insulin to determine the maximal insulin response. Following insulin treatment (forty-eight hours after transfection), total RNA is harvested from all samples, and the amount of total RNA from each sample is determined using a Ribogreen assay (Invitrogen Corporation, Carlsbad, Calif.). Real-time PCR is performed on the total RNA samples using primer/probe sets for three insulin responsive genes: insulin-like growth factor binding protein-1 (IGFBP-1), cytosolic PEPCK (PEPCK-C), and follistatin. Gene expression levels obtained by real-time PCR are normalized for total RNA content in the samples. Gene expression in cells treated with oligomeric compounds is normalized to that from untreated control cells. Values above or below 100% are considered to indicate an increase or decrease in gene expression, respectively.

Oligomeric compounds that interfere with the expression of genes involved in glucose metabolism are candidate therapeutic agents for the treatment of conditions associated with abnormal glucose metabolism, for example, obesity and diabetes.

Inflammation Assays:

In some embodiments, inflammation assays are used. Inflammation assays are designed to identify genes that regulate the activation and effector phases of the adaptive immune response. During the activation phase, T lymphocytes (also known as T-cells) receiving signals from the appropriate antigens undergo clonal expansion, secrete cytokines, and up-regulate their receptors for soluble growth factors, cytokines and co-stimulatory molecules (Cantrell, Annu. Rev. Immunol., 1996, 14, 259-274). These changes drive T-cell differentiation and effector function. Response to cytokines by non-immune effector cells controls the production of inflammatory mediators that can do extensive damage to host tissues. The cells of the adaptive immune systems, their products, as well as their interactions with various enzyme cascades involved in inflammation (e.g., the complement, clotting, fibrinolytic and kinin cascades) all represent potential points for intervention in inflammatory disease.

Dendritic cells treated with oligomeric compounds targeting different genes are used to identify regulators of dendritic cell-mediated T-cell co-stimulation. The level of interleukin-2 (IL-2) production by T-cells, a critical consequence of T-cell activation (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252), is used as an endpoint for T-cell activation. T lymphocytes are important immunoregulatory cells that mediate pathological inflammatory responses. Optimal activation of T lymphocytes requires both primary antigen recognition events as well as secondary or co-stimulatory signals from antigen presenting cells (APC). Dendritic cells are the most efficient APCs known and are principally responsible for antigen presentation to T-cells, expression of high levels of co-stimulatory molecules during infection and disease, and the induction and maintenance of immunological memory (Banchereau and Steinman, Nature, 1998, 392, 245-252). While a number of co-stimulatory ligand-receptor pairs have been shown to influence T-cell activation, a principal signal is delivered by engagement of CD28 on T-cells by CD80 (B7-1) and CD86 (B7-2) on APCs (Boussiotis et al., Curr. Opin. Immunol., 1994, 6, 797-807; Lenschow et al., Annu. Rev. Immunol., 1996, 14, 233-258). In contrast, a B7 counter-receptor, CTLA-4, has been shown to negatively regulate T-cell activation, maintain immunological homeostasis and promote immune tolerance (Walunas and Bluestone, J. Immunol., 1998, 160, 3855-3860). Inhibition of T-cell co-stimulation by APCs holds promise for novel and more specific strategies of immune suppression. In addition, blocking co-stimulatory signals may lead to the development of long-term immunological anergy (unresponsiveness or tolerance) that would offer utility for promoting transplantation or dampening autoimmunity. T-cell anergy is the direct consequence of failure of T-cells to produce the growth factor interleukin-2 (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252). Dendritic cell cytokine production as a measure of the activation phase of the immune response:

In some embodiments, the effects of the oligomeric compounds of the invention are examined on the dendritic cell-mediated costimulation of T-cells. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of dendritic cell-mediated T-cell costimulation. An oligomeric compound targeted to human CD86 is known to inhibit dendritic cell-mediated T-cell stimulation and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 200 nM of oligomeric compound and 6 Î¼g/mL LIPOFECTINâ„¢. Untreated control cells receive LIPOFECTINâ„¢ only. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Following incubation with the oligomeric compounds and LIPOFECTINâ„¢, fresh growth medium with cytokines is added and DC culture is continued for an additional 48 hours. DCs are then co-cultured with Jurkat T-cells in RPMI medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Company, St. Louis, Mo.). Culture supernatants are collected 24 hours later and assayed for IL-2 levels (IL-2 DuoSet, R&D Systems, Minneapolis, Minn.). IL-2 levels in cells treated with oligomeric compounds are normalized to those from untreated control cells. A value greater than 100% indicates an induction of the inflammatory response, whereas a value less than 100% demonstrates a reduction in the inflammatory response.

Oligomeric compounds that inhibit T-cell co-stimulation are candidate therapeutic compounds with applications in the prevention, treatment or attenuation of conditions associated with hyperstimulation of the immune system, including rheumatoid arthritis, irritable bowel disease, asthma, lupus and multiple sclerosis. Oligomeric compounds that induce T-cell co-stimulation are candidate therapeutic agents for the treatment of immunodeficient conditions.

Cytokine Signaling as a Measure of the Effector Phase of the Inflammatory Response:

The cytokine signaling assay further identifies genes that regulate inflammatory responses of non-immune effector cells (initially endothelial cells) to stimulation with cytokines such as interferon-gamma (IFN-Î³). Response to IFN-Î³ is assessed by measuring the expression levels of three genes: intercellular adhesion molecule-1 (ICAM-1), interferon regulatory factor 1 (IRF1) and small inducible cytokine subfamily B (Cys-X-Cys), member 11 (SCYB11). The cytokine signaling assay further identifies genes that regulate inflammatory responses of non-immune effector cells (initially endothelial cells) to stimulation with IL-1Î² or TNF-Î± (Heyninck et al., J Cell Biol, 1999, 145, 1471-1482; Zetoune et al., Cytokine, 2001, 15, 282-298). Response to IL-1Î² or TNF-Î± stimulation is monitored by measuring the expression levels of four genes: A20, intracellular adhesion molecule 1 (ICAM-1), interleukin-9 (IL-8) and macrophage-inflammatory protein 2 (MIP2Î±). As described below, all of these genes regulate numerous parameters of the inflammatory response.

ICAM-1 is an adhesion molecule expressed at low levels on resting endothelial cells that is markedly up-regulated in response to inflammatory mediators like tumor necrosis factor-Î± (TNF-Î±), interleukin-1Î² (IL-1Î²) and interferon-Î³ (IFN-Î³) (Springer, Nature, 1990, 346, 425-434). ICAM-1 expression serves to attract circulating leukocytes into the inflammatory site.

IRF-1 binds to upstream cis-regulatory elements of interferon-inducible genes and functions as a transcriptional activator. IRF-1 directly binds to a functional IFN-Î³-stimulated response element in the cathepsin S promoter and mediates IFN-Î³ dependent transcriptional activation (Storm van's Gravesande et al., J Immunol, 2002, 168, 4488-4494).

SCYB 11 is essential for mediating normal leukocyte recruitment and trafficking during inflammation. SCYB11 induces a chemotactic response in IL-2 activated T-cells, monocytes and granulocytes (Mohan et al., J Immunol, 2002, 168, 6420-6428).

A20 is a zinc-finger protein that limits the transcription of pro-inflammatory genes by blocking TRAF2-stimulated NK-ÎºB signaling. Studies in mice show that TNF-Î± dramatically increases A20 expression in mice, and that A20 expression is crucial for their survival (Lee et al., Science, 2000, 289, 2350-2354).

IL-8 is a member of the chemokine gene superfamily, members of which promote the pro-inflammatory phenotype of macrophages, vascular smooth muscle cells and endothelial cells (Koch et al., Science, 1992, 258, 1798-1801). IL-8 has been known as one of the major inducible chemokines with the ability to attract neutrophils to the site of inflammation. More recently, IL-8 has been implicated as a major mediator of acute neutrophil-mediated inflammation, and is therefore a potential anti-inflammatory target (Mukaida et al., Cytokine Growth Factor Rev, 1998, 9, 9-23).

MIP2Î±, another chemokine known to play a central role in leukocyte extravasation, has more recently been shown to be involved in acute inflammation (Lukacs et al., Chem Immunol, 1999, 72, 102-120). MIP2Î± is expressed in response to microbial infection, to injection of lipopolysaccharides (LPS), and to stimulation of cells with pro-inflammatory mediators such as IL-1Î² and TNF-Î± (Kopydlowski et al., J Immunol, 1999, 163, 1537-1544). Endothelial cells are one of several cell types that are sources of MIP2a (Rudner et al., J Immunol, 2000, 164, 6576-6582).

In some embodiments, the effects of the oligomeric compounds of the invention on the cellular response to cytokines may be examined in HUVECs. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of cytokine signaling.

Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ to achieve a final concentration of 75 nM of oligomeric compound and 2.25 Î¼g/mL LIPOFECTINâ„¢. Untreated control cells receive LIPOFECTINâ„¢ only. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicate wells.

For IFN-Î³ stimulation, following transfection, fresh growth medium is added and DC culture is continued for an additional 44 hours, after which HUVECS are stimulated with 10 ng/ml of IFN-Î³ for a period of 4 hours. For stimulation with IL-1Î² or TNF-Î±, fresh growth medium is added and DC culture is continued for an additional 46 hours, after which HUVECs are stimulated with 0.1 ng/mL of IL-1Î² or 1 ng/mL of TNF-Î± for a period of 2 hours. Total RNA is harvested 48 hours following transfection, and real time PCR is performed using primer/probe sets to detect ICAM-1, IRF-1 and SCYB11 in IFN-Î³-stimulated cells, or ICAM-1, A20, IL-8 and MIP2a in IL-1Î²-stimulated and TNF-Î±-stimulated cells. Expression levels of each gene are normalized to total RNA. Gene expression levels from cells treated with oligomeric compounds are normalized to those from untreated control cells. A value greater than 100% indicates an induction of the inflammatory response, whereas a value less than 100% demonstrates a reduction in the inflammatory response.

Oligomeric compounds that inhibit the inflammatory response are candidate therapeutic compounds with applications in the prevention, treatment or attenuation of conditions associated with hyperstimulation of the immune system, including rheumatoid arthritis, irritable bowel disease, asthma, lupus and multiple sclerosis.

In Vivo Studies

The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

Mouse Model of Tumorigenesis:

Animal models of tumorigenesis are used in some embodiments of the present invention. In this model, tumorigenic cells are injected into immunocompromised mice (i.e. nude mice), and subsequent growth of a tumor is measured.

Serially transplanted MDA-MB-231 (a human breast carcinoma cell line, American Type Culture Collection, Manassas, Va.) tumors are established subcutaneously in nude mice. Beginning two weeks later, one or more of the oligomeric compounds of the invention are administered intravenously daily for 14 days at dosages of 15 mg/kg or 30 mg/kg. Control compounds are also administered at these doses, and a saline control is also given. Tumor growth rates are monitored for the two-week period of oligonucleotide administration. Activity of the oligomeric compounds of the invention is measured by a reduction in tumor growth. Activity is measured by reduced tumor volume compared to saline or control compound. Following death or sacrifice of mice, tumor tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin.

Tumor tissue sections are evaluated for tumor morphology and size.

Human A549 lung tumor cells are also injected into nude mouse to produce tumors. 200 Î¼l of A549 cells (5Ã—10⁶cells) are implanted subcutaneously in the inner thigh of nude mice. Oligomeric compounds of the invention are administered twice weekly for four weeks, beginning one week following tumor cell inoculation. Oligomeric compounds are formulated with cationic lipids (LIPOFECTINâ„¢, Invitrogen Corporation, Carlsbad, Calif.) and given subcutaneously in the vicinity of the tumor. Oligomeric compound dosage is 5 mg/kg with 60 mg/kg cationic lipid. Tumor size is recorded weekly. Activity of the oligomeric compounds of the invention is measured by reduction in tumor size compared to controls.

Xenograft studies are also performed using the U-87 human glioblastoma cell line (American Type Culture Collection, Manassas, Va.). Nude mice are injected subcutaneously with 2Ã—10⁷U-87 cells. Mice are injected intraperitoneally with one or more of the oligomeric compounds of the invention or a control compound at dosages of either 15 mg/kg or 30 mg/kg for 21 consecutive days beginning 7 days after xenografts are implanted. Saline-injected animals serve as a control. Tumor volumes are measured on days 14, 21, 24, 31 and 35. Activity is measured by reduced tumor volume compared to saline or control compound. Following death or sacrifice of mice, tumor tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Tumor tissue sections are evaluated for tumor morphology and size.

Alternatively, intracerebral U-87 xenografts are generated by implanting U-87 glioblastoma cells into the brains of nude mice. Mice are treated via continuous intraperitoneal administration with one or more of the oligomeric compounds of the invention at 20 mg/kg, control compound at 20 mg/kg or saline beginning on day 7 after xenograft implantation. Activity of the oligomeric compounds of the invention is measured by an increase in survival time compared to controls. Following death or sacrifice, brain tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Brain tissue sections are evaluated for tumor growth.

Leptin-Deficient Mice (a Model of Obesity and Diabetes (Ob/Ob Mice)):

Leptin is a hormone produced by fat cells that regulates appetite. Deficiencies in this hormone in both humans and non-human animals leads to obesity. ob/ob mice have a mutation in the leptin gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. ob/ob mice have higher circulating levels of insulin and are less hyperglycemic than db/db mice, which harbor a mutation in the leptin receptor. In accordance with the present invention, the oligomeric compounds of the invention are tested in the ob/ob model of obesity and diabetes.

Seven-week old male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 10-15% and are subcutaneously injected with the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and target RNA expression level quantitation are performed as described by other examples herein.

To assess the physiological effects resulting from modulation of target, the ob/ob mice are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis, or clearing of lipids from the liver, is assessed by measuring the liver triglyceride content. Hepatic steatosis is assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

The effects of target modulation on glucose and insulin metabolism are evaluated in the ob/ob mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following at 2 weeks and at 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of ob/ob mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice are also measured.

The ob/ob mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism. These genes include, but are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone sensitive lipase. mRNA levels in liver and white and brown adipose tissue are quantitated by real-time PCR as described in other examples herein, employing primer/probe sets that are generated using published sequences of each gene of interest.

Leptin Receptor-Deficient Mice (a Model of Obesity and Diabetes (Db/Db Mice)):

db/db mice have a mutation in the leptin receptor gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. db/db mice, which have lower circulating levels of insulin and are more hyperglycemic than ob/ob mice which harbor a mutation in the leptin gene, are often used as a rodent model of type 2 diabetes. In accordance with the present invention, oligomeric compounds of the present invention are tested in the db/db model of obesity and diabetes.

Seven-week old male C57B1/6J-Lepr db/db mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 15-20% and are subcutaneously injected with one or more of the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin receptor wildtype littermates (i.e. lean littermates) and db/db mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the db/db mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the db/db mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.

To assess the metabolic rate of db/db mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.

The db/db mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

Lean Mice on a Standard Rodent Diet:

C57B1/6 mice are maintained on a standard rodent diet and are used as control (lean) animals. In one embodiment of the present invention, the oligomeric compounds of the invention are tested in normal, lean animals.

Seven-week old male C57B1/6 mice are fed a diet with a fat content of 4% and are subcutaneously injected with one or more of the oligomeric compounds of the invention or control compounds at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of the target, the lean mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the lean mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.

To assess the metabolic rate of lean mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.

The lean mice that received treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

Levin Model of Diet-Induced Obesity in Rats:

The Levin Model is a polygenic model of rats selectively bred to develop diet-induced obesity (DIO) associated with impaired glucose tolerance, dyslipidemia and insulin resistance when fed a high-fat diet (Levin, et al., Am. J. Physiol, 1997, 273, R725-30). The advantage of this model is that it displays traits more similar to human obesity and glucose intolerance than in animals that are obese/hyperinsulinemic due to genetic defects e.g. defects in leptin signaling. This model is useful in investigating the oligomeric compounds of the present invention for their ability to affect obesity and related complications, such as impaired glucose tolerance, dyslipidemia and insulin resistance. In accordance with the present invention, the oligomeric compounds of the invention are tested in the Levin model of diet-induced obesity.

Eight-week old male Levin rats (Charles River Laboratories, Wilmington, Mass.), weighing Ëœ500 g, are fed a diet with a fat content of 60% for eight weeks, after which they are subcutaneously injected with one or more of the oligomeric compounds of the invention at a dose of 25 mg/kgÃ—2 per week for 8 weeks. Control groups consist of animals injected with saline or a control compound and lean littermates fed a standard rodent diet. The control compound is injected at the same dose as the target-specific compound.

Throughout the treatment period, the rats are evaluated for food consumption, weight gain, as well as serum levels of glucose, insulin, cholesterol, free fatty acids, triglycerides and liver enzymes.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the Levin rats treated with the oligomeric compounds of the invention. Plasma glucose and insulin are monitored throughout the treatment by analyzing blood samples. Glucose and tolerance are assessed in fed or fasted rats. After blood is collected for baseline glucose and insulin levels, a glucose challenge is administered, after which blood glucose and insulin levels are measured at 15, 20 or 30 minute intervals for up to 3 hours. Insulin tolerance is similarly analyzed, beginning with blood collection for baseline glucose and insulin levels, followed by an insulin challenge, after which blood glucose levels are measured at 15, 20 or 30 minute intervals for up to 3 hours. Plasma insulin and glucose are also measured at study termination.

At the end of the treatment period, the rats are sacrificed. Organs are removed and weighed, including liver, white adipose tissue, brown adipose tissue and spleen. Target RNA expression levels are measured in all tissues that are isolated, using quantitative real-time PCR. Target protein levels are also evaluated by immunoblot analysis using antibodies that specifically recognize the target protein.

Also evaluated at the end of the treatment period are serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The Levin rats that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

C57BL/6 on a High-Fat Diet (a Model of Diet-Induced Obesity (DIO)):

The C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. Consequently, when these mice are fed a high-fat diet, they develop diet-induced obesity. Accordingly these mice are a useful model for the investigation of obesity and treatments designed to treat these conditions. In one embodiment of the present invention, the oligomeric compounds of the invention are tested in a model of diet-induced obesity.

Male C57BL/6 mice (7-weeks old) receive a 60% fat diet for 8 weeks, after which mice are subcutaneously injected with one or more of the oligomeric compounds of the invention at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected or control compound-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the diet-induced obese mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the diet-induced obese mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of diet-induced obese mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.

The diet-induced obese mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

P-407 Mouse Model of Hyperlipidemia:

Poloxamer 407 (P-407), an inert block copolymer comprising a hydrophobic core flanked by hydrophilic polyoxyethelene units has been shown to induce hyperlipidemia in rodents. In the mouse, one injection, intraperitoneally, of P-407 (0.5 g/kg) produced hypercholesterolemia that peaked at 24 hours and returned to control levels by 96 hours following treatment (Palmer, et al., Atherosclerosis, 1998, 136, 115-123). Consequently, these mice are a useful model for the investigation of compounds that modulate hyperlipidemia. In accordance with the present invention, the oligomeric compounds of the invention are tested in the P-407 model of hyperlipidemia.

Seven-week old male C57B1/6 mice are divided into two groups; (1) control and (2) P-407 injected animals (0.5 g/kg every 3 days, following an overnight fast). Animals in each group receive either a saline injection or injection with one or more of the oligomeric compounds of the invention or control compounds at 25 mg/kg three times per week or 50 mg/kg two times per week. All injections are administered intraperitoneally.

After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the P-407 injected animals that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are evaluated in the P-407 injected animals treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of P-407 injected animals treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is measured.

The P-407 injected animals that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

ApoE Knockout Mice (a Model of Dyslipidemia and Obesity):

B6.129P-ApoE^tm1Uneknockout mice (herein referred to as ApoE knockout mice) obtained from The Jackson Laboratory (Bar Harbor, Me.), are homozygous for the Apoe^tm1Uncmutation and show a marked increase in total plasma cholesterol levels that are unaffected by age or sex. These animals present with fatty streaks in the proximal aorta at 3 months of age. These lesions increase with age and progress to lesions with less lipid but more elongated cells, typical of a more advanced stage of pre-atherosclerotic lesion.

The mutation in these mice resides in the apolipoprotein E (ApoE) gene. The primary role of the ApoE protein is to transport cholesterol and triglycerides throughout the body. It stabilizes lipoprotein structure, binds to the low density lipoprotein receptor (LDLR) and related proteins, and is present in a subclass of HDLs, providing them the ability to bind to LDLR. ApoE is expressed most abundantly in the liver and brain. In one embodiment of the present invention, female B6.129P-Apoetm1Unc knockout mice (ApoE knockout mice) are used in the following studies to evaluate the oligomeric compounds of the invention as potential lipid lowering compounds.

Female ApoE knockout mice range in age from 5 to 7 weeks and are placed on a normal diet for 2 weeks before study initiation. ApoE knockout mice are then fed ad libitum a 60% fat diet, with 0.15% added cholesterol to induce dyslipidemia and obesity. Control animals include ApoE knockout mice and ApoE wildtype mice (i.e. lean littermates) maintained on a high-fat diet with no added cholesterol. After overnight fasting, mice from each group are dosed intraperitoneally every three days with saline, 50 mg/kg of a control compound or 5, 25 or 50 mg/kg of one or more of the oligomeric compounds of the invention.

After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target, the ApoE knockout mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism are also evaluated in the ApoE knockout mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of ApoE knockout mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice are measured.

The ApoE knockout mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.

In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloningâ€”A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

EXAMPLESExample 1Oligomeric Compounds Targeting Small Non-Coding RNAs

In accordance with the present invention, a series of oligomeric compounds are designed to target different regions of small non-coding target RNAs. The oligomeric compounds can be investigated for their effect on small non-coding RNA levels by quantitative real-time PCR. The target regions to which these sequences are complementary are herein referred to as â€œsuitable target regionsâ€ .

Example 2Oligomeric Compounds that Mimic or Replace Small Non-Coding RNAs

In accordance with the present invention, a series of oligomeric compounds are designed to mimic the structure and/or function of small non-coding RNAs. These mimics may include isolated single-, double-, or multiple-stranded compounds, any of which may include regions of intrastrand nucleobase complementarity, said regions capable of folding and forming a molecule with fully or partially double-stranded or multiple-stranded character based on regions of precise or imperfect complementarity. The oligomeric compound mimics can then be investigated for their effects on a cell, tissue or organism system lacking endogenous small non-coding RNAs or systems with aberrant expression of small non-coding RNAs using the screening methods disclosed herein or those commonly used in the art. Changes in levels, expression or function of the small non-coding RNA or its downstream target nucleic acid levels can be analyzed by quantitative real-time PCR as described, supra.

Example 3Pri-miRNAs Targeted by Compounds of the Present Invention

In accordance with the present invention, oligomeric compounds were designed to target one or more microRNA (miRNA) genes or gene products. Certain pri-miRNAs have been reported by Lim et al. Science, 2003, 299; 1540; in Brevia (detailed in the supplemental online materials; www.sciencemag.org/cgi/content/full/299/5612/1540/DC1) and these were used as starting targets.

A list of pri-miRNAs targeted is shown in Table 1. The gene name for each of the 188 targets (assigned by Lim et al.) is given in the table. For those pri-miRNAs that did not produce an identifiable miRNA detectable by PCR in the Lim publication, the position and sequence of the miRNAs were identified herein and are referred to as novel or hypothetical miRNAs. Also shown is the Genbank Accession number of the source sequence from which the pri-miRNA was extracted. The sequence is set forth in the Sequence Listing and is written in the 5â€² to 3â€² direction and is represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 1pri-miRNAsGenbankAccession #of sourceSEQpri-miRNAsequenceID NOmir-140NT_037896.14mir-30aNT_007299.115mir-34NT_028054.106mir-29b-1NT_021877.137mir-29b-2NT_007933.108mir-16-3NT_005612.119mir-203NT_026437.910mir-7-1NT_023935.1311mir-10bNT_037537.112mir-128aNT_034487.213mir-153-1NT_005403.1114mir-153-2NT_007741.1015hypothetical miRNA-013NT_010194.1316mir-27bNT_008476.1317mir-96NT_007933.1018mir-17as/mir-91NT_009952.1119mir-123/mir-126asNT_024000.1320mir-132NT_010692.921mir-108-1NT_010799.1122mir-23bNT_008476.1323let-7iNT_009711.1324mir-212NT_010692.925hypothetical miRNA-023NT_004658.1226mir-131-2NT_029973.627let-7bNT_011523.828mir-1dNT_035608.129mir-122aNT_033907.330mir-22NT_010692.931mir-92-1NT_009952.1132hypothetical miRNA-030NT_007933.1033mir-142NT_010783.1134mir-183NT_007933.1035hypothetical miRNA-033NT_011588.1136mir-214NT_029874.737mir-143NT_006859.1138mir-192-1NT_033241.339mir-192-2NT_033241.339mir-192-3NT_033241.339hypothetical miRNA-039NT_028392.442hypothetical miRNA-040NT_023148.943hypothetical miRNA-041NT_023089.1144let-7a-3NT_011523.845hypothetical miRNA-043NT_004902.1246hypothetical miRNA-044NT_009952.1147mir-181aNT_017568.1148let-7a-1NT_008476.1349mir-205NT_021877.1350mir-103-1NT_037665.151mir-26aNT_005580.1352mir-33aNT_011520.853mir-196-2NT_009458.1254mir-107NT_033890.355mir-106NT_011786.1156let-7f-1NT_008476.1357hypothetical miRNA-055NT_006713.1158mir-29cNT_021877.1359mir-130aNT_033903.360hypothetical miRNA-058NT_037537.161mir-218-1NT_006316.1362mir-124a-2NT_008183.1363mir-21NT_035426.264mir-16-1NT_033922.365mir-144NT_010799.1166mir-221NT_011568.1067mir-222NT_011568.1068mir-30dNT_028251.869mir-19b-2NT_011786.1170mir-128bNT_005580.1371hypothetical miRNA-069NT_017568.1172hypothetical miRNA-070NT_005375.1173hypothetical miRNA-071NT_011512.774mir-29b-3NT_007933.1075mir-129-2NT_009237.1376mir-133bNT_007592.1177hypothetical miRNA-075NT_006044.878let-7dNT_008476.1379mir-15bNT_005612.1180mir-29a-1NT_007933.1081hypothetical miRNA-079NT_021907.1382mir-199bNT_017568.1183mir-129-1NT_007933.1084let-7eNT_011109.1385hypothetical miRNA-083NT_024524.1186let-7cNT_011512.787mir-204NT_008580.1188mir-145NT_006859.1189mir-124a-1NT_019483.1390hypothetical miRNA-088NT_011519.991mir-213NT_029862.892hypothetical miRNA-090NT_006171.1393mir-20NT_009952.1194mir-133a-1NT_011044.1195mir-138-2NT_010498.1196mir-98NT_011799.1097mir-196-1NT_010783.1198mir-125b-1NT_033899.399mir-199a-2NT_029874.7100mir-29a-2NT_007933.10101hypothetical miRNA-099NT_016297.12102mir-181bNT_029862.8103hypothetical miRNA-101NT_030828.7104mir-141NT_035206.1105mir-131-1NT_004858.13106mir-133a-2NT_035608.1107hypothetical miRNA-105NT_017795.13108hypothetical miRNA-106NT_017795.13109hypothetical miRNA-107NT_008583.13110mir-1bNT_011044.11111mir-18NT_009952.11112mir-220NT_011588.11113hypothetical miRNA-111NT_004525.13114mir-7-3NT_011255.11115mir-218-2NT_023132.10116mir-24-2NT_031915.4117mir-24-1NT_008476.13118mir-103-2NT_011387.8119mir-211NT_010363.13120mir-101-3NT_008413.13121mir-30bNT_028251.8122hypothetical miRNA-120NT_009952.11123let-7a-4NT_033899.3124mir-10aNT_010783.11125mir-19aNT_009952.11126let-7f-2NT_011799.10127mir-15a-1NT_010393.11128mir-108-2NT_034392.2129mir-137NT_033951.3130mir-219NT_007592.11131mir-148bNT_009458.12132mir-130bNT_011520.8133mir-19b-1NT_009952.11134let-7a-2NT_033899.3135mir-216NT_005375.11136mir-100-1NT_033899.3137mir-100-2NT_033899.3137mir-187NT_010966.11139hypothetical miRNA-137NT_011387.8140hypothetical miRNA-138NT_008902.13141hypothetical miRNA-139NT_008902.13142mir-124a-3NT_011333.5143mir-7-2NT_033276.3144hypothetical miRNA-142NT_033317.3145hypothetical miRNA-143NT_007819.11146hypothetical miRNA-144NT_010783.11147mir-210NT_035113.2148mir-215NT_021953.13149mir-223NT_011669.11150mir-131-3NT_033276.3151mir-199a-1NT_011176.13152mir-30cNT_007299.11153mir-101-1NT_029865.8154mir-101-2NT_029865.8154hypothetical miRNA-153NT_005332.11156hypothetical miRNA-154NT_030828.7157mir-26bNT_005403.11158hypothetical miRNA-156NT_029289.7159mir-152NT_010783.11160mir-135-1NT_005986.13161mir-135-2NT_009681.13162mir-217NT_005375.11163hypothetical miRNA-161NT_004658.12164mir-15a-2NT_033922.3165let-7gNT_005986.13166hypothetical miRNA-164NT_010783.11167mir-33bNT_030843.4168hypothetical miRNA-166NT_011588.11169mir-16-2NT_033922.3170hypothetical miRNA-168NT_011520.8171hypothetical miRNA-169NT_007933.10172hypothetical miRNA-170NT_005151.11173hypothetical miRNA-171NT_006171.13174hypothetical miRNA-172NT_037752.1175hypothetical miRNA-173NT_008413.13176mir-182NT_007933.10177hypothetical miRNA-175NT_006258.12178hypothetical miRNA-176NT_025004.11179hypothetical miRNA-177NT_023098.7180hypothetical miRNA-178NT_037537.1181hypothetical miRNA-179NT_010194.13182hypothetical miRNA-180NT_010363.13183hypothetical miRNA-181NT_033899.3184mir-148aNT_007819.11185hypothetical miRNA-183NT_010363.13186mir-23aNT_031915.4187hypothetical miRNA-185NT_007592.11188hypothetical miRNA-186NT_008705.13189mir-181cNT_031915.4190hypothetical miRNA-188NT_023148.9191

Example 4miRNAs within Pri-miRNAs

miRNAs found within the pri-miRNA structures disclosed above were used in certain embodiments of the present invention. These miRNAs represent target nucleic acids to which the oligomeric compounds of the present invention were designed. The oligomeric compounds of the present invention can also be designed to mimic the miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thereby creating a construct with superior properties over the endogenous miRNA. The miRNA target sequences are shown in Table 2.

TABLE 2miRNAs found within pri-miRNAsmiRNA sequenceSEQ IDPri-miRNA(DNA form; where T replaces U in RNA)NOmir-140AGTGGTTTTACCCTATGGTAG192 mir-30aCTTTCAGTCGGATGTTTGCAGC193 mir-34TGGCAGTGTCTTAGCTGGTTGT194 mir-29b-1TAGCACCATTTGAAATCAGTGTT195 mir-29b-2TAGCACCATTTGAAATCAGTGTT195 mir-16-3TAGCAGCACGTAAATATTGGCG196 mir-203GTGAAATGTTTAGGACCACTAG197 mir-7-1TGGAAGACTAGTGATTTTGTT198 mir-10bTACCCTGTAGAACCGAATTTGT199 mir-128aTCACAGTGAACCGGTCTCTTTT200 mir-153-1TTGCATAGTCACAAAAGTGA201 mir-153-2TTGCATAGTCACAAAAGTGA201 mir-27bTTCACAGTGGCTAAGTTCTG202 mir-96TTTGGCACTAGCACATTTTTGC203 mir-17as/mir-91CAAAGTGCTTACAGTGCAGGTAGT204 mir-123/mir-126asCATTATTACTTTTGGTACGCG205 mir-132TAACAGTCTACAGCCATGGTCGC206 mir-108-1ATAAGGATTTTTAGGGGCATT207 mir-23bATCACATTGCCAGGGATTACCAC208 let-7iTGAGGTAGTAGTTTGTGCT209 mir-212TAACAGTCTCCAGTCACGGCC210 mir-131-2TAAAGCTAGATAACCGAAAGT211 let-7bTGAGGTAGTAGGTTGTGTGGTT212 mir-1dTGGAATGTAAAGAAGTATGTAT213 mir-122aTGGAGTGTGACAATGGTGTTTGT214 mir-22AAGCTGCCAGTTGAAGAACTGT215 mir-92-1TATTGCACTTGTCCCGGCCTGT216 mir-142CATAAAGTAGAAAGCACTAC217 mir-183TATGGCACTGGTAGAATTCACTG218 mir-214ACAGCAGGCACAGACAGGCAG219 mir-143TGAGATGAAGCACTGTAGCTCA220 mir-192-1CTGACCTATGAATTGACAGCC221 mir-192-2CTGACCTATGAATTGACAGCC221 mir-192-3CTGACCTATGAATTGACAGCC221 let-7a-3TGAGGTAGTAGGTTGTATAGTT222 mir-181aAACATTCAACGCTGTCGGTGAGT223 let-7a-1TGAGGTAGTAGGTTGTATAGTT222 mir-205TCCTTCATTCCACCGGAGTCTG224 mir-103-1AGCAGCATTGTACAGGGCTATGA225 mir-26aTTCAAGTAATCCAGGATAGGCT226 mir-33aGTGCATTGTAGTTGCATTG227 mir-196-2TAGGTAGTTTCATGTTGTTGGG228 mir-107AGCAGCATTGTACAGGGCTATCA229 mir-106AAAAGTGCTTACAGTGCAGGTAGC230 let-7f-1TGAGGTAGTAGATTGTATAGTT231 mir-29cCTAGCACCATTTGAAATCGGTT232 mir-130aCAGTGCAATGTTAAAAGGGC233 mir-218-1TTGTGCTTGATCTAACCATGT234 mir-124a-2TTAAGGCACGCGGTGAATGCCA235 mir-21TAGCTTATCAGACTGATGTTGA236 mir-16-1TAGCAGCACGTAAATATTGGCG196 mir-144TACAGTATAGATGATGTACTAG237 mir-221AGCTACATTGTCTGCTGGGTTTC238 mir-222AGCTACATCTGGCTACTGGGTCTC239 mir-30dTGTAAACATCCCCGACTGGAAG240 mir-19b-2TGTGCAAATCCATGCAAAACTGA241 mir-128bTCACAGTGAACCGGTCTCTTTC242 mir-29b-3TAGCACCATTTGAAATCAGTGTT195 mir-129-2CTTTTTGCGGTCTGGGCTTGC243 mir-133bTTGGTCCCCTTCAACCAGCTA244 let-7dAGAGGTAGTAGGTTGCATAGT245 mir-15bTAGCAGCACATCATGGTTTACA246 mir-29a-1CTAGCACCATCTGAAATCGGTT247 mir-199bCCCAGTGTTTAGACTATCTGTTC248 mir-129-1CTTTTTGCGGTCTGGGCTTGC243 let-7eTGAGGTAGGAGGTTGTATAGT249 let-7cTGAGGTAGTAGGTTGTATGGTT250 mir-204TTCCCTTTGTCATCCTATGCCT251 mir-145GTCCAGTTTTCCCAGGAATCCCTT252 mir-124a-1TTAAGGCACGCGGTGAATGCCA235 mir-213ACCATCGACCGTTGATTGTACC253 mir-20TAAAGTGCTTATAGTGCAGGTAG254 mir-133a-1TTGGTCCCCTTCAACCAGCTGT255 mir-138-2AGCTGGTGTTGTGAATC256 mir-98TGAGGTAGTAAGTTGTATTGTT257 mir-196-1TAGGTAGTTTCATGTTGTTGGG228 mir-125b-1TCCCTGAGACCCTAACTTGTGA258 mir-199a-2CCCAGTGTTCAGACTACCTGTTC259 mir-29a-2CTAGCACCATCTGAAATCGGTT247 mir-181bAACATTCATTGCTGTCGGTGGGTT260 mir-141AACACTGTCTGGTAAAGATGG261 mir-131-1TAAAGCTAGATAACCGAAAGT211 mir-133a-2TTGGTCCCCTTCAACCAGCTGT255 mir-1bTGGAATGTAAAGAAGTATGTAT213 mir-18TAAGGTGCATCTAGTGCAGATA262 mir-220CCACACCGTATCTGACACTTT263 mir-7-3TGGAAGACTAGTGATTTTGTT198 mir-218-2TTGTGCTTGATCTAACCATGT234 mir-24-2TGGCTCAGTTCAGCAGGAACAG264 mir-24-1TGGCTCAGTTCAGCAGGAACAG264 mir-103-2AGCAGCATTGTACAGGGCTATGA225 mir-211TTCCCTTTGTCATCCTTCGCCT264 mir-101-3TACAGTACTGTGATAACTGA265 mir-30bTGTAAACATCCTACACTCAGC266 let-7a-4TGAGGTAGTAGGTTGTATAGTT222 mir-10aTACCCTGTAGATCCGAATTTGTG267 mir-19aTGTGCAAATCTATGCAAAACTGA268 let-7f-2TGAGGTAGTAGATTGTATAGTT231 mir-15a-1TAGCAGCACATAATGGTTTGTG269 mir-108-2ATAAGGATTTTTAGGGGCATT207 mir-137TATTGCTTAAGAATACGCGTAG270 mir-219TGATTGTCCAAACGCAATTCT271 mir-148bTCAGTGCATCACAGAACTTTGT272 mir-130bCAGTGCAATGATGAAAGGGC273 mir-19b-1TGTGCAAATCCATGCAAAACTGA241 let-7a-2TGAGGTAGTAGGTTGTATAGTT222 mir-216TAATCTCAGCTGGCAACTGTG274 mir-100-1AACCCGTAGATCCGAACTTGTG275 mir-100-2AACCCGTAGATCCGAACTTGTG275 mir-187TCGTGTCTTGTGTTGCAGCCGG276 mir-124a-3TTAAGGCACGCGGTGAATGCCA235 mir-7-2TGGAAGACTAGTGATTTTGTT198 mir-210CTGTGCGTGTGACAGCGGCTG277 mir-215ATGACCTATGAATTGACAGAC278 mir-223TGTCAGTTTGTCAAATACCCC279 mir-131-3TAAAGCTAGATAACCGAAAGT211 mir-199a-1CCCAGTGTTCAGACTACCTGTTC259 mir-30cTGTAAACATCCTACACTCTCAGC280 mir-101-1TACAGTACTGTGATAACTGA265 mir-101-2TACAGTACTGTGATAACTGA265 mir-26bTTCAAGTAATTCAGGATAGGTT281 mir-152TCAGTGCATGACAGAACTTGG282 mir-135-1TATGGCTTTTTATTCCTATGTGAT283 mir-135-2TATGGCTTTTTATTCCTATGTGAT283 mir-217TACTGCATCAGGAACTGATTGGAT284 mir-15a-2TAGCAGCACATAATGGTTTGTG269 let-7gTGAGGTAGTAGTTTGTACAGT285 mir-33bGTGCATTGCTGTTGCATTG286 mir-16-2TAGCAGCACGTAAATATTGGCG196 mir-182TTTGGCAATGGTAGAACTCACA287 mir-148aTCAGTGCACTACAGAACTTTGT288 mir-23aATCACATTGCCAGGGATTTCC289 mir-181cAACATTCAACCTGTCGGTGAGT290

Example 5Uniform 2â€²-MOE Phosphorothioate (PS) Oligomeric Compounds Targeting miRNAs

In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target miRNA sequences disclosed by Lim et al. Science, 2003, 299, 1540. The compounds are shown in Table 3. â€œPri-miRNAâ€ indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 3 are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides throughout and the internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.

TABLE 3Uniform 2â€²-MOE PS Compounds targeting miRNAsISIS NumberSEQ ID NOSequencePri-miRNA327873291CTACCATAGGGTAAAACCACTmir-140 327874292GCTGCAAACATCCGACTGAAAGmir-30a 327875293ACAACCAGCTAAGACACTGCCAmir-34 327876294AACACTGATTTCAAATGGTGCTAmir-29b-1 327877295CGCCAATATTTACGTGCTGCTAmir-16-3 327878296CTAGTGGTCCTAAACATTTCACmir-203 327879297AACAAAATCACTAGTCTTCCAmir-7-1 327880298ACAAATTCGGTTCTACAGGGTAmir-10b 327881299AAAAGAGACCGGTTCACTGTGAmir-128a 327882300TCACTTTTGTGACTATGCAAmir-153-1 327883301CAGAACTTAGCCACTGTGAAmir-27b 327884302GCAAAAATGTGCTAGTGCCAAAmir-96 327885303ACTACCTGCACTGTAAGCACTTTGmir-17as/mir-91 327886304CGCGTACCAAAAGTAATAATGmir-123/mir-126as 327887305GCGACCATGGCTGTAGACTGTTAmir-132 327888306AATGCCCCTAAAAATCCTTATmir-108-1 327889307GTGGTAATCCCTGGCAATGTGATmir-23b 327890308AGCACAAACTACTACCTCAlet-7i 327891309GGCCGTGACTGGAGACTGTTAmir-212 327892310ACTTTCGGTTATCTAGCTTTAmir-131-2/mir-9 327893311AACCACACAACCTACTACCTCAlet-7b 327894312ATACATACTTCTTTACATTCCAmir-1d 327895313ACAAACACCATTGTCACACTCCAmir-122a 327896314ACAGTTCTTCAACTGGCAGCTTmir-22 327897315ACAGGCCGGGACAAGTGCAATAmir-92-1 327898316GTAGTGCTTTCTACTTTATGmir-142 327899317CAGTGAATTCTACCAGTGCCATAmir-183 327900318CTGCCTGTCTGTGCCTGCTGTmir-214 327901319TGAGCTACAGTGCTTCATCTCAmir-143 327902320GGCTGTCAATTCATAGGTCAGmir-192-1 327903321AACTATACAACCTACTACCTCAlet-7a-3 327904322ACTCACCGACAGCGTTGAATGTTmir-181a 327905323CAGACTCCGGTGGAATGAAGGAmir-205 327906324TCATAGCCCTGTACAATGCTGCTmir-103-1 327907325AGCCTATCCTGGATTACTTGAAmir-26a 327908326CAATGCAACTACAATGCACmir-33a 327909327CCCAACAACATGAAACTACCTAmir-196-2 327910328TGATAGCCCTGTACAATGCTGCTmir-107 327911329GCTACCTGCACTGTAAGCACTTTTmir-106 327912330AACTATACAATCTACTACCTCAlet-7f-1 327913331AACCGATTTCAAATGGTGCTAGmir-29c 327914332GCCCTTTTAACATTGCACTGmir-130a 327915333ACATGGTTAGATCAAGCACAAmir-218-1 327916334TGGCATTCACCGCGTGCCTTAAmir-124a-2 327917335TCAACATCAGTCTGATAAGCTAmir-21 327918336CTAGTACATCATCTATACTGTAmir-144 327919337GAAACCCAGCAGACAATGTAGCTmir-221 327920338GAGACCCAGTAGCCAGATGTAGCTmir-222 327921339CTTCCAGTCGGGGATGTTTACAmir-30d 327922340TCAGTTTTGCATGGATTTGCACAmir-19b-2 327923341GAAAGAGACCGGTTCACTGTGAmir-128b 327924342GCAAGCCCAGACCGCAAAAAGmir-129-2 327925343TAGCTGGTTGAAGGGGACCAAmir-133b 327926344ACTATGCAACCTACTACCTCTlet-7d 327927345TGTAAACCATGATGTGCTGCTAmir-15b 327928346AACCGATTTCAGATGGTGCTAGmir-29a-1 327929347GAACAGATAGTCTAAACACTGGGmir-199b 327930348ACTATACAACCTCCTACCTCAlet-7e 327931349AACCATACAACCTACTACCTCAlet-7c 327932350AGGCATAGGATGACAAAGGGAAmir-204 327933351AAGGGATTCCTGGGAAAACTGGACmir-145 327934352GGTACAATCAACGGTCGATGGTmir-213 327935353CTACCTGCACTATAAGCACTTTAmir-20 327936354ACAGCTGGTTGAAGGGGACCAAmir-133a-1 327937355GATTCACAACACCAGCTmir-138-2 327938356AACAATACAACTTACTACCTCAmir-98 327939357TCACAAGTTAGGGTCTCAGGGAmir-125b-1 327940358GAACAGGTAGTCTGAACACTGGGmir-199a-2 327941359AACCCACCGACAGCAATGAATGTTmir-181b 327942360CCATCTTTACCAGACAGTGTTmir-141 327943361TATCTGCACTAGATGCACCTTAmir-18 327944362AAAGTGTCAGATACGGTGTGGmir-220 327945363CTGTTCCTGCTGAACTGAGCCAmir-24-2 327946364AGGCGAAGGATGACAAAGGGAAmir-211 327947365TCAGTTATCACAGTACTGTAmir-101-3 327948366GCTGAGTGTAGGATGTTTACAmir-30b 327949367CACAAATTCGGATCTACAGGGTAmir-10a 327950368TCAGTTTTGCATAGATTTGCACAmir-19a 327951369CACAAACCATTATGTGCTGCTAmir-15a-1 327952370CTACGCGTATTCTTAAGCAATAmir-137 327953371AGAATTGCGTTTGGACAATCAmir-219 327954372ACAAAGTTCTGTGATGCACTGAmir-148b 327955373GCCCTTTCATCATTGCACTGmir-130b 327956374CACAGTTGCCAGCTGAGATTAmir-216 327957375CACAAGTTCGGATCTACGGGTTmir-100-1 327958376CCGGCTGCAACACAAGACACGAmir-187 327959377CAGCCGCTGTCACACGCACAGmir-210 327960378GTCTGTCAATTCATAGGTCATmir-215 327961379GGGGTATTTGACAAACTGACAmir-223 327962380GCTGAGAGTGTAGGATGTTTACAmir-30c 327963381AACCTATCCTGAATTACTTGAAmir-26b 327964382CCAAGTTCTGTCATGCACTGAmir-152 327965383ATCACATAGGAATAAAAAGCCATAmir-135-1 327966384ATCCAATCAGTTCCTGATGCAGTAmir-217 327967385ACTGTACAAACTACTACCTCAlet-7g 327968386CAATGCAACAGCAATGCACmir-33b 327969387TGTGAGTTCTACCATTGCCAAAmir-182 327970388ACAAAGTTCTGTAGTGCACTGAmir-148a 327971389GGAAATCCCTGGCAATGTGATmir-23a 327972390ACTCACCGACAGGTTGAATGTTmir-181c

Example 6Uniform 2â€²-MOE Phosphorothioate (PS) Oligomeric Compounds Targeting Novel miRNAs

In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target novel miRNAs. The compounds are shown in Table 4. â€œPri-miRNAâ€ indicates the particular pri-miRNA defined herein which contains the miRNA that the oligomeric compound was designed to target. The sequence of the compounds represent the full complement of the novel miRNA defined herein. All compounds in Table 4 are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides throughout and the internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or downstream targets of miRNAs.

TABLE 4Uniform 2â€²-MOE PS Compounds targeting novel pri-miRNAsISISSequenceNumberSEQ ID NO(5â€²-3â€²)Pri-miRNA328089391ACTGTAGGAATATGTTTGATAhypothetical miRNA-013 328090392ATTAAAAAGTCCTCTTGCCCAhypothetical miRNA-023 328091393GCTGCCGTATATGTGATGTCAhypothetical miRNA-030 328092394GGTAGGTGGAATACTATAACAhypothetical miRNA-033 328093395TAAACATCACTGCAAGTCTTAhypothetical miRNA-039 328094396TTGTAAGCAGTTTTGTTGACAhypothetical miRNA-040 328095397TCACAGAGAAAACAACTGGTAhypothetical miRNA-041 328096398CCTCTCAAAGATTTCCTGTCAhypothetical miRNA-043 328097399TGTCAGATAAACAGAGTGGAAhypothetical miRNA-044 328098400GAGAATCAATAGGGCATGCAAhypothetical miRNA-055 328099401AAGAACATTAAGCATCTGACAhypothetical miRNA-058 328100402AATCTCTGCAGGCAAATGTGAhypothetical miRNA-070 328101403AAACCCCTATCACGATTAGCAhypothetical miRNA-071 328102404GCCCCATTAATATTTTAACCAhypothetical miRNA-075 328103405CCCAATATCAAACATATCAhypothetical miRNA-079 328104406TATGATAGCTTCCCCATGTAAhypothetical miRNA-083 328105407CCTCAATTATTGGAAATCACAhypothetical miRNA-088 328106408ATTGATGCGCCATTTGGCCTAhypothetical miRNA-090 328107409CTGTGACTTCTCTATCTGCCThypothetical miRNA-099 328108410AAACTTGTTAATTGACTGTCAhypothetical miRNA-101 328109411AAAGAAGTATATGCATAGGAAhypothetical miRNA-105 328110412GATAAAGCCAATAAACTGTCAhypothetical miRNA-107 328111413TCCGAGTCGGAGGAGGAGGAAhypothetical miRNA-111 328112414ATCATTACTGGATTGCTGTAAhypothetical miRNA-120 328113415CAAAAATTATCAGCCAGTTTAhypothetical miRNA-137 328114416AATCTCATTTTCATACTTGCAhypothetical miRNA-138 328115417AGAAGGTGGGGAGCAGCGTCAhypothetical miRNA-142 328116418CAAAATTGCAAGCAAATTGCAhypothetical miRNA-143 328117419TCCACAAAGCTGAACATGTCThypothetical miRNA-144 328118420TATTATCAGCATCTGCTTGCAhypothetical miRNA-153 328119421AATAACACACATCCACTTTAAhypothetical miRNA-154 328120422AAGAAGGAAGGAGGGAAAGCAhypothetical miRNA-156 328121423ATGACTACAAGTTTATGGCCAhypothetical miRNA-161 328122424CAAAACATAAAAATCCTTGCAhypothetical miRNA-164 328123425TTACAGGTGCTGCAACTGGAAhypothetical miRNA-166 328124426AGCAGGTGAAGGCACCTGGCThypothetical miRNA-168 328125427TATGAAATGCCAGAGCTGCCAhypothetical miRNA-169 328126428CCAAGTGTTAGAGCAAGATCAhypothetical miRNA-170 328127429AACGATAAAACATACTTGTCAhypothetical miRNA-171 328128430AGTAACTTCTTGCAGTTGGAhypothetical miRNA-172 328129431AGCCTCCTTCTTCTCGTACTAhypothetical miRNA-173 328130432ACCTCAGGTGGTTGAAGGAGAhypothetical miRNA-175 328131433ATATGTCATATCAAACTCCTAhypothetical miRNA-176 328132434GTGAGAGTAGCATGTTTGTCThypothetical miRNA-177 328133435TGAAGGTTCGGAGATAGGCTAhypothetical miRNA-178 328134436AATTGGACAAAGTGCCTTTCAhypothetical miRNA-179 328135437ACCGAACAAAGTCTGACAGGAhypothetical miRNA-180 328136438AACTACTTCCAGAGCAGGTGAhypothetical miRNA-181 328137439GTAAGCGCAGCTCCACAGGCThypothetical miRNA-183 328138440GAGCTGCTCAGCTGGCCATCAhypothetical miRNA-185 328139441TACTTTTCATTCCCCTCACCAhypothetical miRNA-188

Example 7Chimeric Phosphorothioate Compounds Having 2â€²-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs

In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target different regions of pri-miRNA structures. The compounds are shown in Table 5. â€œPri-miRNAâ€ indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 5 are chimeric oligonucleotides (â€œgapmersâ€ ) 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.

TABLE 5Chimeric phosphorothioate oligomeric compounds having2â€²-MOE wings and a deoxy gap targeting pri-miRNAsISISNumberSEQ ID NOSequencepri-miRNA328333442AGAACAGCATGACGTAACCTmir-140 328334443GCCCATCTGTGGCTTCACAGmir-30a 328335444GAAGTCCGAGGCAGTAGGCAmir-30a 328336445CTTCCTTACTATTGCTCACAmir-34 328337446GCTAGATACAAAGATGGAAAmir-29b-1 328338447CTAGACAATCACTATTTAAAmir-29b-2 328339448GCAGCGCAGCTGGTCTCCCCmir-29b-2 328340449TAATATATATTTCACTACGCmir-16-3 328341450TGCTGTATCCCTGTCACACTmir-16-3 328342451CAATTGCGCTACAGAACTGTmir-203 328343452TCGATTTAGTTATCTAAAAAmir-7-1 328344453CTGTAGAGGCATGGCCTGTGmir-7-1 328345454TGACTATACGGATACCACACmir-10b 328346455GGAACAAGGCCAATTATTGCmir-128a 328347456AGAAATGTAAACCTCTCAGAmir-128a 328348457AGCTGTGAGGGAGAGAGAGAmir-153-1 328349458CTGGAGTGAGAATACTAGCTmir-153-1 328350459ACTGGGCTCATATTACTAGCmir-153-2 328351460TTGGATTAAATAACAACCTAhypothetical miRNA-013 328352461CCCGGAGACAGGGCAAGACAhypothetical miRNA-013 328353462AAAGCGGAAACCAATCACTGmir-27b 328354463GTCCCCATCTCACCTTCTCTmir-27b 328355464TCAGAGCGGAGAGACACAAGmir-96 328356465TAGATGCACATATCACTACCmir-17as/mir-91 328357466CTTGGCTTCCCGAGGCAGCTmir-17as/mir-91 328358467AGTTTGAAGTGTCACAGCGCmir-123/mir-126as 328359468GCGTTTTCGATGCGGTGCCGmir-123/mir-126as 328360469GAGACGCGGGGGCGGGGCGCmir-132 328361470TACCTCCAGTTCCCACAGTAmir-132 328362471TGTGTTTTCTGACTCAGTCAmir-108-1 328363472AGAGCACCTGAGAGCAGCGCmir-23b 328364473TCTTAAGTCACAAATCAGCAmir-23b 328365474TCTCCACAGCGGGCAATGTClet-7i 328366475GGCGCGCTGTCCGGGCGGGGmir-212 328367476ACTGAGGGCGGCCCGGGCAGmir-212 328368477GTCCTCTTGCCCAAGCAACAhypothetical miRNA-023 328369478GAAGACCAATACACTCATACmir-131-2 328370479CCGAGGGGCAACATCACTGClet-7b 328371480TCCATAGCTTAGCAGGTCCAmir-1d 328372481TTTGATAGTTTAGACACAAAmir-122a 328373482GGGAAGGATTGCCTAGCAGTmir-122a 328374483AGCTTTAGCTGGGTCAGGACmir-22 328375484TACCATACAGAAACACAGCAmir-92-1 328376485TCACAATCCCCACCAAACTCmir-92-1 328377486TCACTCCTAAAGGTTCAAGThypothetical miRNA-030 328378487CACCCTCCAGTGCTGTTAGTmir-142 328379488CTGACTGAGACTGTTCACAGmir-183 328380489CCTTTAGGGGTTGCCACACChypothetical miRNA-033 328381490ACAGGTGAGCGGATGTTCTGmir-214 328382491CAGACTCCCAACTGACCAGAmir-143 328383492AGAGGGGAGACGAGAGCACTmir-192-1 328384493TCACGTGGAGAGGAGTTAAAhypothetical miRNA-039 328385494AGTGCTAATACTTCTTTCAThypothetical miRNA-040 328386495ACCTGTGTAACAGCCGTGTAhypothetical miRNA-041 328387496TTATCGGAACTTCACAGAGAhypothetical miRNA-041 328388497TCCCATAGCAGGGCAGAGCClet-7a-3 328389498GGCACTTCATTGCTGCTGCChypothetical miRNA-043 328390499GGAGCCTTGCGCTCAGCATThypothetical miRNA-043 328391500ATGGTAATTTCATTTCAGGChypothetical miRNA-044 328392501GATTGCACATCCACACTGTChypothetical miRNA-044 328393502GCTGGCCTGATAGCCCTTCTmir-181a 328394503GTTTTTTCAAATCCCAAACTmir-181a 328395504CCCAGTGGTGGGTGTGACCClet-7a-1 328396505CTGGTTGGGTATGAGACAGAmir-205 328397506TTGATCCATATGCAACAAGGmir-103-1 328398507GCCATTGGGACCTGCACAGCmir-26a 328399508ATGGGTACCACCAGAACATGmir-33a 328400509AGTTCAAAACTCAATCCCAAmir-196-2 328401510GCCCTCGACGAAAACCGACTmir-196-2 328402511TTGAACTCCATGCCACAAGGmir-107 328403512AGGCCTATTCCTGTAGCAAAmir-106 328404513GTAGATCTCAAAAAGCTACCmir-106 328405514CTGAACAGGGTAAAATCACTlet-7f-1 328406515AGCAAGTCTACTCCTCAGGGlet-7f-1 328407516AATGGAGCCAAGGTGCTGCChypothetical miRNA-055 328408517TAGACAAAAACAGACTCTGAmir-29c 328409518GCTAGTGACAGGTGCAGACAmir-130a 328410519GGGCCTATCCAAAGTGACAGhypothetical miRNA-058 328411520TACCTCTGCAGTATTCTACAhypothetical miRNA-058 328412521TTTACTCATACCTCGCAACCmir-218-1 328413522AATTGTATGACATTAAATCAmir-124a-2 328414523CTTCAAGTGCAGCCGTAGGCmir-124a-2 328415524TGCCATGAGATTCAACAGTCmir-21 328416525ACATTGCTATCATAAGAGCTmir-16-1 328417526TAATTTTAGAATCTTAACGCmir-16-1 328418527AGTGTCTCATCGCAAACTTAmir-144 328419528TGTTGCCTAACGAACACAGAmir-221 328420529GCTGATTACGAAAGACAGGAmir-222 328421530GCTTAGCTGTGTCTTACAGCmir-30d 328422531GAGGATGTCTGTGAATAGCCmir-30d 328423532CCACATATACATATATACGCmir-19b-2 328424533AGGAAGCACACATTATCACAmir-19b-2 328425534GACCTGCTACTCACTCTCGTmir-128b 328426535GGTTGGCCGCAGACTCGTAChypothetical miRNA-069 328427536GATGTCACTGAGGAAATCAChypothetical miRNA-070 328428537TCAGTTGGAGGCAAAAACCChypothetical miRNA-071 328429538GGTAGTGCAGCGCAGCTGGTmir-29b-3 328430539CCGGCTATTGAGTTATGTACmir-129-2 328431540ACCTCTCAGGAAGACGGACTmir-133b 328432541GAGCATGCAACACTCTGTGChypothetical miRNA-075 328433542CCTCCTTGTGGGCAAAATCClet-7d 328434543CGCATCTTGACTGTAGCATGmir-15b 328435544TCTAAGGGGTCACAGAAGGTmir-29a-1 328436545GAAAATTATATTGACTCTGAmir-29a-1

Example 8Chimeric Phosphorothioate Compounds Having 2â€²-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs

In accordance with the present invention, a second series of oligomeric compounds were designed and synthesized to target different regions of pri-miRNA structures. The compounds are shown in Table 6. â€œPri-miRNAâ€ indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 6 are chimeric oligonucleotides (â€œgapmersâ€ ) 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.

TABLE 6Chimeric phosphorothioate oligomeric compounds having2â€²-MOE wings and a deoxy gap targeting pri-miRNAsISISNumberSEQ ID NOSequencepri-miRNA328637546GGTTCCTAATTAAACAACCChypothetical miRNA-079 328638547CCGAGGGTCTAACCCAGCCCmir-199b 328639548GACTACTGTTGAGAGGAACAmir-129-1 328640549TCTCCTTGGGTGTCCTCCTClet-7e 328641550TGCTGACTGCTCGCCCTTGChypothetical miRNA-083 328642551ACTCCCAGGGTGTAACTCTAlet-7c 328643552CATGAAGAAAGACTGTAGCCmir-204 328644553GACAAGGTGGGAGCGAGTGGmir-145 328645554TGCTCAGCCAGCCCCATTCTmir-124a-1 328646555GCTTTTAGAACCACTGCCTChypothetical miRNA-088 328647556GGAGTAGATGATGGTTAGCCmir-213 328648557ACTGATTCAAGAGCTTTGTAhypothetical miRNA-090 328649558GTAGATAACTAAACACTACCmir-20 328650559AATCCATTGAAGAGGCGATTmir-133a-1 328651560GGTAAGAGGATGCGCTGCTCmir-138-2 328652561GGCCTAATATCCCTACCCCAmir-98 328653562GTGTTCAGAAACCCAGGCCCmir-196-1 328654563TCCAGGATGCAAAAGCACGAmir-125b-1 328655564TACAACGGCATTGTCCTGAAmir-199a-2 328656565TTTCAGGCTCACCTCCCCAGhypothetical miRNA-099 328657566AAAAATAATCTCTGCACAGGmir-181b 328658567AGAATGAGTTGACATACCAAhypothetical miRNA-101 328659568GCTTCACAATTAGACCATCCmir-141 328660569AGACTCCACACCACTCATACmir-131-1 328661570ATCCATTGGACAGTCGATTTmir-133a-2 328662571GGCGGGCGGCTCTGAGGCGGhypothetical miRNA-105 328663572CTCTTTAGGCCAGATCCTCAhypothetical miRNA-106 328664573TAATGGTATGTGTGGTGATAhypothetical miRNA-107 328665574ATTACTAAGTTGTTAGCTGTmir-1b 328666575GATGCTAATCTACTTCACTAmir-18 328667576TCAGCATGGTGCCCTCGCCCmir-220 328668577TCCGCGGGGGCGGGGAGGCThypothetical miRNA-111 328669578AGACCACAGCCACTCTAATCmir-7-3 328670579TCCGTTTCCATCGTTCCACCmir-218-2 328671580GCCAGTGTACACAAACCAACmir-24-2 328672581AAGGCTTTTTGCTCAAGGGCmir-24-1 328673582TTGACCTGAATGCTACAAGGmir-103-2 328674583TGCCCTGCTCAGAGCCCTAGmir-211 328675584TCAATGTGATGGCACCACCAmir-101-3 328676585ACCTCCCAGCCAATCCATGTmir-30b 328677586TCCTGGATGATATCTACCTChypothetical miRNA-120 328678587TCTCCCTTGATGTAATTCTAlet-7a-4 328679588AGAGCGGAGTGTTTATGTCAmir-10a 328680589TCATTCATTTGAAGGAAATAmir-19a 328681590TCCAAGATGGGGTATGACCClet-7f-2 328682591TTTTTAAACACACATTCGCGmir-15a-1 328683592AGATGTGTTTCCATTCCACTmir-108-2 328684593CCCCCTGCCGCTGGTACTCTmir-137 328685594CGGCCGGAGCCATAGACTCGmir-219 328686595CTTTCAGAGAGCCACAGCCTmir-148b 328687596GCTTCCCAGCGGCCTATAGTmir-130b 328688597CAGCAGAATATCACACAGCTmir-19b-1 328689598TACAATTTGGGAGTCCTGAAmir-199b 328690599GCCTCCTTCATATATTCTCAmir-204 328691600CCCCATCTTAGCATCTAAGGmir-145 328692601TTGTATGGACATTTAAATCAmir-124a-1 328693602TTTGATTTTAATTCCAAACTmir-213 328694603CAAACGGTAAGATTTGCAGAhypothetical miRNA-090 328695604GGATTTAAACGGTAAACATCmir-125b-1 328696605CTCTAGCTCCCTCACCAGTGhypothetical miRNA-099 328697606GCTTGTCCACACAGTTCAACmir-181b 328698607GCATTGTATGTTCATATGGGmir-1b 328699608TGTCGTAGTACATCAGAACAmir-7-3 328700609AGCCAGTGTGTAAAATGAGAmir-24-1 328701610TTCAGATATACAGCATCGGTmir-101-3 328702611TGACCACAAAATTCCTTACAmir-10a 328703612ACAACTACATTCTTCTTGTAmir-19a 328704613TGCACCTTTTCAAAATCCACmir-15a-1 328705614AACGTAATCCGTATTATCCAmir-137

Example 9Chimeric Phosphorothioate Compounds Having 2â€²-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs

In accordance with the present invention, a third series of oligomeric compounds were designed and synthesized to target different pri-miRNA structures. The compounds are shown in Table 7. â€œPri-miRNAâ€ indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 7 are chimeric oligonucleotides (â€œgapmersâ€ ) 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.

TABLE 7Chimeric phosphorothioate oligomeric compounds having2â€²-MOE wings and a deoxy gap targeting pri-miRNAsISISNumberSEQ ID NOSequencepri-miRNA328706615CGTGAGGGCTAGGAAATTGCmir-216 328707616GCAACAGGCCTCAATATCTTmir-100-1 328708617ACGAGGGGTCAGAGCAGCGCmir-187 328709618GGCAGACGAAAGGCTGACAGhypothetical miRNA-137 328710619CTGCACCATGTTCGGCTCCChypothetical miRNA-138 328711620GGGGCCCTCAGGGCTGGGGCmir-124a-3 328712621CCGGTCCACTCTGTATCCAGmir-7-2 328713622GCTGGGAAAGAGAGGGCAGAhypothetical miRNA-142 328714623TCAGATTGCCAACATTGTGAhypothetical miRNA-143 328715624CTGGGGAGGGGGTTAGCGTChypothetical miRNA-144 328716625TGGGTCTGGGGCAGCGCAGTmir-210 328717626TTGAAGTAGCACAGTCATACmir-215 328718627TCTACCACATGGAGTGTCCAmir-124a-3 328719628AGTGCCGCTGCCGCGCCGTGmir-7-2 328720629ACACATTGAGAGCCTCCTGAhypothetical miRNA-142 328721630GTCGCTCAGTGCTCTCTAGGhypothetical miRNA-143 328722631AGGCTCCTCTGATGGAAGGThypothetical miRNA-144 328723632GCTGTGACTTCTGATATTAThypothetical miRNA-153 328724633GACATCATGTGATTTGCTCAhypothetical miRNA-154 328725634CACCCCAAGGCTGCAGGGCAmir-26b 328726635TGTCAAGCCTGGTACCACCAhypothetical miRNA-156 328727636CTGCTCCAGAGCCCGAGTCGmir-152 328728637ACCCTCCGCTGGCTGTCCCCmir-135-1 328729638TAGAGTGAATTTATCTTGGTmir-135-2 328730639TGGTGACTGATTCTTATCCAmir-217 328731640CAATATGATTGGATAGAGGAhypothetical miRNA-161 328732641TTTAAACACACATTCGCGCCmir-15a-2 328733642ACCGGGTGGTATCATAGACClet-7g 328734643TGCATACCTGTTCAGTTGGAhypothetical miRNA-164 328735644GCCCGCCTCTCTCGGCCCCCmir-33b 328736645TCGCCCCCTCCCAGGCCTCThypothetical miRNA-166 328737646ACAACTGTAGAGTATGGTCAmir-16-2 328738647GCTGACCATCAGTACTTTCChypothetical miRNA-168 328739648TTATAGAACAGCCTCCAGTGhypothetical miRNA-169 328740649TTCAGGCACTAGCAGTGGGThypothetical miRNA-170 328741650AGTACTGCGAGGTTAACCGChypothetical miRNA-171 328742651GGACCTTTAAGATGCAAAGThypothetical miRNA-172 328743652TTCATATTATCCACCCAGGThypothetical miRNA-173 328744653CGGATCCTGTTACCTCACCAmir-182 328745654TGGTGCCTGCCACATCTTTGhypothetical miRNA-175 328746655TGGGAGGCTGAATCAAGGAChypothetical miRNA-176 328747656TGACAACCAGGAAGCTTGTGhypothetical miRNA-177 328748657GCCAGGCAGCGAGCTTTTGAhypothetical miRNA-178 328749658CAGCCTGCCACCGCCGCTTThypothetical miRNA-179 328750659CTGCCCCCGTGGACCGAACAhypothetical miRNA-180 328751660TCGTGCACCTGAGGAGTCTGhypothetical miRNA-181 328752661CAAACGTGCTGTCTTCCTCCmir-148a 328753662AAGGACTCAGCAGTGTTTCAhypothetical miRNA-183 328754663TCCTCGGTGGCAGAGCTCAGmir-23a 328755664AGACAATGAGTACACAGTTChypothetical miRNA-185 328756665CTGCAAGCACTGGTTCCCAThypothetical miRNA-186 328757666TTGCCTGAGCTGCCCAAACTmir-181c 328758667TCCATCACACTGTCCTATGAhypothetical miRNA-188 328759668GAGGGATTGTATGAACATCTmir-216 328760669GCTTGTGCGGACTAATACCAmir-100-1 328761670GCAGGCTAAAAGAAATAAGChypothetical miRNA-138 328762671ATTGTATAGACATTAAATCAmir-124a-3 328763672GTTGAGCGCAGTAAGACAACmir-7-2 328764673AGATGTTTCTGGCCTGCGAGhypothetical miRNA-142 328765674GACAAACTCAGCTATATTGTmir-215 328766675ACGGCTCTGTGGCACTCATAmir-131-3 328767676GCTTTCTTACTTTCCACAGCmir-30c 328768677TACCTTTAGAATAGACAGCAmir-101-1 328769678AGGCTGGACAGCACACAACCmir-26b 328770679AGCAGGAGCCTTATCTCTCChypothetical miRNA-156 328771680ATGAGTGAGCAGTAGAATCAmir-135-1 328772681TGAGACTTTATTACTATCACmir-135-2 328773682TACTTTACTCCAAGGTTTTAmir-15a-2 328774683GCACCCGCCTCACACACGTGmir-33b 328775684TTCCCGACCTGCCTTTACCThypothetical miRNA-166 328776685TCCTGTAATTATAGGCTAGChypothetical miRNA-169 328777686GGATCATATCAATAATACCAhypothetical miRNA-172 328778687TGCTGAGACACACAATATGThypothetical miRNA-176 328779688TGTTTGTCTCCAAGAAACGThypothetical miRNA-177 328780689TGTCATGGACAGGATGAATAhypothetical miRNA-179 328781690TCTATCATACTCAGAGTCGGmir-148a 328782691TTGTGACAGGAAGCAAATCCmir-23a 328783692CATCAGAGTCACCAACCCCAhypothetical miRNA-185 328784693CAAGAGATGTCTCGTTTTGChypothetical miRNA-186

Example 10Chimeric Phosphorothioate Compounds Having 2â€²-MOE Wings and a Deoxy Gap Targeted to the Stem Loop of Pri-miRNA Structures

In accordance with the present invention, a fourth series of oligomeric compounds were designed to target the stem loop of different pri-miRNA structures. In some cases, these oligomeric compounds contain mismatches, and thus hybridize with partial complementarity to the stemloop structure of the pri-miRNA targeted. The compounds are shown in Table 8. â€œPri-miRNAâ€ indicates the particular pri-miRNA that the oligomeric compound was designed to target. All compounds in Table 8 are chimeric oligonucleotides (â€œgapmersâ€ ), composed of a central â€œgapâ€ region consisting of 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or downstream nucleic acid targets.

TABLE 8Chimeric phosphorothioate compounds having2â€²-MOE wings and a deoxy gap targetedto the stem loop of pri-miRNA structuresCompoundSEQ IDNumberNO.SequencePri-miRNARG1694GTGGTAGAACAGCATGACGTCmir-140 RG2695AGCTGTGAAGCCACGATGGGCmir-30a RG3696AGATACAAAGATGGAAAAATCmir-29b-1 RG4697CTTCCTTACTATTGCTCACAAmir-34 RG5698TGTTTAATATATATTTCACTCmir-16-3 RG6699TGTCAAGACATCGCGTTAACAmir-203 RG7700TGTCGATTTAGTTATCCAACAmir-7-1 RG8701GTGACTATACGGATACCACACmir-10b RG9702ACCTCTCCAAATGTAAAGAmir-128a RG10703CAAAGCGGAAACCAATCACTGmir-27b RG11704CTGCAGTACATGCACATATCAmir-91 RG12705AACAATGACACCCTTGACCTmir-132 RG13706TTTTAATCTTAAGTCACAAAmir-23b RG14707ATCTCCACAGCGGGCAATGTClet-7i RG15708TATGAAGACCAATACACTCCAmir-131-2 RG16709GGGGCAACATCACTGCCClet-7b RG17710CCATGTTAGCAGGTCCATATGmir-1d RG18711GTTTGATAGTTTAGACACAAAmir-122a RG19712TGGGTCAGGACTAAAGCTTCmir-22 RG20713AATACCATACAGAAACACAGCmir-92-1 RG21714TTCGTGATGATTGTCGTGCCmir-142 RG22715ACTGCGAGACTGTTCACAGTTmir-183 RG23716TACAGGTGAGCGGATGTTCTGmir-214 RG24717TCTCAGCTCCCAACTGACCAGmir-143 RG25718ACCGCAGATATTACAGCCACTlet-7a-3 RG26719CCTGATAGCCCTTCTTAAGGAmir-181a RG27720CTTGATCCATATGCAACAAGGmir-103-1 RG28721GCCATTGGGACCTGCACACCmir-26a RG29722GCATGGGTACCACCCCATGCmir-33a RG30723CGAGTTCAAAACTCAATCCCAmir-196-2 RG31724CTTGAACTCCATGCCACAAGGmir-107 RG32725GTAGATCTCAAAAAGCTAGCmir-106 RG33726GAACAGGGTAAAATCACTAClet-7f-1 RG34727AGACAAAAACAGACTCTGAAmir-29c RG35728GCTAGTGACAGGTCCAGACAGmir-130a RG36729TTTACTCATACCTCGCAACCAmir-218-1 RG37730TTAATTGTATGACATTAAATCAmir-124a-2 RG38731TGCCATGAGATTCAACAGTCAmir-21 RG39732GATAATATTTAGAATCTTAACmir-16-1 RG40733TAGTGTCTCATCGCAAACTTAmir-144 RG41734CTGTTGCCTAACGAACACAGAmir-221 RG42735TGCTGATTACGAAAGACAGGATmir-222 RG43736GCTTAGCTGTGTCTTACAGCTmir-30d

Example 11Effects of Oligomeric Compounds Targeting miRNAs on Apoptosis in Caspase Assay

Programmed cell death or apoptosis involves the activation of proteases, a family of intracellular proteases, through a cascade which leads to the cleavage of a select set of proteins. The caspase family contains at least 14 caspases, with differing substrate preferences. The caspase activity assay uses a DEVD peptide to detect activated caspases in cell culture samples. The peptide is labeled with a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC). Activated caspases cleave the DEVD peptide resulting in a fluorescence shift of the AFC. Increased fluorescence is indicative of increased caspase activity and consequently increased cell death. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.

The effect of several oligomeric compounds of the present invention was examined in cells expressing miRNA targets. The cells expressing the targets used in these experiments were T47D, a breast carcinoma cell line. Other cell lines can also be employed in this assay and these include normal human mammary epithelial cells (HMECs) as well as two breast carcinoma cell lines, MCF7 and T47D. All of the cell lines were obtained from the American Type Culture Collection (Manassas, Va.). The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. MCF-7 cells are routinely cultured in DMEM low glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. T47D cells were cultured in Gibco DMEM High glucose media supplemented with 10% Fetal Bovine Serum (FBS).

Cells were plated at 10,000 cells per well for HMEC cells or 20,000 cells per well for MCF7 and T47D cells, and allowed to attach to wells overnight. Plates used were 96 well Costar plate 1603 (black sides, transparent bottom). DMEM high glucose medium, with and without phenol red, were obtained from Invitrogen (San Diego, Calif.). MEGM medium, with and without phenol red, were obtained from Biowhittaker (Walkersville, Md.). The caspase-3 activity assay kit was obtained from Calbiochem (Cat. #HTS02) (EMD Biosciences, San Diego, Calif.).

Before adding to cells, the oligomeric compound cocktail was mixed thoroughly and incubated for 0.5 hrs. The oligomeric compound or the LIPOFECTINâ„¢-only vehicle control was added (generally from a 3 Î¼M stock of oligonucleotide) to a final concentration of 200 nM with 6 Î¼g/ml LIPOFECTINâ„¢. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 Î¼l of PBS (150 Î¼L HBSS for HMEC cells). The wash buffer in each well was replaced with 100 Î¼L of the oligomeric compound/OPTI-MEMâ„¢/LIPOFECTINâ„¢ cocktail (this was T=0 for oligomeric compound treatment). The plates were incubated for 4 hours at 37Â° C., after which the medium was dumped and the plate was tapped on sterile gauze. 100 Î¼l of full growth medium without phenol red was added to each well. After 48 hours, 50 Î¼l of oncogene buffer (provided with Calbiochem kit) with 1004 DTT was added to each well. 20 Î¼l of oncogene substrate (DEVD-AFC) was added to each well. The plates were read at 400Â±25 nm excitation and 508Â±20 nm emission at t=0 and t=3 time points. The t=0Ã—(0.8) time point was subtracted from the t=3 time point, and the data are shown as percent of LIPOFECTINâ„¢-only (untreated control) treated cells.

Four experiments were performed and the results are shown in Tables 9-12. The concentration of oligomeric compound used was 200 nM. All compounds in Tables 9-12 are chimeric oligomeric compounds (â€œgapmersâ€ ) 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the compound. All cytidine residues are 5-methylcytidines. As a control a 20-mer oligonucleotide random-mer, ISIS-29848 (NNNNNNNNNNNNNNNNNNNN; where N is A, T, C or G; herein incorporated as SEQ ID NO: 737) was used. In addition, two positive controls targeting expressed genes known to induce apoptosis when inhibited were included. These were ISIS-148715 (TTGTCCCAGTCCCAGGCCTC; herein incorporated as SEQ ID NO: 738) which targets human Jagged2 and ISIS-226844 (GCCCTCCATGCTGGCACAGG; herein incorporated as SEQ ID NO: 739) which targets human Notch1. Both positive controls have the same chemistry and gap structure as the compounds being tested. An increase in fluorescence indicates that the compound, by inhibiting its target, induces apoptosis as compared to untreated controls (UTC).

TABLE 9Effects of oligomeric compounds targetingmiRNAs on Apoptosis in Caspase AssaySEQ IDFold IncreaseISIS NumberNO.Pri-miRNAover UTCUTCN/AN/A1.0Untreated controlISIS-29848737N/A3.5n-merISIS-148715738Jagged21.5Positive controlISIS-226844739Notch13.6Positive control328371480mir-1d1.2328400509mir-196-21.3328420529mir-2221.0328692601mir-124a-11.2328381490mir-2141.1328691600mir-1450.9328391500hypothetical miRNA-0440.8328415524mir-211.1328433542let-7d1.0328643552mir-2040.9328377486hypothetical miRNA-0300.7328405514let-7f-11.0328372481mir-122a1.0328403512mir-1061.0328424533mir-19b-20.9328648557hypothetical miRNA-0901.1328397506mir-103-11.2328656565hypothetical miRNA-0991.1328392501hypothetical miRNA-0441.0328421530mir-30d1.2328417526mir-16-11.0328647556mir-2130.9328378487mir-1421.0328416525mir-16-10.9

TABLE 10Effects of oligomeric compounds targetingmiRNAs on Apoptosis in Caspase AssaySEQ IDFold IncreaseISIS NumberNO.Pri-miRNAover UTCUTCN/AN/A0.9Untreated controlISIS-29848737N/A3.0n-merISIS-148715738Jagged21.0Positive controlISIS-226844739Notch13.1Positive control328375484mir-92-10.9328382491mir-1430.9328383492mir-192-11.2328385494hypothetical miRNA-0400.9328395504let-7a-11.0328398507mir-26a0.9328399508mir-33a1.0328402511mir-1071.2328408517mir-29c0.9328409518mir-130a0.7328422531mir-30d1.0328423532mir-19b-20.6328425534mir-128b0.8328431540mir-133b0.9328436545mir-29a-10.9328646555hypothetical miRNA-0881.1328649558mir-201.0328651560mir-138-20.9328652561mir-981.2328657566mir-181b0.8328672581mir-24-10.9328694603hypothetical miRNA-0900.8328696605hypothetical miRNA-0991.5328700609mir-24-10.8

TABLE 11Effects of oligomeric compounds targetingmiRNAs on Apoptosis in Caspase AssaySEQ IDFold IncreaseISIS NumberNO.Pri-miRNAover UTCUTCN/AN/A0.9Untreated controlISIS-29848737N/A3.2n-merISIS-148715738Jagged21.1Positive controlISIS-226844739Notch13.1Positive control328374483mir-221.1328376485mir-92-10.7328384493hypothetical miRNA-0391.0328386495hypothetical miRNA-0410.7328390499hypothetical miRNA-0430.9328393502mir-181a1.5328404513mir-1060.9328406515let-7f-11.0328407516hypothetical miRNA-0551.2328410519hypothetical miRNA-0581.5328411520hypothetical miRNA-0580.8328413522mir-124a-20.8328426535hypothetical miRNA-0691.3328427536hypothetical miRNA-0700.8328435544mir-29a-11.3328637546hypothetical miRNA-0791.0328638547mir-199b0.8328639548mir-129-10.8328645554mir-124a-12.2328653562mir-196-11.1328654563mir-125b-11.0328655564mir-199a-20.7328689598mir-199b0.8328695604mir-125b-10.8

TABLE 12Effects of oligomeric compounds targetingmiRNAs on Apoptosis in Caspase AssaySEQ IDFold IncreaseISIS NumberNO.Pri-miRNAover UTCUTCN/AN/A1.0Untreated controlISIS-29848737N/A3.5n-merISIS-148715738Jagged21.3Positive controlISIS-226844739Notch13.5Positive control328373482mir-122a0.9328379488mir-1831.1328387496hypothetical miRNA-0411.4328388497let-7a-30.9328389498hypothetical miRNA-0431.1328394503mir-181a0.8328396505mir-2050.8328401510mir-196-20.8328412521mir-218-11.2328414523mir-124a-20.9328418527mir-1441.0328419528mir-2210.7328430539mir-129-21.3328432541hypothetical miRNA-0750.6328434543mir-15b0.8328640549let-7e0.9328641550hypothetical miRNA-0831.1328642551let-7c1.0328644553mir-1450.7328650559mir-133a-10.8328658567hypothetical miRNA-1011.2328690599mir-2040.8328693602mir-2131.0328697606mir-181b1.0

From these data, it is evident that SEQ ID NOs. 480, 509, 601, 490, 524, 557, 506, 565, 530, 605, 492, 561, 511, 555, 483, 502, 535, 562, 544, 519, 516, 554, 496, 567, 521, 539, 488, 498, and 550 induce apoptosis in T47D cells, while SEQ ID NOs. 500, 486, 518, 532, 534, 566, 603, 609, 485, 495, 520, 522, 536, 547, 548, 564, 598, 604, 503, 505, 510, 528, 541, 543, 553, 559, and 599 prevent or have a protective effect from apoptosis in the same system.

Example 12Oligomeric Compounds Targeting the Mir-30a Pri-miRNA Structure

In one embodiment of the invention, oligomeric compounds targeting the hairpin structure of mir-30a pri-miRNA were designed and tested for their effects on miRNA signaling in 293T cells (American Type Culture Collection (Manassas, Va.)).

A synthetic DNA fragment comprised of four tandem repeats of the target site for mir-30a was cloned into the vector pGL3-C (purchased from Promega Corp., Madison Wis.) at the unique XbaI site (pGL3C-M30-4Ã—). This places the target site in the 3â€²UTR of the luciferase reporter vector. An oligomeric compound mimicking the mir-30a pri-miRNA (AATTTAATACGACTCACTATAGGGCGACTGTAAACATCCTCGACTGGAAGCTGTGAAG CCACAGATGGGCTTTCAGTCGGATGTTTGCAGCTGC, herein incorporated as SEQ ID NO: 1749) was in vitro transcribed using T7 RNA polymerase and a DNA template produced by PCR (the T7 promoter is shown in bold).

On the day prior to the experiment 24-well plates were seeded with 293T cells at 50% confluency. The following morning cells were treated with oligomeric compounds targeted to the mir-30a pri-miRNA mimic. The oligomeric compounds used in this study are shown in Table 13. All of the compounds are 20 nucleobases in length having either a phosphorothioate backbone throughout (PS) or a phosphodiester backbone throughout (PO). As designated in the table, ISIS 328076, 328078, 328081, 328084, 328086, 328088 are chimeric oligomeric compounds (â€œgapmersâ€ ) 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The remaining compounds in the table have 2â€²-methoxyethoxy (MOE) nucleotides throughout with either a phosphorothioate (PS) or phosphodiester (PO) internucleoside linkages.

If the compound targeted the pre-loop of the mir-30a pri-miRNA structure, that designation is also noted in the table.

TABLE 13Oligomeric compounds targeting the mir-30a pri-miRNAIsisSEQ IDNumberSequenceChemistryNO328075GCTTCACAGCTTCCAGTCGA(PS/MOE)740 328076GCTTCACAGCTTCCAGTCGA(PS/MOE 5-10-5 gapmer)740 328077CCCATCTGTGGCTTCACAGC(PS/MOE); pre-loop741 328078CCCATCTGTGGCTTCACAGC(PS/MOE 5-10-5 gapmer); pre-loop741 328079CCCATCTGTGGCTTCACAGC(PO/MOE); pre-loop741 328080TGAAAGCCCATCTGTGGCTT(PS/MOE); pre-loop742 328081TGAAAGCCCATCTGTGGCTT(PS/MOE 5-10-5 gapmer); pre-loop742 328082TGAAAGCCCATCTGTGGCTT(PO/MOE); pre-loop742 328083GCAGCTGCAAACATCCGACT(PS/MOE)743 328084GCAGCTGCAAACATCCGACT(PS/MOE 5-10-5 gapmer)743 328085CATCTGTGGCTTCACAGCTT(PS/MOE)744 328086CATCTGTGGCTTCACAGCTT(PS/MOE 5-10-5 gapmer)744 328087AAGCCCATCTGTGGCTTCAC(PS/MOE)745 328088AAGCCCATCTGTGGCTTCAC(PS/MOE 5-10-5 gapmer)745

Cells were washed once with PBS then oligomeric compounds were added to triplicate wells at 150 nM in OPTI-MEMâ„¢ media and 4.5 Î¼l/ml LIPOFECTINâ„¢ reagent (Invitrogen Corporation, Carlsbad, Calif.). After 3 hours, the media was removed, and the cells were treated with the mir-30a pri-miRNA mimic at 200 nM in OPTI-MEMâ„¢ with 6 Î¼l/ml LIPOFECTINâ„¢ reagent. After 3 hours the media was removed from the cells. The reporter plasmid, pGL3C-M30-4Ã—, was then transfected using SuperFect reagent. 20 Î¼g of pGL3C-M30-4Ã— and 2 Î¼g of pRL-CMV, a plasmid expressing Renilla luciferase, were suspended in 600 Î¼l of serum-free DMEM to which 120 Î¼l of Superfect was added. After a 5 minute incubation, 6 mls of DMEM+10% FCS was added. 125 Î¼l of the plasmid/SuperFect suspension was added to each well. After a 2 hour incubation cells were washed and fresh growth media added. Cells were incubated overnight.

The following morning the media was removed and the cells were lysed in 120 Î¼l passive lysis buffer (PLB; Promega). 40 Î¼l of the lysate was then assayed for Photinus (PL) and Renilla (RL) luciferases using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are given as percent pGL3C-M30-4X expression (PL) normalized to pRL-CMV expression (RL). The 20-nucleobase oligonucleotide random-mer ISIS Number 29848 was used as a negative control. The data are shown in Table 14.

TABLE 14Effects of oligomeric compounds targeting themir-30a pri-miRNA on reporter gene expressionpercent controlSEQ IDluciferaseNOISIS NumberexpressionN/AUntreated control100N/AMir-30a pri-miRNA only6273729848 control added after63mir-30a pri-miRNA2923278746674032807555740328076577413280777074132807863742328080727423280818074332808475744328085727443280869574532808783745328088107

Upon administration of the mir-30a pri-miRNA mimic, the pri-miRNA is believed to be processed in the cell by the endogenous Drosha RNase III enzyme into a pre-miRNA, which is then processed by human Dicer into a mature miRNA, which is then able to hybridize to the target site, thus effectively reducing luciferase reporter expression.

Upon treatment of the system with the oligomeric compounds targeting the mir-30a pri-miRNA, the processing and/or production of the mir-30a mature miRNA is inhibited, and the mir-30a miRNA is no longer able to bind its target site, thus allowing luciferase reporter expression to increase.

Cells treated with mir-30a pri-miRNA mimic show an approximately 38% reduction in luciferase expression compared to the untreated controls. Treatment with ISIS 328086, 328087 and 328088 had the most dramatic effect in reversing the mir-30a miRNA-mediated silencing, restoring luciferase reporter expression to near control levels. Thus, it was demonstrated that the oligomeric compound mimicking the mir-30a pri-miRNA silences luciferase activity from the reporter vector, and that oligomeric compounds targeting the mir-30a pri-miRNA can inhibit its silencing activity, possibly by interfering with its processing into the pre-miRNA or mature miRNA molecules.

ISIS 328085 to ISIS 328088 were designed to target the mir-30a pri-miRNA as pseudo half-knot compounds. Methods for the preparation of pseudo half-knot compounds are disclosed in U.S. Pat. No. 5,512,438 which is incorporated herein by reference. This motif has been used to disrupt the structure of regulatory RNA stem loops in larger viral genomic structures. (Ecker et al, Science. 1992; 257:958-61). However, this is the first example of the pseudo half-knot motif being used to regulate a small non-coding RNA, more specifically a miRNA such as those disclosed herein. It is also the first demonstration of apoptotic modulation in a cell by pseudo half-knot structured oligomeric compounds.

Example 13Effects of Oligomeric Compounds Targeting miRNAs on Expression of Adipocyte Differentiation Markers

The effect of several oligomeric compounds of the present invention targeting miRNAs on the expression of markers of cellular differentiation was examined in preadipocytes.

One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. An excessive recruitment and differentiation of preadipocytes into mature adipocytes is a characteristic of human obesity, which is a strong risk factor for type 2 diabetes, hypertension, atherosclerosis, cardiovascular disease, and certain cancers. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and PPARÎ³ (Peroxisome proliferator-activated receptor gamma). These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. For example, HSL is involved in the mobilization of fatty acids from adipose tissue into the bloodstream; studies suggest that increased free fatty acid levels are one of the causative factors in type 2 diabetes. aP2 is believed to play a role in athersclerosis. Glut4 is important in insulin signaling. PPARÎ³ is believed to be involved in adipocyte differentiation, insulin sensitivity, and colonic tumor development.

Leptin is also a marker for differentiated adipocytes. In the adipocyte assay, leptin secretion into the media above the differentiated adipocytes was measured by protein ELISA. Cell growth, transfection and differentiation procedures were carried out as described for the Triglyceride accumulation assay (see below). On day nine post-transfection, 96-well plates were coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, Minn.) and left at 4Â° C. overnight. The plates were blocked with bovine serum albumin (BSA), and a dilution of the media was incubated in the plate at RT for 2 hours. After washing to remove unbound components, a second monoclonal antibody to human leptin (conjugated with biotin) was added. The plate was then incubated with strepavidin-conjugated horseradish peroxidase (HRP) and enzyme levels are determined by incubation with 3,3â€²,5,5â€²-Tetramethlybenzidine, which turns blue when cleaved by HRP. The OD₄₅₀was read for each well, where the dye absorbance is proportional to the leptin concentration in the cell lysate. Results are expressed as a percentÂ±standard deviation relative to transfectant-only controls.

An increase in triglyceride content is another well-established marker for adipocyte differentiation. The triglyceride accumulation assay measures the synthesis of triglyceride by adipocytes. Triglyceride accumulation was measured using the Infinityâ„¢ Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.). Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, N.C.) were grown in preadipocyte media (ZenBio Inc.). One day before transfection, 96-well plates were seeded with 3000 cells/well. Cells were transfected according to standard published procedures with 250 nM oligomeric compound in LIPOFECTINâ„¢ (Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomeric compounds were tested in triplicate on each 96-well plate, and the effects of TNF-Î±, a positive drug control that inhibits adipocyte differentiation, were also measured in triplicate. Negative and transfectant-only controls may be measured up to six times per plate. After the cells have reached confluence (approximately three days), they were exposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-Î³ agonist, IBMX, dexamethasone, and insulin for three days. Cells were then fed adipocyte media (Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals. On day nine post-transfection, cells were washed and lysed at room temperature, and the triglyceride assay reagent was added. Triglyceride accumulation was measured based on the amount of glycerol liberated from triglycerides by the enzyme lipoprotein lipase. Liberated glycerol is phosphorylated by glycerol kinase, and hydrogen peroxide is generated during the oxidation of glycerol-1-phosphate to dihydroxyacetone phosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP) uses H₂O₂to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye. Dye absorbance, which is proportional to the concentration of glycerol, was measured at 515 nm using an UV spectrophotometer. Glycerol concentration was calculated from a standard curve for each assay, and data were normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Results are expressed as a percentÂ±standard deviation relative to transfectant-only control.

For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARÎ³, as well as triglyceride (TG) accumulation and leptin secretion were measured in adipocytes transfected with the uniform 2â€²-MOE phosphorothioate (PS) oligomeric compounds previously described. Cells are lysed on day nine post-transfection, in a guanadinium-containing buffer and total RNA is harvested. Real-time PCR is performed (Applied Biosystems, Prism 7700) on the total RNA using the following primer/probe sets for the adipocyte differentiation hallmark genes: (aP2): forward 5â€²-GGTGGTGGAATGCGTCATG-3â€² (SEQ ID NO: 746), reverse 5â€²-CAACGTCCCTTGGCTTATGC-3â€² (SEQ ID NO: 747), probe 5â€²-FAM-AAGGCGTCACTTCCACGAGAGTTTATGAGA-TAMRA-3â€² (SEQ ID NO: 748); (Glut4): forward 5â€²-GGCCTCCGCAGGTTCTG-3â€² (SEQ ID NO: 749), reverse 5â€²-TTCGGAGCCTATCTGTTGGAA-3â€² (SEQ ID NO: 750), probe 5â€²-FAM-TCCAGGCCGGAGTCAGAGACTCCA-TAMRA-3â€² (SEQ ID NO: 751); (HSL): forward 5â€²-ACCTGCGCACAATGACACA-3â€² (SEQ ID NO: 752), reverse 5â€²-TGGCTCGAGAAGAAGGCTATG-3â€² (SEQ ID NO: 753), probe 5â€²-FAM-CCTCCGCCAGAGTCACCAGCG-TAMRA-3â€² (SEQ ID NO: 754); (PPAR-Î³): forward 5â€²-AAATATCAGTGTGAATTACAGCAAACC-3â€² (SEQ ID NO: 755), reverse 5â€²-GGAATCGCTTTCTGGGTCAA-3â€² (SEQ ID NO: 756), probe 5â€²-FAM-TGCTGTTATGGGTGAAACTCTGGGAGATTCT-TAMRA-3â€² (SEQ ID NO: 757). The amount of total RNA in each sample is determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.), and expression levels of the adipocyte differentiation hallmark genes were normalized to total RNA. Leptin protein and triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed relative to control levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). Results of two experiments are shown in Tables 15 and 16.

TABLE 15Effects of oligomeric compounds targeting miRNAs onexpression of adipocyte differentiation markersISISSEQPPARNumberID NOTGAP2HSLGlut4gamma3278762940.470.750.470.360.573278782960.650.850.930.690.973278802980.520.970.801.110.533278883060.981.181.381.371.363278893070.470.690.590.550.713278903080.920.910.861.101.183278923100.420.310.250.070.323279013190.540.420.330.190.303279033211.201.151.231.721.193279053230.691.141.110.840.543279133310.590.990.920.840.723279193370.580.790.570.320.523279223401.090.990.951.751.373279253430.720.770.781.990.603279333511.481.461.352.521.523279343520.991.201.021.220.973279393570.921.081.210.870.833279413591.311.781.732.070.803279543720.580.951.030.920.73

TABLE 16Effects of oligomeric compounds targeting miRNAs onexpression of adipocyte differentiation markersISISSEQPPARNumberID NOTGLeptinAP2HSLGlut4gamma3278883060.441.380.470.500.170.663278893070.461.050.570.540.460.823278903080.611.360.690.670.670.943278933110.951.140.970.851.471.033279013190.531.020.470.470.290.723279033210.581.610.920.801.120.983279053230.581.620.680.690.400.833279193370.401.440.480.370.180.573279223400.431.250.750.720.430.803279253430.631.400.770.750.610.833279263441.061.470.850.821.100.933279303480.970.950.860.891.010.983279313491.111.121.000.991.371.563279343520.621.250.660.640.440.723279383561.051.350.860.850.800.903279393570.592.670.690.630.300.703279413590.420.540.880.810.440.863279423600.852.030.820.790.660.873279553730.811.220.740.820.450.923279673850.901.220.860.970.560.89

From these data, values above 1.0 for triglyceride accumulation (column â€œTGâ€ in the tables) indicate that the compound has the ability to stimulate triglyceride accumulation, whereas values at or below 1.0 indicate that the compound inhibits triglyceride accumulation. With respect to leptin secretion (column â€œLeptinâ€ in the tables), values above 1.0 indicate that the compound has the ability to stimulate secretion of the leptin hormone, and values at or below 1.0 indicate that the compound has the ability to inhibit secretion of leptin. With respect to the four adipocyte differentiation hallmark genes (columns â€œAP2,â€ â€œHSL,â€ â€œGlut4,â€ and â€œPPAR gammaâ€ in the tables), values above 1.0 indicate induction of cell differentiation, whereas values at or below 1.0 indicate that the compound inhibits differentiation.

Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b; ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131-1, mir-131-2 and mir-131-3 (also known as mir-9); ISIS Number 327942 (SEQ ID NO: 360) targeted to mir-141 and ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143 were shown to significantly reduce the expression levels of 5 of 6 markers of adipocyte differentiation (excepting leptin levels), indicating that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as pharmaceutical agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.

The compound ISIS Number 327939 (SEQ ID NO: 357), targeted to mir-125b-1, for example, produced surprising results in that it demonstrates a significant increase in leptin secretion but a concomitant decrease in triglyeride accumulation and a decrease in the expression of all four adipocyte differentiation hallmark genes, indicating that this oligomeric compound may be useful as a pharmaceutic agent in the treatment of obesity, as well as having applications in other metabolic diseases.

The oligomeric compound ISIS Number 327931 (SEQ ID NO: 349), targeted to let-7c is an example of a compound which demonstrates an increase in four out of six markers of adipocyte differentiation, including a significant increase in the expression of PPAR-Î³. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of cell differentiation is desirable.

The oligomeric compound ISIS Number 327933 (SEQ ID NO: 351), targeted to mir-145 is an example of a compound which demonstrates an increase in all six markers of adipocyte differentiation. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue (such as from pluripotent stem cells) would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

In some embodiments, differentiating adipocytes were treated with uniform 2â€²-MOE phosphorothioate oligomeric compounds according to the methods described above, and the expression of the four hallmark genes, HSL, aP2, Glut4, and PPARÎ³, as well as triglyceride (TG) accumulation were measured. TG levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of control levels (control=treatment with ISIS 342673; AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), a uniform 2â€²-MOE phosphorothioate oligomeric compound containing 15 mismatches with respect to the mature mir-143 miRNA). Undifferentiated adipocytes were also compared as a negative control. As a positive control, differentiating adipocytes were treated with ISIS 105990 (AGCAAAAGATCAATCCGTTA; herein incorporated as SEQ ID NO: 759), a 5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA, previously demonstrated to inhibit adipocyte differentiation. The effects of TNF-Î±, also known to inhibit adipocyte differentiation, were also measured. Results of these experiments are shown in Tables 17 and 18.

TABLE 17Effects of oligomeric compounds targeting miRNAs onexpression of adipocyte differentiation markersISISSEQPPARNumberID NOTGAP2HSLGlut4gammaUntreatedN/A88.587.888.6102.794.9control10599075928.251.649.259.551.8342673758100.0100.0100.0100.0100.0TNF-alphaN/A10.05.50.70.518.8Undiff.N/A2.70.00.30.19.2adipocytes32811641882.187.775.875.278.432811741955.065.461.768.164.132811842069.392.785.376.680.232811942190.299.998.595.282.732812042282.781.077.794.870.5328121423134.8127.0126.0140.8103.632812242478.979.372.785.977.8328123425120.8106.785.4162.474.732812442699.1101.8103.6122.790.432812542781.786.975.899.576.132812642898.990.983.2100.775.032812742974.586.989.780.877.632812843098.7100.794.1101.984.032812943153.867.656.560.071.8328130432122.486.676.583.899.432813143389.195.481.8103.688.2328132434114.190.273.772.190.032813343561.269.563.091.963.832813443685.780.174.788.378.432813543763.680.676.790.370.032813643847.073.065.066.772.732813743983.299.686.388.585.7328138440100.685.389.886.883.832813944189.198.392.6106.3115.0

TABLE 18Effects of oligomeric compounds targeting miRNAs onexpression of adipocyte differentiation markersSEQPPARISIS #ID NOTGAP2HSLGlut4gammaUntreatedN/A102.290.894.9117.8103.3control10599075932.849.852.068.160.1342673758100100100100100TNF-alphaN/A14.59.63.11.927.9Undiff.N/A2.80.01.40.310.7adipocytes327912330107.490.190.689.076.932796938746.059.866.460.669.232809940193.985.988.486.881.932810040271.561.972.074.266.7328101403108.683.291.884.779.332810240495.987.997.079.293.7328103405110.283.282.594.374.3328104406122.6102.298.2119.190.432810540793.188.294.294.293.332810640890.588.894.9105.790.732810740966.767.561.072.579.332810841089.683.790.194.984.032810941184.984.986.9106.696.132811041297.793.391.0104.791.2328111413101.971.569.559.674.932811241498.199.1101.2122.5102.432811341580.884.590.699.993.8328114416117.394.493.3114.989.3328115417108.780.089.0132.095.834180376085.977.375.586.871.234180476160.970.871.673.674.134180576278.181.981.888.280.434180676383.275.873.469.472.6341807764114.174.896.8119.586.2

Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 328117 (SEQ ID NO: 419), targeted to hypothetical miRNA-144, ISIS Number 328129 (SEQ ID NO: 431), targeted to hypothetical miRNA-173, ISIS Number 328136 (SEQ ID NO: 438), targeted to hypothetical miRNA-181, and ISIS Number 327969 (SEQ ID NO: 387), targeted to mir-182, were each shown to reduce the expression levels of triglycerides by at least 50%, and treatment with ISIS 328117, 328129, or 328136 also each resulted in a reduction of expression of the other four hallmark genes, indicating that these oligomeric compounds targeted to hypothetical miRNA-144, hypothetical miRNA-173, hypothetical miRNA-181, and mir-182, may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases.

The oligomeric compound ISIS Number 328121 (SEQ ID NO: 423), targeted to hypothetical miRNA-161 is an example of a compound which stimulates an increase in all five markers of adipocyte differentiation. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

Example 14Expression of Mir-143 in Human Tissues and Cell Lines

Total RNA from spleen, kidney, testicle, heart and liver tissues as well as total RNA from human promyelocytic leukemia HL-60 cells, human embryonic kidney 293 (HEK293) cells, and T47D human breast carcinoma cells was purchased from Ambion, Inc. (Austin, Tex.). RNA from preadipocytes and differentiated adipocytes was purchased from Zen-Bio, Inc. (Research Triangle Park, N.C.). RNA was prepared from the HeLa, NT2, T-24, and A549 cell lines cultured as described above, using the following protocol: cell monolayers were washed twice with cold PBS, and cells were lysed in 1 mL TRIZOLâ„¢ (Invitrogen) and total RNA prepared using the manufacturer's recommended protocols.

Fifteen to twenty micrograms of total RNA was fractionated by electrophoresis through 10% acrylamide urea gels using a TBE buffer system (Invitrogen). RNA was transferred from the gel to HYBONDâ„¢-N+nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by electroblotting in an Xcell SureLockâ„¢ Minicell (Invitrogen). Membranes were fixed by UV cross-linking using a STRATALINKERâ„¢ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hyb buffer solution (Amersham) using manufacturer's recommendations for oligonucleotide probes.

To detect mir-143, a target specific DNA oligonucleotide probe with the sequence TGAGCTACAGTGCTTCATCTCA (SEQ ID NO: 319) was synthesized by IDT (Coralville, Iowa). The oligo probe was 5â€² end-labeled with T4 polynucleotide kinase with (Î³-³²P) ATP (Promega). To normalize for variations in loading and transfer efficiency membranes can be stripped and probed for U6 RNA. Hybridized membranes were visualized and quantitated using a Storm 860 PHOSPHORIMAGERâ„¢ and IMAGEQUANTâ„¢ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.).

Using this probe, the mir-143 miRNA was found to be most highly expressed in human heart, thymus and kidney, and was also expressed to a lesser extent in lung, spleen, liver, and brain tissues. For example, as compared to expression levels in liver, mir-143 was expressed approximately 24-fold higher in heart, 17-fold higher in thymus, and 8-fold higher in kidney.

The mir-143 miRNA was also found to be expressed in adipocytes and preadipocytes, and levels of mir-143 were found to be dramatically upregulated in differentiated adipocytes as compared to preadipocytes, indicating that this miRNA may be important in adipocyte differentiation. These data, taken together with the finding that the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143, was shown to inhibit the adipocyte differentiation markers (described above, Example 13), supports the conclusion that mir-143 is involved in cellular differentiation pathways.

Example 15Effects of Oligomeric Compounds Targeting miRNAs on Apoptosis in the Caspase Assay in Preadipocytes

The effect of oligomeric compounds of the present invention targeting miRNAs was examined in preadipocytes (Zen-Bio, Inc., Research Triangle Park, N.C.) using the fluorometric caspase assay previously described in Example 11. The oligonucleotide random-mer, ISIS-29848 (SEQ ID NO: 737) was used as a negative control, and ISIS-148715 (SEQ ID NO: 738), targeting the human Jagged2 mRNA, known to induce apoptosis when inhibited, was used as a positive control. The measurement obtained from the untreated control cells is designated as 100% activity and was set equal to 1.0. Results are shown in Table 19.

TABLE 19Effects of targeting miRNAs on apoptosis in preadipocytesSEQ IDISIS NumberNO.Pri-miRNAFold Increase over UTCUTCN/AN/A1.0Untreated controlISIS-29848737N/A1.2n-merISIS-148715738Jagged236.9Positive control327888306mir-108-11.1327889307mir-23b1.1327890308let-7i1.3327893311let-7b1.3327901319mir-1432.0327903321let-7a-31.6327905323mir-2051.5327919337mir-2211.3327922340mir-19b-21.0327925343mir-133b2.0327926344let-7d1.8327930348let-7e1.4327931349let-7c1.5327934352mir-2132.0327938356mir-981.0327939357mir-125b-12.2327941359mir-181b1.3327942360mir-1411.0327955373mir-130b4.3327967385let-7g1.5

From these data, it is evident that the oligomeric compounds of the present invention generally do not induce the activity of caspases involved in apoptotic pathways in preadipocytes. In particular, the oligomeric compound targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319), does not result in a significant increase in caspase activity as compared to the Jagged2 positive control. Taken together with the results from the adipocyte differentiation assay (Example 13) and the expression analysis of mir-143 (Example 14), these data suggest that the mir-143 miRNA plays a role in stimulating cellular differentiation, employing pathways other than the caspase cascades activated during apoptosis.

It was recently reported that bone marrow cells may contribute to the pathogenesis of vascular diseases, and that cell differentiation appears to be important in models of postangioplasty restenosis, graft vasculopathy, and hyperlipidemia-induced atherosclerosis. Bone marrow cells have the potential to give rise to vascular progenitor cells that home in on damaged vessels and differentiate into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation (Sata, M. Trends Cardiovasc Med. 2003 13(6):249-53). Thus, the ability to modulate cell differentiation may provide the basis for the development of new therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation, and proliferation of circulating vascular progenitor cells.

Example 16Comparison of Effects of Oligomeric Compounds Targeting the Mir-143 Pri-miRNA or Mature Mir-143 miRNA on Adipocyte Differentiation

Two oligomeric compounds targeting the mature mir-143 miRNA and two oligomeric compounds targeting the 110-nucleotide mir-143 pri-miRNA were compared for their effects on adipocyte differentiation using the same adipocyte differentiation assay as described in Example 13.

The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), 22-nucleotides in length, targets the mature mir-143 miRNA and is composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides and phosphorothioate (Pâ• S) internucleoside (backbone) linkages throughout. The oligomeric compound ISIS Number 338664 (CAGACTCCCAACTGACCAGA; SEQ ID NO: 491) is also a uniform 2â€²-MOE oligonucleotide, which is designed to target the mir-143 pri-miRNA. Another oligomeric compound targeting the mir-143 pri-miRNA, ISIS Number 328382 (SEQ ID NO: 491) is a chimeric oligonucleotide, 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwingsâ€ having 2â€²-MOE substituents in the wing nucleosides (a â€œ5-10-5 gapmerâ€ ), and ISIS Number 340927 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319) is a 5-10-7 gapmer designed to target mature mir-143. The internucleoside (backbone) linkages are phosphorothioate (P=5) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The effect of these oligomeric compounds targeting the mir-143 miRNA and the mir-143 pri-miRNA on expression of the 5 hallmark genes indicating cellular differentiation was examined in preadipocytes using the same methods described in Example 13. Results are shown in Table 20.

TABLE 20Comparison of uniform 2â€²-MOE and chimeric oligomericcompounds targeting the mir-143 miRNA and pri-miRNAson expression of adipocyte differentiation markersISISSEQPPARNumberID NOTGAP2HSLGlut4gamma3279013190.540.420.330.190.303283824910.720.890.750.850.963386644911.421.010.761.810.863409273190.650.770.730.540.36

From these data, it was observed that while the gapmer oligomeric compound targeting the mature mir-143 (ISIS Number 340972) results in reduced expression of the adipocyte differentiation markers, the uniform 2â€²-MOE oligomeric compound targeting mature mir-143 (ISIS Number 327901) was more effective. For the oligomeric compounds targeting the mir-143 pri-miRNA, the gapmer compound (ISIS Number 328382) appeared to be more effective in blocking adipocyte differentiation than was the uniform 2â€²-MOE oligomeric compound (ISIS Number 338664).

Dose Responsiveness:

In one embodiment, the oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143 was selected for additional dose response studies in the adipocyte differentiation assay. Differentiating adipocytes (at day 10 post-induction of differentiation) were treated with 50, 100, 200, and 300 nM ISIS 327901, or the scrambled control ISIS Number 342673 (SEQ ID NO: 758) containing 15 mismatches with respect to the mature mir-143 miRNA. ISIS Numbers 327901 and 342673 are uniform 2â€²-MOE phosphorothioate oligomeric compounds 22 nucleotides in length. Differentiating adipocytes treated with ISIS Number 29848 (SEQ ID NO: 737) served as the negative control to which the data were normalized. Differentiating adipocytes treated with ISIS 105990 (SEQ ID NO: 759), a 5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA which has been demonstrated previously to inhibit adipocyte differentiation, served as the positive control. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes (PPAR-gamma, aP2, HSL, and GLUT4) were measured 24 hours after treatment as described above. Untreated cells were compared to cells treated with oligomeric compounds, and results of these dose response studies are shown in Table 21, where levels of the markers is expressed as a percentage of untreated control (% UTC) levels. Where present, â€œN.D.â€ indicates â€œno data.â€

TABLE 21Effects of oligomeric compounds targeting mir-143on expression of adipocyte differentiation markers% UTCHallmarkDose of oligomeric compoundMeasured:Isis #:50 nM100 nM200 nM300 nMTriglycer-34267394.2105.3â€‚98.3108.2idesnegative control105990N.D.N.D.N.D.16.6positive control32790185.368.934.023.0PPAR-gamma34267377.589.994.685.8mRNAnegative control105990N.D.N.D.N.D.43.9positive control32790174.670.851.839.3AP2 mRNA34267382.490.381.170.9negative control105990N.D.N.D.N.D.17.9positive control32790178.364.639.022.4HSL mRNA34267392.095.697.385.2negative control105990N.D.N.D.N.D.7.4positive control32790189.573.540.211.9GLUT4 mRNA34267394.990.797.6102.7negative control105990N.D.N.D.N.D.11.8positive control32790174.249.732.817.4

From these data, it was observed that treatment of differentiating adipocytes with the uniform 2â€²-MOE oligomeric compound, ISIS Number 327901 targeting mir-143 results in a dose responsive reduction of expression of all five markers of differentiation. Thus, this oligomeric compound may be useful in the treatment of diseases associated with increased expression of these hallmark genes, such as obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.

Example 17Human Let 7 Homologs

Let-7 is one of the two miRNAs originally identified in C. elegans as an antisense translational repressor of messenger RNAs encoding key developmental timing regulators in nematode larva. Several genes predicted to encode let-7-like miRNAs have been identified in a wide variety of species, and these let-7-like homologs are believed to control temporal transitions during development across animal phylogeny. Oligomeric compounds of the present invention were designed to target several human let-7-like genes. Additionally, a series of target-specific DNA oligonucleotide probes were synthesized by IDT (Coralville, Iowa) and used in Northern analyses to assess the expression of let-7-like miRNA homologs in various tissues. These let-7 homolog specific probes are shown in Table 22.

TABLE 22Probes for Northern analyses ofmRNA expression of let-7 homologsSEQISIS NumberID NOSequencepri-miRNA327890308AGCACAAACTACTACCTCAlet-7i 327893311AACCACACAACCTACTACCTCAlet-7b 327903321AACTATACAACCTACTACCTCAlet-7a-3 327926344ACTATGCAACCTACTACCTCTlet-7d 327930348ACTATACAACCTCCTACCTCAlet-7e 327931349AACCATACAACCTACTACCTCAlet-7c 327967385ACTGTACAAACTACTACCTCAlet-7g

For Northern analyses with let-7 homolog probes, total RNA from spleen, kidney, testes, heart, and liver tissues as well as total RNA from HEK293, T47D, T-24, MCF7, HepG2, and K-562 Leukemia cell lines was either prepared as described above or purchased from Ambion, Inc. (Austin, Tex.). Northern blotting was performed as described above (Example 14). The let-7c miRNA was observed to be expressed in spleen, kidney, testes, heart and liver tissues, as well as in HEK293 and T47D cell lines. The let-7e miRNA was observed to be expressed in T-24, MCF7, T47D, 293T, HepG2, and K-562 cell lines.

In one embodiment, expression of let-7-like pri-miRNA homologs was detected in total RNA from brain, liver and spleen tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HeLa, HEK-293, and T-24 cell lines by real-time RT-PCR. Primer/probe sets were designed to distinguish between and amplify specific let-7-like pri-miRNA homologs. These primer/probe sets are shown in Table 23.

TABLE 23Primer/probe sets for assaying expression of let-7 miRNA homologsPrimer orIsisSEQ IDPri-miRNAprobenumberNO.sequencelet-7bforward341672765GAGGTAGTAGGTTGTGTGGTTTCAreverse341673766AGGGAAGGCAGTAGGTTGTATAGTTprobe341674767CAGTGATGTTGCCCCTCGGAAGA let-7cforward341675768TGCATCCGGGTTGAGGTAreverse341676769AGGAAAGCTAGAAGGTTGTACAGTTAAprobe341677770AGGTTGTATGGTTTAGAGTTACACCCTGGGA let-7dforward341678771CCTAGGAAGAGGTAGTAGGTTGCAreverse341679772CAGCAGGTCGTATAGTTACCTCCTTprobe341680773AGTTTTAGGGCAGGGATTTTGCCCA let-7gforward341681774TTCCAGGCTGAGGTAGTAGTTTGreverse341682775TTATCTCCTGTACCGGGTGGTprobe341683776ACAGTTTGAGGGTCTAT let-7iforward341684777TGAGGTAGTAGTTTGTGCTGTTGGTreverse341685778AGGCAGTAGCTTGCGCAGTTAprobe341686779TTGTGACATTGCCCGCTGTGGAG let-7a-1forward341687780GGATGAGGTAGTAGGTTGTATAGTTTTAGGreverse341688781CGTTAGGAAAGACAGTAGATTGTATAGTTATCprobe341689782TCACACCCACCACTGG let-7a-3forward341690783GGGTGAGGTAGTAGGTTGTATAGTTTGGreverse341691784CACTTCAGGAAAGACAGTAGATTGTATAGTTprobe341692785CTCTGCCCTGCTATGG

Using these primer/probe sets, the let-7-like pri-miRNA homologs were found to be expressed in human brain, liver and spleen, as well as preadipocytes, differentiated adipocytes, and HeLa, T-24 and HEK-293 cells lines. In particular, the let-7b pri-miRNA exhibited approximately 100-fold higher expression in differentiated adipocytes as compared to preadipocytes. Furthermore, the let-7b, let-7c, let-7d, let-71, and let-7a-3 pri-miRNAs were highly expressed in brain and spleen tissues.

In summary, the let-7-like homologs have been found to be widely expressed in various human tissues and several cell lines. Furthermore, some oligomeric compounds targeted to human let-7 pri-miRNAs generally appeared to result in the induction of cell differentiation, consistent with the functional role of let-7 as a regulator of developmental timing in nematode larva. Specifically, the oligomeric compounds targeted to let-7c (ISIS Number 327931; SEQ ID NO: 349) and let-7a-3 (ISIS Number 327903; SEQ ID NO: 321) resulted in an increase in expression levels for several markers of adipocyte differentiation. Furthermore, inhibition of the let-7-like homologs by oligomeric compounds of the present invention did not appear to induce caspases activated in apoptotic pathways (performed in Example 15). Thus, the oligomeric compounds of the present invention targeting let-7-like pri-miRNA homologs appear to stimulate adipocyte differentiation and do not promote cell death by apoptosis. Thus, the oligomeric compounds of the present invention may be useful as pharmaceutical agents in the treatment of anorexia or diseases, conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which neural regeneration would be beneficial.

Example 18Effects of Oligomeric Compounds Targeting miRNAs on Insulin Signaling in HepG2 Cells

Insulin is secreted from pancreatic Î²-cells in response to increasing blood glucose levels. Through the regulation of protein expression, localization and activity, insulin ultimately stimulates conversion of excess glucose to glycogen, and results in the restoration of blood glucose levels. Insulin is known to regulate the expression of over 100 gene products in multiple cell types. For example, insulin completely inhibits the expression of hepatic insulin-like growth factor binding protein-1 (IGFBP-1), a protein which can sequester insulin-like growth factors, and phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-c) which is a rate-controlling enzyme of hepatic gluconeogenesis. Levels of the follistatin mRNA are also believed to decrease in response to insulin treatment. IGFBP-1 and PEPCK-c are overexpressed in diabetes, and PEPCK-c overexpression in animals promotes hyperglycemia, impaired glucose tolerance and insulin-resistance. Thus, the IGFBP-1, PEPCK-c and follistatin genes serve as marker genes for which mRNA expression can be monitored and used as an indicator of an insulin-resistant state. Oligomeric compounds with the ability to reduce expression of IGFBP-1, PEPCK-c and follistatin are highly desirable as agents potentially useful in the treatment of diabetes and hypertension.

Oligomeric compounds of the present invention were tested for their effects on insulin signaling in HepG2 cells. HepG2 cells were plated at 7500 cells/well in collagen coated 96-well plates. The following day, cells were transfected with oligomeric compounds targeting miRNAs using 100 nM oligomeric compound in LIPOFECTINâ„¢ (Invitrogen Corporation, Carlsbad, Calif.) in two 96-well plates. The oligomeric compounds were tested in triplicate on each 96-well plate, except for positive and negative controls, which were measured up to six times per plate. At the end of transfection, the transfection medium was replaced by regular growth medium. Twenty-eight hours post-transfection, the cells were subjected to overnight (sixteen to eighteen hours) serum starvation using serum free growth medium. Forty-four hours post-transfection, the cells in the transfected wells were treated with either no insulin (â€œbasalâ€ Experiment 1, for identification of insulin-mimetic compounds) or with 1 nM insulin (â€œinsulin treatedâ€ Experiment 2, for identification of insulin sensitizers) for four hours. At the same time, in both plates, cells in some of the un-transfected control wells are treated with 100 nM insulin to determine maximal insulin response. At the end of the insulin or no-insulin treatment (forty-eight hours post-transfection), total RNA is isolated from both the basal and insulin treated (1 nM) 96-well plates, and the amount of total RNA from each sample is determined using a Ribogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR is performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and valuesÂ±standard deviation are expressed relative to the transfectant only untreated control (UTC) and negative control compounds. Results of these experiments are shown in Tables 24 and 25.

TABLE 24Experiment 1: Effects of oligomeric compounds targeting miRNAson insulin-repressed gene expression in HepG2 cellsSEQFolli-ISISIDIGFBP-1PEPCK-cstatinNumberNOPri-miRNA(% UTC)(% UTC)(% UTC)UTCN/AN/A10010010029848 n-mer737N/A958794327876294mir-29b-193119104327878296mir-20316245124327880298mir-10b137110107327889307mir-23b5613756327890308let-7I998578327892310mir-131-2/1087591mir-9327901319mir-14313311993327903321let-7a-3717160327905323mir-205107129104327913331mir-29c123229115327919337mir-221967174327922340mir-19b-21097757327925343mir-133b152145110327933351mir-145125118112327934352mir-21323199140327939357mir-125b-1125125104327941359mir-181b8310180327954372mir-148b11879100338664491mir-143907593pri-miRNA340927319mir-14320187111

Under â€œbasalâ€ conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 327878 targeting mir-203 and ISIS Number 327922 targeting mir-19b-2, for example, result in a 55% and a 23% decrease, respectively, in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, these oligomeric compounds may be useful as pharmaceutic agents comprising insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Conversely, the results observed with the oligomeric compound targeting mir-29c (ISIS Number 327913), for example, exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, suggest that the mir-29c miRNA target may be involved in the regulation of these insulin-responsive genes. When the mir-29c miRNA is inactivated by an oligomeric compound, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed.

TABLE 25Experiment 2: Effects of oligomeric compounds targeting miRNAson insulin-sensitization of gene expression in HepG2 cellsSEQFolli-ISISIDIGFBP-1PEPCK-cstatinNumberNOPri-miRNA(% UTC)(% UTC)(% UTC)UTC(1 nmN/AN/A100100100insulin)29848 n-mer737N/A929497327876294mir-29b-1118176138327878296mir-20318529150327880298mir-10b136125149327890307let-7i88113115327892308mir-131-2/13910496mir-9327901310mir-143135117135327903319let-7a-3818789327905321mir-205115147148327913323mir-29c147268123327919331mir-221154105178327922337mir-19b-21047661327925340mir-133b166182148327933343mir-145179115185327934351mir-213244105103327939352mir-125b-1175153192327941357mir-181b809868327954359mir-148b120102105327889372mir-23b7320272338664491mir-1431007684pri-miRNA340927319mir-143285103128

For HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 327878 targeting mir-203 and ISIS Number 327922 targeting mir-19b-2, for example, were observed to result in a 71% and a 24% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Thus, these oligomeric compounds may be useful as pharmaceutic agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Conversely, the results observed with the oligomeric compounds targeting mir-29c (ISIS Number 327913), mir-133b (ISIS Number 327925), and mir-125b-1 (ISIS Number 327939), all exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, support the conclusion that the mir-29c, mir-133b, and mir-125b-1 miRNAs may be involved in the regulation of insulin-responsive genes. When these miRNAs are inactivated by the oligomeric compounds of the present invention, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed or insulin-sensitive.

A caspase assay was also performed (as in Example 11 above) in HepG2 cells treated with oligomeric compounds of the present invention, and it was determined that oligomeric compounds targeting the mir-29c, mir-133b, and mir-125b-1 miRNAs were not toxic to the cells and that the observed reduction in mRNA expression levels of insulin-responsive genes was not due to a general toxicity of the compounds or an induction of apoptotic pathways.

Example 19Analysis of Expression of Mir-143 Pri-miRNA and Mature Mir-143Ribonuclease Protection Assays:

The ribonuclease protection assay (RPA) is known in the art to be a sensitive and accurate method of measuring and/or following temporal changes in the expression of one or more RNA transcripts in a complex mixture of total RNA. Briefly, this method employs a radioactive probe that specifically hybridizes to a target transcript RNA. The probe is added to a sample of total RNA isolated from tissues or cells of interest, and, upon hybridization to its target, the probe forms a double-stranded RNA region. If the region of hybridization is shorter than the entire length of either the probe or the target RNA molecule, the molecule will be a hybrid molecule with partial double-stranded and partial single-stranded character. These hybrid molecules are then digested with single-strand-specific RNases such as RNase A and/or T1, which remove any non-hybridized single stranded portions of the hybrid molecules, leaving only the â€œprotectedâ€ dsRNA fragments. The RNase protected fragments are then electrophoresed on a denaturing gel, causing the strands to dissociate, and the intensity of radioactive probe signal observed is directly proportional to the amount of specific target transcript RNA in the original total RNA sample.

In an embodiment of the present invention, small non-coding RNAs in a sample were detected by RPA using probes that hybridize to pri-miRNAs, pre-miRNAs or mature miRNAs. Probes were in vitro transcribed using the mirVanaâ„¢ miRNA Probe Construction Kit (Ambion Inc., Austin, Tex.) according to the manufacturer's protocol, beginning with a DNA oligonucleotide representing sense strand of the mature miRNA to be detected plus four thymidylate residues plus an 8-base sequence complementary to the 3â€²-end of the T7 promoter primer supplied with the kit. When the T7 primer is annealed to this DNA oligonucleotide, the Klenow DNA polymerase is used to generate a double-stranded DNA, and then in vitro transcription is performed using the T7 RNA polymerase and radiolabeled nucleotides to generate a radioactive RNA probe for detection of the miRNA.

In one embodiment, a probe specifically hybridizing to the murine mir-143 miRNA was used in a RPA of 5 Î¼g total RNA from kidney, liver, heart, lung, brain, spleen, and thymus tissues from mouse as well as adipose tissue from db/db obese mice, total RNA from an 1â€²-day-old embryo, and total RNA from undifferentiated and differentiated 3T3-L1 cells. All signals were normalized to the levels of 5.8S rRNA. Expression levels of mir-143 were highest in lung, heart, spleen, thymus and kidney tissues from wildtype mice. Notably, mir-143 expression levels were significantly elevated in adipose tissue from db/db mice (approximately 4 times higher than expression levels in kidney, 2.4 times higher than levels in heart and 1.6 times higher than levels in lung tissues from wildtype mice).

In one embodiment, a probe hybridizing to the mir-143 pri-miRNA molecule was used in a RPA of 2-5 Î¼g total RNA from human spleen, thymus, testes, heart, liver, kidney, skeletal muscle, brain, lung and adipose tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HepG2 cells. A probe hybridizing to the Î²-actin mRNA was used as a control. The highest levels of mir-143 pri-miRNA were observed in heart, kidney, thymus and adipose tissues, as well as in differentiated adipocytes.

In one embodiment, a probe hybridizing to the mature mir-143 miRNA was also used in a RPA of 2 Î¼g total RNA from human spleen, thymus, heart, liver, kidney and brain, tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and total RNA from HepG2, A549, T-24, HEK293, HuVEC (human umblical vein endothelial cells), HL-60 and T47D cell lines. A probe hybridizing to the Î²-actin mRNA was used as a control, and all signals were normalized to the levels of mir-143 expression in preadipocytes. The results are shown in Table 26.

TABLE 26RNase protection of mature mir-143 intotal RNA from tissues and cell linesFold Increase overTissue or cell linepreadipocytesSpleen2.6Thymus3.8Heart8.2Liver0Kidney10.0Brain0.9Preadipocytes1.0Differentiated2.6adipocytesHepG20.5A549N.D.T-240.4HEK2930.5HuVEC0.3HL-600.4T47D0.3

From these data, the highest levels of expression of the mature mir-143 miRNA were observed in total RNA from kidney and heart tissues. High levels of expression of the mature mir-143 miRNA were also observed in total RNA from lymphoid tissues such as spleen and thymus. Expression of the mature mir-143 miRNA is increased in differentiated adipocytes as compared to levels in preadipocytes. These data also suggest that the mir-143 miRNA plays a role in cellular differentiation.

In one embodiment, a uniform 2â€²-MOE phosphorothioate oligomeric compound with a sequence antisense to the mature mir-143 miRNA was spiked into the RPA mixture above. This antisense mir-143 compound was found to block the ribonuclease protection expression pattern previously observed, suggesting that this antisense mir-143 oligomeric compound specifically hybridizes to and inhibits the activity of mir-143. This oligomeric compound targeting the mir-143 miRNA is predicted to form a double stranded molecule that blocks endogenous mir-143 miRNA activity when employed in vivo.

It was also noted that, while expression of the mir-143 miRNA can be detected in non-transformed cells, such as HuVECs, in general, transformed cell lines have not been observed to exhibit high levels expression of mir-143. When taken together with the observation that the mir-143 miRNA is upregulated as adipocytes differentiate as well as the observation that oligomeric compounds targeting mir-143 inhibit adipocyte differentiation, these data suggest that mir-143 normally promotes adipocyte differentiation and mir-143 may have an inhibitory effect on cellular transformation that is consistent with its role in promoting cellular differentiation. Lack of expression or downregulation of mir-143 in transformed cell lines may be a cause or consequence of the undifferentiated state. Thus, mir-143 mimics may be useful as pharmaceutical agents in the treatment of hyperproliferative disorders such as cancer.

In one embodiment, the expression of human mir-143 was assessed during adipocyte differentiation. A probe hybridizing to the human mir-143 miRNA was used in a RPA of 5 Î¼g total RNA from pre-adipocytes, and differentiated adipocytes sampled at one, four, and ten days post-differentiation. All signals were normalized to the levels of 5.8S rRNA. mir-143 expression levels were 2.5 to 3-fold higher by day 10 post-differentiation when compared to mir-143 expression levels in pre-adipocytes by ribonuclease protection assay.

Real-Time RT-PCR Analysis of Mir-143 Pri-miRNA Expression:

Expression levels of mir-143 pri-miRNA were compared in total RNAs from various tissues and total RNA from several cell lines. Total RNA from spleen, heart, liver, and brain tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HepG2, T-24 and HeLa cell lines was purchased or prepared as described supra. 80 ng of total RNA from each source was used to perform real-time RT-PCR using a primer/probe set specific for the mir-143 pri-miRNA molecule. ISIS 339314 (TCCCAGCCTGAGGTGCA; SEQ ID NO: 786) was used as the forward primer, ISIS 342897 (GCTTCATCTCAGACTCCCAACTG; SEQ ID NO: 787) was used as the reverse primer, and ISIS 342898 (TGCTGCATCTCTG; SEQ ID NO: 788) was used as the probe. RNA levels from all sources were compared to RNA levels from preadipocytes. Greater than 32-fold higher levels of mir-143 pri-miRNA were observed in heart tissue as compared to preadipocytes; 19-fold higher levels of mir-143 pri-miRNA were observed in differentiated adipocytes relative to levels in preadipocytes; 5-fold higher levels of mir-143 pri-miRNA were observed in spleen as compared to preadipocytes.

Northern blot analyses were performed in differentiating adipocytes as described in Example 14 using the mir-143-specific DNA oligonucleotide probe (SEQ ID NO: 319) to detect the mir-143 target and a probe for the U6 RNA to normalize for variations in loading and transfer efficiency, and it was confirmed by Northern analysis that expression of mature mir-143 increases from day 1 through day 10 after induction of differentiation.

In human pre-adipocytes and adipocytes sampled one, four, seven and ten days post-differentiation, expression levels of mir-143 pri-miRNA were also assessed using real-time RT-PCR analysis as described herein. 80 ng of total RNA from pre-adipocytes or differentiated adipocytes was used to perform real-time RT-PCR using the same primer/probe set specific for the mir-143 pri-miRNA molecule described supra (ISIS 339314, SEQ ID NO: 786 was used as the forward primer, ISIS 342897, SEQ ID NO: 787 was used as the reverse primer, and ISIS 342898, SEQ ID NO: 788 was used as the probe). RNA levels from all sources were normalized to 5.8S rRNA levels. mir-143 pri-miRNA levels in preadipocytes were 94% of the level of the 5.8S rRNA. At day 1 post-differentiation, mir-143 pri-miRNA levels had decreased to 38% of the level of the 5.8S rRNA. By day 4 post-differentiation, mir-143 pri-miRNA levels had decreased to 26%, by day 7 post-differentiation, mir-143 pri-miRNA levels were at 25%, and by day 10 post-differentiation, mir-143 pri-miRNA levels had dropped to 23% of the level of the 5.8S rRNA. Taken together with the results from RPA analysis, it appears that levels of the mature mir-143 miRNA increases approximately 2- to 3-fold by day 10 post-differentiation in differentiated adipocytes, accompanied by a concomitant approximately 4-fold decrease in the levels of unprocessed mir-143 pri-miRNA, indicating that adipocyte differentiation coincides with either an increase in processing of the mir-143 miRNA from the mir-143 pri-miRNA or an overall decrease in mir-143 pri-miRNA production.

Effects of Oligomeric Compounds on Expression of Pri-miRNAs:

Mature miRNAs originate from long endogenous primary transcripts (pri-miRNAs) that are often hundreds of nucleotides in length. It is believed that a nuclear enzyme in the RNase III family, known as Drosha, processes pri-miRNAs (which can range in size from about 110 nucleotides up to about 450 nucleotides in length) into pre-miRNAs (from about 70 to 110 nucleotides in length) which are subsequently exported from the nucleus to the cytoplasm, where the pre-miRNAs are processed by human Dicer into double-stranded intermediates resembling siRNAs, which are then processed into mature miRNAs. Using the real-time RT-PCR methods described herein, the expression levels of several pri-miRNAs were compared in differentiating adipocytes. Total RNA from preadipocytes and differentiating adipocytes was prepared as described herein.

In one embodiment, modified oligomeric compounds can be transfected into preadipocytes or other undifferentiated cells, which are then induced to differentiate (as described in detail, herein), and it can be determined whether these modified oligomeric compounds act to inhibit or promote cellular differentiation. Real-time RT-PCR methods can then be used to determine whether modified oligomeric compounds targeting miRNAs can affect the expression or processing of the pre-miRNAs from the pri-miRNA (by the Drosha enzyme), the processing of the mature miRNAs from the pre-miRNA molecules (by the Dicer enzyme), or the RISC-mediated binding of a miRNA to its target nucleic acid.

Here, oligomeric compounds targeting mir-143 were transfected into preadipocytes which were then induced to differentiate, in order to assess the effects of these compounds on mir-143 pri-miRNA levels during differentiation. mir-143 pri-miRNA levels were assessed on days 3 and 9 after differentiation.

In addition to the uniform 2â€²-MOE phosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143, a 5-10-7 gapmer oligomeric compound, ISIS Number 340927 (SEQ ID NO: 319), was designed to target mature mir-143. As negative controls, â€œscrambledâ€ oligomeric compounds were also designed; ISIS Number 342672 (ATACCGCGATCAGTGCATCTTT; incorporated herein as SEQ ID NO: 789) contains 13 mismatches with respect to the mature mir-143 miRNA, and ISIS Number 342673 (SEQ ID NO: 758) contains 15 mismatches with respect to the mature mir-143 miRNA. ISIS 342672 and ISIS 342673 are uniform 2â€²-MOE phosphorothioate oligomeric compounds 22 nucleotides in length. ISIS Number 342677 (SEQ ID NO: 789) and ISIS Number 342678 (SEQ ID NO: 758) are the corresponding 5-10-7 scrambled 2â€²-MOE gapmer oligomeric compounds. All cytidine residues are 5-methylcytidines. Additionally, ISIS Number 342683 (CCTTCCCTGAAGGTTCCTCCTT; herein incorporated as SEQ ID NO: 790), representing the scrambled sequence of an unrelated PTP1B antisense oligonucleotide, was also used as a negative control.

These compounds were transfected into differentiating adipocytes and their effects on levels of the mir-143 pri-miRNA molecule were assessed in pre-adipocytes vs. differentiated adipocytes, by real-time RT-PCR using the primer/probe set specific for the mir-143 pri-miRNA (forward primer=ISIS 339314, SEQ ID NO: 786; reverse primer=ISIS 342897, SEQ ID NO.: 787; probe=ISIS 342898, SEQ ID NO.: 788). Thus, it was observed that in the presence of the oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), levels of the mir-143 pri-miRNA are enhanced approximately 4-fold in differentiated adipocytes 9 days post-differentiation as compared to 3 days post-differentiation. These results suggest that ISIS Number 327901, the uniform 2â€²-MOE Pâ• S oligomeric compound targeted to mature mir-143, interferes with the processing of the mir-143 pri-miRNA into the pre-miRNA by the Drosha RNase III enzyme. Alternatively, the compound interferes with the processing of the mir-143 pre-miRNA into the mature mir-143 miRNA by the Dicer enzyme. The decrease in levels of mature mir-143 miRNA in differentiating cells treated with ISIS Number 327901 (SEQ ID NO: 319) may also trigger a feedback mechanism that signals these cells to increase production of the mir-143 pri-miRNA molecule. Not mutually exclusive with the processing interference or the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-143 pri-miRNA or pre-miRNA molecules. Oligomeric compounds of the present invention are predicted to disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder Drosha and/or Dicer cleavage, respectively. Furthermore, oligomeric compounds which are capable of binding to the mature miRNA are also predicted to prevent the RISC-mediated binding of a miRNA to its target nucleic acid, either by cleavage or steric occlusion of the miRNA.

Example 20Identification of RNA Transcripts Bound by miRNAs

The RACE-PCR method (Rapid Amplification of cDNA Ends) was used as a means of identifying candidate RNA transcripts bound and/or potentially regulated by miRNAs. RNA was prepared and isolated from preadipocytes, and, using the SMART RACE cDNA Amplification kit (BD Biosciences, Clontech, Palo Alto, Calif.) according to manufacturer's protocol, synthetic adaptor sequences were incorporated into both the 5â€²- and 3â€²-ends of the amplified cDNAs during first strand cDNA synthesis. 5â€² RACE-PCR was then performed using the mature miRNA as the 3â€²-end primer along with the 5â€² adapter primer from the kit to amplify the 5â€²-end of the candidate RNA transcript. 3â€²RACE-PCR was performed using the antisense sequence of the miRNA as a primer along with the 3â€² adapter primer from the kit to amplify the 3â€²-end of the candidate RNA transcript. In some embodiments, the primers 2-nucleotides shorter than the corresponding miRNA were used in order to identify targets with some mismatching nucleotides at the end of the miRNA (these primers are indicated by â€œ3â€²-RACE-2 ntâ€ in Table 27 below).

For example, the antisense sequences of the mature mir-43, let-7 g, mir-23b, mir-29c, mir-131, mir-143, mir-130b and mir-213 miRNAs were used as primers in 3â€² RACE-PCR, and the mature mir-143 or mir-15a sequences were used in 5â€² RACE-PCR. The RACE-PCR products employing the mir-143 miRNA, the mir-143 antisense sequence, the mir-131 antisense sequence or the mir-15a miRNA as primers were electrophoresed and gel purified, prominent bands were excised from the gel, and these products were subcloned using standard laboratory methods. The subcloned products from the RACE-PCR were then were sent to Retrogen, Inc. (San Diego, Calif.) for sequencing. Candidate RNA transcripts targeted by miRNAs were thereby identified.

Candidate RNA targets identified by RACE-PCR methods are shown in Table 27, where the miRNA-specific primer used to identify each transcript is indicated in the column entitled â€œprimerâ€ . (In some cases, the target was identified multiple times by more than one RACE-PCR method, and thus appears in the table more than once).

TABLE 27Predicted RNA targets of mir-143SEQGenBankIDPrimerMethodAccessionRNA transcript targeted by miRNANOmir-1435â€²RACENM_001753.2caveolin 1, caveolae protein, 22 kDa791mir-1435â€²RACENM_004652.1ubiquitin specific protease 9, X-792linked (fat facets-like, Drosophila)mir-1435â€²RACENM_007126.2valosin-containing protein793mir-1435â€²RACENM_000031.1aminolevulinate, delta-, dehydratase794mir-1435â€²RACENM_007158.1NRAS-related gene795mir-1435â€²RACENM_015396.1HSPC056 protein796mir-1435â€²RACENM_001219.2calumenin797mir-1435â€²RACEBC051889.1RNA binding motif, single stranded798interacting protein 1mir-1435â€²RACEBX647603.1Homo sapiens mRNA; cDNA799DKFZp686L01105 (from cloneDKFZp686L01105)mir-1435â€²RACEAB051447.1KIAA1660 protein800mir-1435â€²RACENM_007222.1zinc-fingers and homeoboxes 1801mir-1435â€²RACENM_001855.1collagen, type XV, alpha 1802mir-1433â€²RACENM_007222.1zinc-fingers and homeoboxes 1801mir-1433â€²RACENM_006732FBJ murine osteosarcoma viral803oncogene homolog Bmir-1433â€²RACENM_003718.2cell division cycle 2-like 5804(cholinesterase-related celldivision controller)mir-1433â€²RACENM_005626.3splicing factor, arginine/serine-805rich 4mir-1433â€²RACENM_002355.1mannose-6-phosphate receptor (cation806dependent)mir-1433â€²RACENM_000100.1cystatin B (stefin B)807mir-1433â€²RACENM_015959.1CGI-31 protein808mir-1433â€²RACENM_006769.2LIM domain only 4809mir-1433â€²RACENM_003184.1TAF2 RNA polymerase II, TATA box810binding protein (TBP)-associatedfactor, 150 kDamir-1433â€²RACENM_025107.1myc target in myeloid cells 1811mir-1433â€²RACENM_003113.1nuclear antigen Sp100812mir-1433â€²RACENM_002696.1polymerase (RNA) II (DNA directed)813polypeptide Gmir-1433â€²RACENM_004156.1protein phosphatase 2 (formerly 2A),814catalytic subunit, beta isoformmir-1433â€²RACENM_031157heterogeneous nuclear815ribonucleoprotein A1mir-1433â€²RACENM_004999.1myosin VI817mir-1433â€²RACENM_018036.1chromosome 14 open reading frame 103818mir-1433â€²RACENM_018312.2chromosome 11 open reading frame 23819mir-1433â€²RACENM_002950.1ribophorin I820mir-1433â€²RACENM_006708.1glyoxalase I821mir-1433â€²RACENM_014953.1mitotic control protein dis3 homolog822mir-1433â€²RACENM_004926.1zinc finger protein 36, C3H type-823like 1mir-1433â€²RACENM_004530.1matrix metalloproteinase 2824(gelatinase A, 72 kDa gelatinase,72 kDa type IV collagenase)mir-1433â€²RACENM_015208.1KIAA0874 protein825mir-1433â€²RACENM_002582.1poly (A)-specific ribonuclease826(deadenylation nuclease)mir-1433â€²RACENM_000297.2polycystic kidney disease 2827(autosomal dominant)mir-1433â€²RACENM_001175Rho GDP dissociation inhibitor (GDI)828betamir-1433â€²RACEXM_166529glucocorticoid induced transcript 1,837GLCCI1mir-1433â€²RACE-NM_001753.2caveolin 1, caveolae protein, 22 kDa7912ntmir-1433â€²RACE-NM_006732FBJ murine osteosarcoma viral8032ntoncogene homolog Bmir-1433â€²RACE-NM_000100.1cystatin B (stefin B)8072ntmir-1433â€²RACE-NM_015959.1CGI-31 protein8082ntmir-1433â€²RACE-NM_004156.1protein phosphatase 2 (formerly 2A),8142ntcatalytic subunit, beta isoformmir-1433â€²RACE-NM_031157heterogeneous nuclear8152ntribonucleoprotein A1mir-1433â€²RACE-NM_002582.1poly(A)-specific ribonuclease8262nt(deadenylation nuclease)mir-1433â€²RACE-NM_000297.2polycystic kidney disease 28272nt(autosomal dominant)mir-1433â€²RACE-NM_006325.2RAN, member RAS oncogene family8292ntmir-1433â€²RACE-NM_004627.1tryptophan rich basic protein8302ntmir-1433â€²RACE-NM_012210.1tripartite motif-containing 328312ntmir-1433â€²RACE-AJ131244.1SEC24 related gene family, member A8322nt(S. cerevisiae)mir-1433â€²RACE-NM_031267.1cell division cycle 2-like 58332nt(cholinesterase-related celldivision controller)mir-1433â€²RACE-AL049367.1guanine nucleotide binding protein8352nt(G protein), gamma 12mir-1433â€²RACE-NM_001344defender against cell death 18362ntmir-1313â€²RACEAK001214.1hypothetical protein FLJ103521735mir-1313â€²RACENM_001614actin, gamma 1 (ACTG1), mRNA1736mir-1313â€²RACENM_001948.1dUTP pyrophosphatase (DUT), mRNA1737mir-1313â€²RACENM_002387.1mutated in colorectal cancers (MCC),1738mRNAmir-1313â€²RACENM_004109.1ferredoxin 1 (FDX1), nuclear gene1739encoding mitochondrial protein, mRNAmir-1313â€²RACENM_004342.4caldesmon 1 (CALD1), transcript1740variant 2, mRNAmir-1313â€²RACENM_005572.2lamin A/C (LMNA), transcript variant17412, mRNAmir-1313â€²RACENM_015640.1PAI-1 mRNA-binding protein (PAI-1742RBP1), mRNAmir-1313â€²RACENM_017789.1semaphorin 4C (SEMA4C), mRNA1743mir-1313â€²RACENM_144697.1hypothetical protein BC0173971744(LOC148523), mRNAmir-1313â€²RACENM_173710NADH dehydrogenase 3 (MTND3), mRNA1745mir-15a5â€²RACEAF220018.1Homo sapiens tripartite motif1746protein (TRIM2) mRNAmir-15a5â€²RACEM98399.1Human antigen CD36 mRNA1747mir-15a5â€²RACEY00281.1Human mRNA for ribophorin I1748

Because these RNA transcripts in Table 27 were identified as being bound by one of the mir-143, mir-131, or mir-15a miRNAs, these miRNAs are predicted to serve a regulatory role in expression or activity of these transcripts identified by RACE-PCR. Additional candidate human RNA targets can be identified in the same manner.

Example 21Effects of Oligomeric Compounds on Adipocyte Differentiation Hallmark Genes in Differentiated Adipocytes

The effect of the oligomeric compounds of the present invention targeting miRNAs on the expression of markers of cellular differentiation was examined in differentiated adipocytes.

The effects of the oligomeric compounds of the present invention on the hallmark genes known to be upregulated during adipocyte differentiation assayed in Example 13 were also assayed in differentiated adipoctyes. As previously described, the HSL, aP2, Glut4, and PPARÎ³ genes play important rolls in the uptake of glucose and the metabolism and utilization of fats. Also as previously described, an increase in triglyceride content is another well-established marker for adipocyte differentiation. Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, N.C.) were grown in preadipocyte media (ZenBio Inc.). After the cells reached confluence (approximately three days), they were exposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-Î³ agonist, IBMX, dexamethasone, and insulin for three days. Cells were then fed Adipocyte Medium (Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals. One day before transfection, 96-well plates were seeded with 3000 cells/well. Cells were then transfected on day nine post-differentiation, according to standard published procedures with 250 nM oligonucleotide in LIPOFECTINâ„¢ (Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomeric compounds were tested in triplicate on each 96-well plate, and the effect of TNF-Î±, known to inhibit adipocyte differentiation, was also measured in triplicate. Oligomeric compound treatments and transfectant-only negative controls may be measured up to six times per plate. On day twelve post-differentiation, cells were washed and lysed at room temperature, and the expression of the four hallmark genes, HSL, aP2, Glut4, and PPARÎ³, as well as triglyceride (TG) accumulation were measured in adipocytes transfected with the uniform 2â€²-MOE phosphorothioate (PS) previously described in Example 13 as well as the chimeric gapmer oligomeric compounds targeting the mir-143 miRNA and the mir-143 pri-miRNA described in Example 16. On day twelve post-differentiation, cells were lysed in a guanadinium-containing buffer and total RNA was harvested. The amount of total RNA in each sample was determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.). Real-time PCR was performed on the total RNA using primer/probe sets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2, and PPARÎ³. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed relative to control levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). The results of this experiment are shown in Table 28.

TABLE 28Effects of oligomeric compounds targeting miRNAs onexpression of adipocyte differentiation markersISISSEQPPARNumberID NOTGaP2HSLGlut4gamma3278762941.160.670.813.531.283278782961.080.130.190.170.853278802981.121.140.930.761.863278883061.130.730.840.561.693278893071.091.120.770.991.633278903081.130.350.420.371.053278923101.230.810.620.421.013279013191.121.281.472.201.343279033211.120.560.530.360.913279053231.180.850.650.581.313279133311.121.051.091.521.293279193371.151.200.831.821.803279223401.480.911.010.610.993279253431.330.781.200.741.303279333511.631.581.302.121.603279343521.431.501.971.521.543279393571.331.161.080.721.893279413591.330.901.170.901.663279543721.461.231.350.611.463283824911.330.920.530.750.973386644911.720.771.011.081.063409273191.610.710.640.961.21

From these data, it was observed that the compound targeting the mir-203 miRNA (ISIS Number 327878), exhibited a sustained reduction in the hallmark marker genes at the 12^thday post differentiation. Treatment with this compound resulted in decreased expression of the aP2, HSL, Glut4 and PPARÎ³ marker genes, indicating that this oligomeric compound may lead to reduced levels of mobilization of fatty acids from adipose tissue, and has the potential to ameliorate some of the symptoms of type 2 diabetes, obesity, hypertension, atherosclerosis, cardiovascular disease, insulin resistance, and certain cancers. Notably, the effect of treatment of differentiated adipocytes with this oligomeric compound targeting the mir-203 miRNA mirrors the effect of treating cells with the TNF-Î± positive control that inhibits adipocyte differentiation. This evidence suggests that the oligomeric compound targeting the mir-203 miRNA can act as a TNF-Î±, mimetic compound, and potentially may be used in the suppression of cellular differentiation and the maintenance of cells in a quiescent state.

The oligomeric compound targeting the mir-203 miRNA was also tested in the insulin assay (see Example 18) and was observed to reduce expression of PEPCK-c, indicating that it may also be useful as an insulin mimetic and/or antidiabetic drug.

As an extension of these conclusions, one having ordinary skill in the art would appreciate that further modified oligomeric compounds could be designed to also target the mir-203 mature miRNA, or the pri-miRNA and pre-miRNA precursors. Such compounds are noted to be within the scope of the present invention.

Example 22Effects of Oligomeric Compounds on Lymphocytic Leukemia Cells

Mir15-a-1 and mir-16-3 have been recently shown to reside in human chromosomal region (13q14) that is deleted in about 50% of chronic lymphocytic leukemia (CLL) patients. Mir-15 and 16 were found to be down-regulated in about 68% of CLL cases (Cahn et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15524-15529, which is incorporated herein by reference in its entirety). CLL B-cells develop chemotherapy resistance over time, possibly due to a defective apoptosis pathway.

Using the 5â€²RACE method (described in Example 20), the CD36 mRNA was identified as one target regulated by mir-15 and/or mir-16 miRNAs. CD36 is a scavenger receptor involved in fat uptake by macrophages and adipocytes. CD36 is reported to be upregulated in some CLL cell lines, and its expression may correlate with tumor invasiveness.

If the apoptosis pathway is defective and the deletion or down-regulation of mir-15 and/or mir-16 play a role in CLL chemo-resistance, then addition of mir-15 and/or mir-16 should be able to induce apoptosis in CLL and increase drug-induced apoptosis. RNA oligonucleotide molecules ISIS Number 338963 (TAGCAGCACATAATGGTTTGTG; SEQ ID NO: 269) representing mir-15a-1/mir-15a-2, ISIS Number 338961 (TAGCAGCACATCATGGTTTACA; SEQ ID NO: 246) representing mir-15b, and ISIS Number 338965 (TAGCAGCACGTAAATATTGGCG; SEQ ID NO: 196) representing mir-16-1/mir-16-2/mir-16-3 were synthesized and deprotected. Additionally, RNA oligonucleotides bearing imperfect complementarity to these miRNA mimics (mimicking the imperfect complementarity found in the pri-miRNA) were also synthesized and deprotected. These imperfect complements were ISIS Number 338964 (TGCAGGCCATATTGTGCTGCCT; SEQ ID NO: 840), which is partially complementary to ISIS Number 338963 and represents the imperfect complement of mir-15a-1/mir-15a-2; ISIS Number 338962 (TGCGAATCATTATTTGCTGCTC; SEQ ID NO: 841), which is partially complementary to ISIS Number 338961 and represents the imperfect complement of mir-15b; ISIS Number 338966 (CTCCAGTATTAACTGTGCTGCTG; SEQ ID NO: 842), which is partially complementary to ISIS Number 338965 and represents the imperfect complement of mir-16-1 and mir-16-2; and ISIS Number 338967 (CACCAATATTACTGTGCTGCTT; SEQ ID NO: 843), which is partially complementary to ISIS Number 338965 and represents the imperfect complement of mir-16-3. These RNA molecules were diluted in water, and the concentration determined by A₂₆₀. Equimolar amounts of each of the miRNAs and their imperfect complementary RNA sequences were mixed together in the presence of Dharmacon 5Ã— Universal buffer to form four â€œnaturalâ€ double-stranded miRNA mimics. ISIS Number 338965 (SEQ ID NO: 196) was used twice; once, it was hybridized to ISIS Number 338966, and once it was hybridized to ISIS Number 338967, to form two different â€œnaturalâ€ double-stranded miRNA mimics, Mir-16-1/Mir-16-2 and Mir-16-3, with imperfect complementarity. The mixture of four â€œnaturalâ€ miRNA mimics was incubated for 1-5 minutes at 90Â° C. (the time depends on the volume of the mixture) and then incubated at 37Â° C. for one hour. A₂₆₀readings were taken on the mixture for final concentration determination.

Heparinized peripheral blood from CLL patients was separated on a Ficoll density gradient to obtain greater than 95% pure CLL B-cells. These cells are tested for expression of the CD5/CD19/CD23 antigens. Positive expression of these three antigens indicates that the cells are CLL B-cells (Pederson et al., Blood, 2002, 100, 2965, which is incorporated herein by reference in its entirety). Additionally, cytogenetic analysis can be performed to ascertain that the cells have the 13q deletion. A mixture of all four â€œnaturalâ€ miRNA mimics at 2 Î¼M each was electroporated into the cells. The cells were cultured in the presence or absence of apoptosis-inducing agents fludarabine A, or Dexamethasone (which are known to employ the intrinsic mitochondrial apoptotic pathway) or the antitumor agent CDDO-Im (reported to function through an alternative extrinsic apoptotic pathway) for 24 hours. Following incubation, apoptosis was monitored by annexin/PI double staining as outlined in FIG. 1 of Pederson et al., Blood, 2002, 100, 2965. The double-stranded RNA oligomeric compounds representing mir-15 and mir-16 miRNA mimics were observed to play a role in the induction of spontaneous as well as drug-induced apoptosis. Thus, oligomeric compounds of the present invention may be useful in the treatment of CD36-related diseases and conditions such as chronic lymphocytic leukemia and other cancers.

Example 23Effect of Oligomeric Compounds Targeting miRNAs In Vivo

As described herein, leptin-deficient (ob/ob) mice, leptin receptor-deficient (db/db) mice and diet-induced obesity (DIO) mice are used to model obesity and diabetes. In accordance with the present invention, oligomeric compounds targeting mir-143, mir-131 (also known as mir-9) and mir-203 were tested in the ob/ob and db/db models. The ob/ob mice were fed a high fat diet and were subcutaneously injected with the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 6 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet served as controls. The physiological effects resulting from inhibition of target RNA, such as the effects of target inhibition on glucose and insulin metabolism and the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, were assessed by methods disclosed herein. In brief, plasma levels of liver transaminases, cholesterol, triglycerides, free fatty acids and glucose were assessed weekly by tail bleed, with the tail bleed on week three taken under fasting conditions. After the treatment period, mice were sacrificed and liver, spleen, pancreas, muscle, kidney and heart, as well as brown adipose tissue (BAT) and white adipose tissue (WAT) tissues were collected. mRNA expression levels of the Glut4, aP2, HSL and PPARÎ³ marker genes were evaluated. RNA isolation and target RNA expression level quantitation are performed as described.

Two oligomeric compounds targeting the mir-143 miRNA were compared for their effects on the physiological indications of obesity and diabetes. The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), 22-nucleotides in length, targets the mature mir-143, and is a uniform 2â€²-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 340927 (SEQ ID NO: 319) is a 5-10-7 gapmer also designed to target the mature mir-143 miRNA. The effects of these oligomeric compounds targeting mir-143 on several physiological parameters and markers of obesity and/or diabetes were examined in vivo. Potential effects on food consumption were also monitored.

Plasma cholesterol levels were observed to slightly decrease over time in ob/ob mice treated with the gapmer oligomeric compound ISIS Number 340927 (SEQ ID NO: 319) targeted to mir-143. Similarly, plasma triglyceride and plasma glucose levels were generally slightly lower in ob/ob mice treated with this compound as compared to untreated mice, or mice treated with control compounds. mRNA expression levels of the Glut4, aP2 and HSL marker genes were slightly reduced by both oligomeric compounds ISIS Number 327901 and ISIS Number 340927 targeting mir-143. Thus, these oligomeric compounds targeting mir-143 may be useful compounds in the treatment of obesity or diabetes.

In addition, Northern blot analyses were performed to quantitate the expression of mature mir-143 in kidney samples of ob/ob mice treated with oligomeric compounds of the present invention. The mir-143 specific DNA oligonucleotide probe (SEQ ID NO: 319) described above was used to detect expression levels of the mir-143 miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISIS Numbers 327901, the uniform 2â€²-MOE oligomeric compound, or ISIS Number 340927, the 5-10-7 gapmer compound, both targeted to mir-143, versus saline treated animals or animals treated with ISIS 342672 (SEQ ID NO: 789), a uniform 2â€²-MOE scrambled negative control oligomeric compound. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Most notably, in kidney samples from ob/ob mice treated with ISIS Number 327901, the uniform 2â€²-MOE oligomeric compound targeted to mir-143 exhibited a nearly 40% decrease in in vivo expression levels of the mature mir-143 miRNA. In kidney samples from mice treated with the gapmer oligomeric compound targeting mir-143, ISIS Number 340927, a 23% reduction in in vivo expression levels of the mature mir-143 miRNA was observed.

Oligomeric compounds targeting the mir-131/mir-9 and the mir-203 miRNAs were also tested for their effects on the physiological indicators or markers of obesity and diabetes. The oligomeric compound, ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131/mir-9,21-nucleotides in length, is a uniform 2â€²-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 340926 (SEQ ID NO: 310) is a 5-1-6 gapmer oligomeric compound also designed to target the mir-131/mir-9 miRNA. The oligomeric compound ISIS Number 327878 (SEQ ID NO: 296) targeted to mir-203, 22-nucleotides in length, is a uniform 2â€²-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 345349 (SEQ ID NO: 296) is a 5-10-7 gapmer oligomeric compound also designed to target the mir-203 miRNA. The effects of these oligomeric compounds were examined in vivo in the ob/ob model. Potential effects on food consumption were also monitored.

Fed plasma glucose levels in ob/ob mice treated with the oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9, and ISIS Number 327878 (SEQ ID NO: 296) and ISIS Number 345349 (SEQ ID NO: 296) targeted to mir-203 were observed to be reduced beginning at approximately four weeks after the start of treatment and continuing to decrease on week five as compared to untreated mice, or mice treated with control compounds. Triglyceride levels were also observed to be reduced over time in mice treated with ISIS 340926 and 345349, the gapmer oligomeric compounds targeted to mir-131/mir-9 and mir-203, respectively. No signs of liver toxicity were indicated by weekly measurements of plasma transaminases upon treatment of ob/ob mice with any of the oligomeric compounds targeting mir-143, mir-203 or mir-131/mir9.

ob/ob mice in the fasted state on day 19 after treatment with the oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 also exhibited significant reductions in plasma glucose levels. Notably, the gapmer oligomeric compound ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 was even more potent than the corresponding uniform 2â€²-MOE oligonucleotide ISIS Number 327892 (SEQ ID NO: 310).

Furthermore, a decrease in food consumption was observed by the third week and this reduced level was maintained in the fourth week post-treatment of ob/ob mice with these oligomeric compounds. Therefore, the oligomeric compounds targeting the mir-131/mir-9 and mir-203 miRNAs have potential use as appetite suppressants, as well as in the treatment of obesity or diabetes.

The oligomeric compounds ISIS Number 327901 and ISIS Number 340927 both targeting mir-143, ISIS Number 327892 and ISIS Number 340926 both targeting mir-131/mir-9, and ISIS Number 327878 and ISIS Number 345349 both targeting mir-203 were also tested in db/db mice. Although treatment of db/db mice with the gapmer compounds targeting mir-143, mir-203 or mir-131/mir9 resulted in an approximately 2-fold increase in liver transaminases in db/db mice, the uniform 2â€²-MOE oligomeric compounds targeting mir-143, mir-203 or mir-131/mir-9 were not found to cause liver toxicity in db/db mice, as assessed by weekly measurements of plasma transaminase levels.

Additional oligomeric compounds targeting miRNAs were studied in ob/ob mice. Six week old ob/ob mice were treated (dose=25 mg/kg, twice weekly for four weeks) with uniform 2â€²-MOE and gapmer oligomeric compounds targeting mir-143, mir-23b, mir-221, let-7a, and mir-29b, and compared to saline treated animals or animals treated with ISIS 342672 (SEQ ID NO: 789), a uniform 2â€²-MOE scrambled negative control oligomeric compound bearing 13 base mismatches to mir-143. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Fed plasma samples were taken bi-weekly by tail bleed, and plasma levels of liver transaminases, cholesterol, triglycerides, free fatty acids and glucose were assessed, with the tail bleed on week three taken under fasting conditions. Ob/ob mice were treated with ISIS Numbers 327901 and 340927, the uniform 2â€²-MOE and gapmer oligomeric compounds, respectively, targeting mir-143 are described above. Additionally, ob/ob mice were also treated with the following compounds: ISIS Number 327889 (SEQ ID NO: 307), a phosphorothioate uniform 2â€²-MOE oligomeric compound, and ISIS Number 340925 (SEQ ID NO: 307), a 2â€²-MOE 5-10-8 gapmer oligomeric compound, each targeting mir-23b; ISIS Number 327919 (SEQ ID NO: 337), a uniform 2â€²-MOE oligomeric compound, and ISIS Number 345384 (SEQ ID NO: 337), a phosphorothioate 2â€²-MOE 5-10-8 gapmer oligomeric compound, each targeting mir-221; ISIS Number 327903 (SEQ ID NO: 321), a uniform 2â€²-MOE oligomeric compound, and ISIS Number 345370 (SEQ ID NO: 321), a phosphorothioate 2â€²-MOE 5-10-7 gapmer oligomeric compound, each targeting let-7a; and ISIS Number 327876 (SEQ ID NO: 294), a uniform 2â€²-MOE oligomeric compound, and ISIS Number 345347 (SEQ ID NO: 294), a phosphorothioate 2â€²-MOE 5-10-8 gapmer oligomeric compound, each targeted to mir-29b-1.

Ob/ob mice treated with the gapmer compounds ISIS 340925 and ISIS 345384, targeting mir-23b and mir-221, respectively, exhibited reductions in plasma glucose levels in the fed state at weeks two and four, as compared to untreated mice, or mice treated with control compounds. Furthermore, mice treated with ISIS 340925 exhibited a decrease in triglycerides in the fourth week. Ob/ob mice treated with ISIS 340925 did not exhibit an increase in plasma transaminases at weeks two or four. Thus, the oligomeric compounds ISIS Numbers 340925 and 345384 may be useful as agents for the treatment of obesity and/or diabetes.

In addition, Northern blot analyses were performed to quantitate the expression of mir-23b in kidney samples of ob/ob mice treated with oligomeric compounds of the present invention. To detect the mir-23b target, a target-specific DNA oligonucleotide probe with the sequence GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) was synthesized by IDT (Coralville, Iowa). The oligo probes were 5â€² end-labeled with T4 polynucleotide kinase with (Î³-³²P) ATP (Promega). The mir-23b specific DNA oligonucleotide probe was used to detect expression levels of the mir-23b miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISIS Numbers 327889, the uniform 2â€²-MOE oligomeric compound, or ISIS Number 340925, the 5-10-8 gapmer compound, both targeted to mir-23b, versus saline treated animals or animals treated with a control oligomeric compound, ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), a uniform 5-10-5 2â€²-MOE gapmer targeting an unrelated gene, PTEN. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Most notably, in kidney samples from ob/ob mice treated with ISIS Number 327889, the uniform 2â€²-MOE oligomeric compound targeted to mir-23b exhibited a nearly 64% decrease in in vivo expression levels of the mir-23b miRNA. In kidney samples from mice treated with the gapmer oligomeric compound targeting mir-23b, ISIS Number 340925, a 41% reduction in in vivo expression levels of the mir-23b miRNA was observed.

As described, supra, the C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation, and when these mice are fed a high-fat diet, they develop diet-induced obesity (DIO). Accordingly, the DIO mouse model is useful for the investigation of obesity and development of agents designed to treat these conditions. In one embodiment of the present invention, oligomeric compounds targeting miRNAs were tested in the DIO model. Normal C57/BL6 male mice were fed a high fat diet (40% fat, 41% carbohydrate, 18% protein) for 12 weeks before the study began. DIO mice were then randomized by weight and insulin values. Initial body fat composition was determined by Dual X-ray Absorptiometry (DEXA) Scan. DIO mice were then subcutaneously injected with oligomeric compounds of the invention at a dose of 25 mg/kg, twice weekly. DIO mice were treated with oligomeric compounds ISIS Numbers 327901 and 340927 targeting mir-143, ISIS Numbers 327892 and 340926 targeting mir-131/mir-9, ISIS Numbers 327878 and ISIS Number 345349 targeting mir-203, and ISIS Numbers 327889 and 340925, targeting mir-23b. As negative controls, â€œscrambledâ€ oligomeric compounds were also designed: ISIS Number 342672 contains 13 mismatches with respect to the mature mir-143 miRNA; ISIS Number 353607 (ACTAGTTTTTCTTACGTCTGA; herein incorporated as SEQ ID NO: 845) is a phosphorothioate 5-10-6 2â€²-MOE gapmer oligomeric compound containing 12 mismatches with respect to mir-131/mir-9; ISIS Number 353608 (CTAGACATTAGCTTTGACATCC; herein incorporated as SEQ ID NO: 846) is a phosphorothioate 5-10-7 2â€²-MOE gapmer oligomeric compound containing 16 mismatches with respect to mir-203. DEXA scans were also performed at weeks 0, 3 and 5 after treatment with the oligomeric compounds to assess the fat mass to lean mass ratio. The effects of target inhibition on levels of plasma glucose and insulin, liver transaminases, cholesterol and triglycerides, were also assessed weekly by tail bleed, and after the treatment period, mice were sacrificed and liver and kidney heart, as well as white adipose tissue (WAT) tissues collected. The mRNA expression levels of the Glut4, aP2, HSL and PPARÎ³ marker genes are also assessed. Treatment of DIO mice with the uniform 2â€²-MOE oligomeric compounds ISIS 327901 targeting mir-143, ISIS 327892 targeting mir-131/mir9, ISIS 327878 targeting mir-203, and ISIS 327889 targeting mir-23b did not appear to cause liver toxicity in these mice as assessed by weekly measurements of plasma transaminase levels. Similarly, the gapmer oligomeric compounds ISIS 340927 targeting mir-143, and ISIS 340926 targeting mir-131/mir-9, 340925 did not cause significant increases in liver toxicity, and the gapmer compound ISIS 340925 targeting mir-23b caused only an approximately 2-fold increase in the liver transaminase AST. Interestingly, the gapmer compounds ISIS Numbers 340927 targeting mir-143, 340926 targeting mir-131/mir-9, 345349 targeting mir-203, and 340925, targeting mir-23b were all effective at reducing insulin levels at the two and four week time points, as compared to saline-treated control mice. Furthermore, some improvement in body composition (a reduction in body weight and fat mass) was observed. These data from the DIO model suggest that oligomeric compounds targeting mir-143, mir-131/mir-9, mir-203 and mir-23b may be useful as agents for the treatment of obesity and/or diabetes.

Having the information disclosed herein, one of ordinary skill in the art would comprehend that of other classes of inhibitors targeting mir-143, mir-209, mir-131/mir-9 and mir-23b miRNAs, such as antibodies, small molecules, and inhibitory peptides, can be assessed for their effects on the physiological indicators of diseases in in vivo models, and these inhibitors can be developed for the treatment, amelioration or improvement of physiological conditions associated with a particular disease state or condition. Such inhibitors are envisioned as within the scope of the instant invention.

Example 24Effects of Oligomeric Compounds on Cell CyclingCell Cycle Assay:

Cell cycle regulation is the basis for various cancer therapeutics. Cell cycle checkpoints are responsible for surveillance of proper completion of certain steps in cell division such as chromosome replication, spindle microtubule attachment and chromosome segregation, and it is believed that checkpoint functions are compromised in some cancerous cells. Furthermore, because the shift from quiescence to an actively growing state as well as the passage through mitotic checkpoints are essential transitions in cancer cells, most current chemotherapy agents target dividing cells. For example, by blocking the synthesis of new DNA required for cell division, an anticancer drug can block cells in S-phase of the cell cycle. These chemotherapy agents impact many healthy organs as well as tumors. In some cases, a cell cycle regulator will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected. Loss of tumor supressors such as p53 sensitizes cells to certain anticancer drugs; however, cancer cells often escape apoptosis. Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected. A goal of these assays is to determine the effects of oligomeric compounds on the distribution of cells in various phases of the cell cycle.

In some embodiments, the effects of several oligomeric compounds of the present invention were examined in the normal human foreskin fibroblast BJ cell line, the mouse melanoma cell line B16-F10 (also known as B16 cells), as well as the breast carcinoma cell line, T47D. These cell lines can be obtained from the American Type Culture Collection (Manassas, Va.). BJ cells were routinely cultured in MEM high glucose with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate (all media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). B16-F10 cells were routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). T47D cells were cultured in DMEM High glucose media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum. Cells were routinely passaged by trypsinization and dilution when they reached 80 to 90% confluence. Cells were plated on collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well and allowed to attach to wells overnight.

As a negative control, a random-mer oligomeric compound, 20 nucleotides in length, ISIS 29848 (SEQ ID NO: 737) was used. In addition, a positive control, ISIS 183891 (CCGAGCTCTCTTATCAACAG; herein incorporated as SEQ ID NO: 847) was included; ISIS 183891 targets kinesin-like 1 (also known as Eg5) and inhibits cell cycle progression. Eg5 is known to induce apoptosis when inhibited. ISIS 29248 and ISIS 183891 are chimeric oligomeric compounds (â€œgapmersâ€ ) 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwingsâ€ (a â€œ5-10-5 gapmerâ€ ). The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the compound. All cytidine residues are 5-methylcytidines. ISIS 340348 (CTACCTGCACGAACAGCACTTT; herein incorporated as SEQ ID NO: 848) is a uniform 2â€²-MOE phosphorothioate oligomeric compound targeting the mir-93 miRNA, and ISIS 340365 (TACTTTATATAGAACACAAG; herein incorporated as SEQ ID NO: 849) is a 5-10-5 gapmer phosphorothioate oligomeric compound targeting the mir-92-2 miRNA.

Oligomeric compounds were mixed with LIPOFECTINâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEMâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 150 nM of oligomeric compound and 4.5 Î¼g/ml LIPOFECTINâ„¢. Before adding to cells, the oligomeric compound, LIPOFECTINâ„¢ and OPTI-MEMâ„¢ were mixed thoroughly and incubated for 0.5 hrs. The medium was removed from the plates and each well was washed in 250 of phosphate-buffered saline. The wash buffer in each well was replaced with 250 Î¼L of the oligomeric compound/OPTI-MEMâ„¢/LIPOFECTIN cocktail. Control cells received LIPOFECTINâ„¢ only. The plates were incubated for 4 hours at 37Â° C., after which the medium was removed. 100 Î¼l of full growth medium was added to each well. After 72 hours, routine procedures were used to prepare cells for flow cytometry analysis and cells were fixed with ethanol and stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces an increase in cells with a hypodiploid DNA content. Cells with a hypodiploid DNA content are categorized as â€œsubG1.â€ The cells in the G1, G2/M and S phases are considered to be cycling, and cells in the subG1 and aneuploid categories are considered to have left the cell cycle. An increase in cells in G1 phase is indicative of a cell cycle arrest prior to entry into S phase; an increase in cells in S phase is indicative of cell cycle arrest during DNA synthesis; and an increase in cells in the G2/M phase is indicative of cell cycle arrest just prior to or during mitosis. Data are shown in Table 29 and expressed as percentage of cells in each phase of the cell cycle.

TABLE 29Effects of oligomeric compounds targeting miRNAs on cell cyclingSEQaneu-ISIS #ID #Pri-miRNASubG1G1SG2/MploidUTCN/AN/A8.159.627.512.97.3ISIS-29848737N/A9.657.826.515.612n-merISIS-183891847Kinesin-20.833.139.227.611.5Positivelike 1/Eg5control327878296mir-20317.339.140.82011.9327888306mir-108-113.353.729.516.712.9327889307mir-23b8.253.132.514.410.5327901319mir-1431234.744.920.313.6327902320mir-192-110.650.733.915.313.4327903321let-7a-31153.730.915.413.4327904322mir-181a8.654.429.516.215.6327905323mir-2058.556.928.11514.7327906324mir-103-115.246.13320.915.8327907325mir-26a17.849.532.817.617.8327908326mir-33a5.655.429.215.313.1327909327mir-196-27.952.630.117.316.3327910328mir-1079.349.53317.513.1327911329mir-10610.949.930.12016.5327914332mir-130a8.555.828.915.316.2327919337mir-22110.854.330.315.416327922340mir-19b-21050.430.718.916.8327928346mir-29a-16.65627.91615.9327933351mir-14510.249.631.319.115.9327934352mir-2136.654.428.217.417327941359mir-181b8.257.229.912.915.8327951369mir-15a-14.360.924.814.316.7328342451mir-2034.862.324.912.815.2328362471mir-108-19.151.233.615.112.9328364473mir-23b1.961.524.214.315.1328382491mir-1432.959.825.714.414.8328388497let-7a-34.057.52814.614.5328394503mir-181a2.459.524.51618.3328396505mir-2054.656.828.21519.8328419528mir-2216.051.232.516.317.9328423532mir-19b-24.952.932.414.815.3328424533mir-19b-23.161.923.714.416.9328436545mir-29a-13.559.226.913.917.4328644553mir-1457.258.427.61417.5328691600mir-1457.760.524.415.116.6328697606mir-181b2.457.626.41613.5328773682mir-15a-22.756.426.916.711.7340348848mir-9314.153.931.814.312.3340365849mir-92-24.355.229.415.418.3

From these data, it is evident that treatment with the oligomeric compounds targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319); mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324); mir-106, ISIS Number 327911 (SEQ ID NO: 329); and mir-145, ISIS Number 327933 (SEQ ID NO: 351) resulted in an increased percentage of cells in the G2/M phase, indicating that these oligomeric compounds arrest or delay the cell cycle at or just prior to mitosis, potentially activating a mitotic checkpoint.

Treatment with the oligomeric compounds targeting mir-26a, ISIS Number 327907 (SEQ ID NO: 325); mir-205, ISIS Number 328396 (SEQ ID NO: 505); mir-181a, ISIS Number 328394 (SEQ ID NO: 503); and mir-92-2, ISIS Number 340365 (SEQ ID NO: 849) resulted in higher than average percentages of aneuploid cells, indicating that these oligomeric compounds interfere with proper chromosome segregation.

Treatment with the oligomeric compounds targeting mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324); mir-26a, ISIS Number 327907 (SEQ ID NO: 325); and mir-93, ISIS Number 340348 (SEQ ID NO: 848) resulted in an increased percentage of cells with hypodiploid DNA content (SubG1 phase) indicating that the oligomeric compound treatment may induce apoptotic events.

The effects of several oligomeric compounds of the present invention were also examined in the HeLa and A549 human carcinoma cell lines, both of which can be obtained from the American Type Culture Collection (Manassas, Va.).

In some embodiments, HeLa cells were plated on collagen-coated 24-well plates at 50,000-60,000 cells per well, and allowed to attach to wells overnight. In some embodiments, HeLa cells were synchronized by double thymidine block (cells were washed three times with PBS, then grown in 10% FBS containing 2 mM thymidine; then 19 hours later, cells were washed three times in PBS, 10% FBS for 9 hours; cells were then incubated in 10% FBS, 2 mM thymidine for 15 hours; then washed three times with PBS, 10% FBS and samples were taken every two hours over a 16 hour period). A portion of each time sample was fixed with ethanol and treated with propidium iodide and subjected to FACs analysis for determination of the percentage of cells in each phase of the cell cycle. Distinctive peaks were observed for G0-, S-, Early G2/M-, Late G2/M-, and G1-phases of the cell cycle at 0-, 4-, 6-, 8-, and 12-hours, respectively, indicating that the cells were synchronized. HeLa cells treated with 10 Î¼M cisplatin or 100 ng/ml nocodazole were used as controls for G1-phase and late G2/M-phases, respectively. From the remaining portion of each of these time samples, total RNA was isolated and used to assess the expression of cell cycle marker mRNAs using the real-time RT-PCR methods and/or used to screen microarrays to assess the expression of miRNAs over the course of the cell cycle. It was observed that several miRNAs are expressed in a cell-cycle-dependent manner. Shown in Table 30 are the mRNA levels of the E2F1 transcription factor and topoisomerase 2A (Top2A), which vary over the course of the cell cycle and can be used for comparison to the experimental groups for the confirmation of cell cycle phase. Data are an average of three trials.

TABLE 30Expression levels of cell cycle markerstreatmentE2F1 mRNATop2A mRNA10 uM cisplatin10215100 ng/ml nocodazole231760 hrs (G0-phase)1001004 hrs (S-phase)811056 hrs (early G2/M-phase)392218 hrs (late G2/M-phase)5025412 hrs (G1-phase)61124

In some embodiments, HeLa cells were also treated with oligomeric compounds targeting miRNAs. As described above, oligomeric compounds were mixed with LIPOFECTINâ„¢ in OPTI-MEMâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.) to a final concentration of 150 nM of oligomeric compound and 6 Î¼g/ml LIPOFECTINâ„¢. Before adding to cells, the oligomeric compound, LIPOFECTINâ„¢ and OPTI-MEMâ„¢ were mixed thoroughly and incubated for 0.5 hrs. The medium was removed from the plates. Each well was washed in 250 Î¼l of PBS. The wash buffer in each well was replaced with 250 Î¼L of the oligomeric compound/OPTI-MEMâ„¢/LIPOFECTIN cocktail. Control cells received LIPOFECTINâ„¢ only. The plates were incubated for 4 hours at 37Â° C., after which the medium was removed. 1000 Î¼l of full growth medium was added to each well. After 24 hours (Table 31) or 48 hours (Table 32), cells were prepared for flow cytometry analysis to generate a cell cycle profile. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).

The random-mer ISIS 29848 (SEQ ID NO: 737) was used as a negative control, and ISIS 183891 (SEQ ID NO: 847), targeting kinesin-like 1/Eg5, was included as a positive control. Results of these experiments are shown in Tables 31 and 32. Data are expressed as percentage of cells in each phase relative to the untreated control (UTC); values above 100 are considered to indicate a delay or arrest in that phase of the cell cycle. Table 31 shows the results from cells sampled 24 hours after oligomeric compound treatment, and Table 32 shows the results from cells sampled 48 hours after oligomeric compound treatment. In some cases, the same oligomeric compound was tested in repeated experiments.

TABLE 31Effects of oligomeric compounds targetingmiRNAs on cell cycling (24 hours)SEQ% cells in cell cycle phasePri-miRNAISIS #ID #subG1G1SG2/ManeuploidUTCN/AN/A100100100100100n-mer298487371201168110876Kinesin-like18389184725121109231951/Eg5collagen,33879762419710179148193type I,alpha 1/hypotheticalmiRNA-144hypothetical33866649323512363158102miRNA-039hypothetical32811141362127759950miRNA-111hypothetical3387505771071487697166miRNA-111hypothetical328115417177908714759miRNA-142hypothetical328119421751009411283miRNA-154hypothetical3287246331559190135197miRNA-154hypothetical32874965831212682110138miRNA-179hypothetical3287806891249687136149miRNA-179hypothetical328136438330125818851miRNA-181hypothetical33883366023215056142185miRNA-181let-7a-33279033211189210410698let-7a-33283883751201108311585mir-100-1327957497197918814566mir-100-13287076161883693195166mir-103-13279063242281534710765mir-103-1328397506134938614291mir-10632791132915813062122104mir-106328403512284708519753mir-106328403512189867517982mir-1073279103281741544211873mir-108-13283624711141018712666mir-10a327949367194828417268MiR-125a,34178785222111375144165Mousemir-127,34178885330315454140114Mousemir-130b3286875962318098131149mir-130b33876959618817161103133mir-131-2/3278923101538611110380mir-9mir-131-2/328369310841008812571mir-9mir-131-2/340926478286989112183mir-9mir-133b3387135409315272101187mir-14133874156815714173112166mir-1433279013191081019411090mir-143328382491811187611678mir-14332838249122610280144202mir-1433409273191181217511188mir-1433409273191311287110687mir-1453279333511921028313192mir-145327933351190909114047mir-145328644553711138410968mir-145345395351247548222277mir-149,3417858541251529253158Mousemir-15232872763624513381105161mir-1523388096361061598269210mir-16-33278772951541076615962mir-17/3278853031511296312155mir-91mir-181a-1327904322114991029989mir-18232874465322931108167111mir-1823388266531451487990138mir-192-13279023201785710617666mir-192-13279023201754412116398mir-192-1328383492314558222292mir-192-1328383492289639718398mir-192-13386653401738576175193mir-19b-23279224921319796114104mir-19b-2328424533601108511274mir-203327878296124969412273mir-203328342451192339523867mir-205327905323144998812950mir-205327905323149949512198mir-20532839650597948713988mir-20533867850516212275131202mir-211327946364225908415643mir-211328674583564125938469mir-2113387565831371477599166mir-213/327934352278878516055mir-181a-2mir-213/3279343522041186613777mir-181a-2mir-213/32864755614010192119140mir-181a-2mir-2163279563741201246812061mir-2163287596682398878168184mir-223278963141218310312865mir-223283744831985411516297mir-2203279443621658511011150mir-221327919337859210310996mir-221328419528871097912477mir-23a33883666315318553105150mir-23b3278893071221041028782mir-23b3409253071511038911773mir-26a-13279073252241197711175mir-26a-1345373325196669417668mir-29b-1327876294103981049566mir-29b-1327876294149939213175mir-29b-132833744610710688113104mir-29b-1328337446991088810964mir-29b-23283394482357710214361mir-29c33869051714912478123194mir-30a32808458538143104163101mir-30b3286765851399986134169mir-30b33875874311312981108190mir-30d3284215302884710520070mir-33a3279083261389899106114mir-92-13278973151431148011569mir-92-13278973151801287410054mir-92-23403658491091257111484mir-953403508552181835410494(Mourelatos)mir-96338637464881707084188

TABLE 32Effects of oligomeric compounds targetingmiRNAs on cell cycling (48 hours)SEQ% cells in cell cycle phasePri-miRNAISIS #ID #subG1G1SG2/ManeuploidUTCN/AN/A100100100100100n-mer298487378687121117109Kinesin-like18389184717319124331721/Eg5collagen,33879762481366124168175type I,alpha 1/hypotheticalmiRNA-144hypothetical338666493183244136217125miRNA-039hypothetical3281114133718412611990miRNA-111hypothetical33875057720199101103190miRNA-111hypothetical3281154171959211410786miRNA-142hypothetical3281194217677514512481miRNA-154hypothetical32872463365370134140155miRNA-154hypothetical3287496589623712924665miRNA-179hypothetical32878068991783130110133miRNA-179hypothetical3281364381408313311385miRNA-181hypothetical338833660109144106258154miRNA-181let-7a-332790332174102959894let-7a-332838837511299101102126mir-100-13279574978646516912785mir-100-1328707616148646134213155mir-103-1327906324571009810383mir-103-1328397506749710110996mir-106327911329659996109101mir-1063284035128636117713185mir-1063284035121088214810680mir-10732791032853999111192mir-108-1328362471879610410897mir-10a3279493677736615713871MiR-125a,34178785270755126197153Mousemir-127,34178885374876105163116Mousemir-130b328687596111955174131171mir-130b33876959648276116149194mir-131-2/3278923101217415012979mir-9mir-131-2/328369310729995109109mir-9mir-131-2/3409264786883120131106mir-9mir-133b33871354042695104109194mir-14133874156818510010199170mir-1433279013199398104103104mir-1433283824917110292103109mir-14332838249135083122120133mir-1433409273199591107121113mir-1433409273198391107122108mir-145327933351917613513886mir-1453279333514388013312375mir-145328644553521011019882mir-1453453953512135119215787mir-149,341785854114882126116166Mousemir-15232872763684668152124140mir-15233880963634586110129157mir-16-33278772957555915216880mir-17/3278853034567812913376mir-91mir-181a-13279043221168712611480mir-18232874465317743178334171mir-18233882665369661124182137mir-192-132790232011763917120881mir-192-13279023202024416620587mir-192-13283834923035321712490mir-192-13283834929405417815090mir-192-133866534016294089292149mir-19b-2327922492819610510991mir-19b-23284245338910391101111mir-203327878296508911911492mir-20332834245118955115225107mir-2053279053237194819415067mir-2053279053231007814312299mir-2053283965058889114119129mir-20533867850511588178188179mir-2113279463644317215012976mir-211328674583166369160109134mir-21133875658331190121100169mir-213/3279343527526215615292mir-181a-2mir-213/32793435215566148155117mir-181a-2mir-213/32864755658969153118136mir-181a-2mir-21632795637418491106121110mir-21632875966817445031343148mir-223278963148865514019466mir-223283744837876515714371mir-2203279443624907512914478mir-22132791933710480122139104mir-221328419528839996107112mir-23a33883666381152152169165mir-23b32788930713378137130101mir-23b340925307898713010993mir-26a-13279073251169211111594mir-26a-134537332511675132145119mir-29b-13278762944187120119100mir-29b-13278762942517614112669mir-29b-13283374466692105119108mir-29b-13283374466627314313574mir-29b-23283394486787315312392mir-29c33869051741391110112190mir-30a32808458510282016824157mir-30b32867658536686118118172mir-30b3387587432671039992153mir-30d32842153011033020219864mir-33a32790832661999810593mir-92-13278973151341001039584mir-92-13278973151259411410563mir-92-2340365849719994109129mir-95340350855114476126134125(Mourelatos)mir-9633863746423990109117210

Several oligomeric compounds were observed to result in an arrest or delay of the cell cycle, in some cases correlating with a cell-cycle-dependent expression profile as determined by miRNA microarray analysis.

For example, from these data, it was observed that treatment of HeLa cells with oligomeric compounds (MOE-gapmers and fully modified MOEs) targeting miRNAs caused an increase in the percentage of cells exhibiting a subG1-phase or aneuploid DNA content, indicating aberrant chromosome segregation. Treatment with oligomeric compounds ISIS Number 338797 (SEQ ID NO: 624) targeted to hypothetical miRNA-144, ISIS Number 338833 (SEQ ID NO: 660) targeted to hypothetical miRNA-181, and ISIS Number 328759 (SEQ ID NO: 668) targeted to mir-216, each appeared to cause an induce chromosome missegregation events at both the 24-hour and 48-hour timepoints. Thus, these compounds may be useful in triggering a checkpoint arrest in rapidly dividing cells, potentially useful in the treatment of hyperproliferative disorders such as cancer.

It was also observed that other oligomeric compounds (MOE-gapmers and fully modified MOEs) targeting miRNAs appeared to induce an arrest or delay in the G1-, S-, or G2/M-phases of the cell cycle. By miRNA microarray analysis, expression levels of the mir-205 miRNA were observed to increase in the S- and G1-phases of the cell cycle in HeLa cells. Treatment of HeLa cells with the oligomeric compound ISIS Number 327905 (SEQ ID NO: 323), targeting the mir-205 miRNA, was observed to arrest or delay the cell cycle in S-phase at the 48-hour time point, suggesting that the mir-205 miRNA may play a role in regulating DNA replication. It was also observed that treatment of HeLa cells with the oligomeric compound ISIS Number 338678 (SEQ ID NO: 505), targeted to the mir-205 pri-miRNA, resulted in an arrest or delay primarily in the G2/M-phase of the cell cycle, suggesting that this oligomeric compound may interfere with processing of the miRNA precursor into a mature miRNA, which appears to have an impact on mitosis.

Treatment of HeLa cells with oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310), targeting mir-131/mir-9, and ISIS Number 327934 (SEQ ID NO: 352), targeting mir-213/mir-181a-2, was observed to arrest or delay the cell cycle in G2/M- and S-phases at the 48-hour time point, suggesting that the mir-131/mir-9 and mir-213/mir-181a-2 miRNAs are involved in regulating DNA replication and entry into mitosis.

Treatment of HeLa cells with oligomeric compound ISIS Number 345373 (SEQ ID NO: 325), targeting miR-26a-1, was observed to arrest or delay cells mainly in the G2/M-phase at 24 hrs after oligonucleotide-treatment, and at 48 hrs after oligonucleotide-treatment to arrest or delay cells mainly in S-phase of the cell cycle, suggesting that miR-26a-1 is involved in mitosis and that cells making it through a first round of cell division may harbor errors that cause them to arrest during a new round of DNA replication.

By miRNA microarray analysis, expression levels of the mir-145 miRNA were observed to increase in the G2/M-phase of the cell cycle in HeLa cells, and treatment of HeLa cells with the oligomeric compounds ISIS Number 327933 (SEQ ID NO: 351), a uniform 2â€²-MOE compound, and ISIS Number 345395 (SEQ ID NO: 351), a chimeric 2â€²-MOE gapmer compound, both targeting the mir-145 miRNA, were observed to arrest or delay the cell cycle in G2/M-phase at the 24-hour time point and at subG1-phase at the 48-hour time point, suggesting that the mir-145 miRNA plays a role in mitosis and that cells making it through a first round of cell division may harbor errors that cause them to arrest before a new round of DNA replication.

By miRNA microarray analysis, expression levels of the mir-192-1 miRNA were observed to increase in the G2/M-phase of the cell cycle in HeLa cells, and treatment of HeLa cells with the oligomeric compounds ISIS Number 327902 (SEQ ID NO: 320), a uniform 2â€²-MOE compound, and ISIS Number 328383 (SEQ ID NO: 492), a chimeric 2â€²-MOE gapmer compound, targeted against the mir-192-1 miRNA and the mir-192-1 precursor, respectively, were observed to arrest or delay the cell cycle in the G2/M-phase at 24-hours after oligonucleotide treatment, and at both the Sâ€” and G2/M-phases at the 48-hour time point, suggesting that the mir-192 miRNA is involved in mitosis, and that cells making it through a first round of cell division may harbor errors that cause them to arrest during a new round of DNA replication. A uniform 2â€²-MOE oligomeric compound ISIS Number 338665 targeting the mir-192-1 precursor was also observed to induce a G2/M-phase arrest at both time points.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328744 (SEQ ID NO: 653), targeting the mir-182 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at 48-hours after oligonucleotide treatment, suggesting that the mir-182 miRNA plays a role in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328421 (SEQ ID NO: 530), targeting miR-30d was also observed to arrest or delay cells mainly in the G2/M-phase at the 24-hour time point and at both the S- and G2/M-phases at the 48-hour time point after oligonucleotide treatment, suggesting that the mir-30d miRNA is involved in mitosis, and that a cell division error arising from the first round of division may allow cells to pass through mitosis and initiate a second round of division, but then a cell cycle checkpoint is set off before the cells are able to complete DNA synthesis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328403 (SEQ ID NO: 512), targeting mir-106 was also observed to arrest or delay cells in the G2/M-phase at the 24-hour time point and at both the Sâ€” and G2/M-phases at the 48-hour time point after oligonucleotide treatment, suggesting that the mir-106 miRNA is involved in mitosis, and that a cell division error arising from the first round of division may allow cells to pass through mitosis and initiate a second round of division, but then a cell cycle checkpoint is set off before the cells are able to complete DNA synthesis. Interestingly, the cell cycle regulatory transcription factor E2F1 mRNA is reported to be a target of the mir-106 miRNA (Lewis et al., Cell, 2003, 115, 787-798).

Treatment of HeLa cells with the oligomeric compound ISIS Number 328759 (SEQ ID NO: 668), targeting the mir-216 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the mir-216 miRNA plays a role in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328342 (SEQ ID NO: 451), targeting the mir-203 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the mir-203 miRNA plays a role in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328707 (SEQ ID NO: 616), targeting miR-100-1 was also observed to arrest or delay cells mainly in the G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the miR-100-1 miRNA plays a role in regulating mitosis.

Dose Responsiveness:

In accordance with the present invention, certain oligomeric compounds targeting miRNAs were selected for dose response studies. Using the cell cycle assay described above, the cell cycle profiles of HeLa or A549 cells treated with varying concentrations of oligomeric compounds of the present invention were assessed.

HeLa cells were treated with 25-, 50-, 100- or 150 nM of the oligomeric compounds ISIS Numbers 327902 (SEQ ID NO: 320) and 328383 (SEQ ID NO: 492), both targeted against mir-192, and ISIS 327905 (SEQ ID NO: 323), targeting mir-205, and ISIS 328403 (SEQ ID NO: 512), targeting mir-106. Cells treated with increasing concentrations of oligomeric compounds were compared to untreated cells, to assess the dose-dependency of the observed delay or arrest. The random-mer ISIS 29848 was used as a negative control. Cells were prepared for flow cytometry 48-hours after oligonucleotide treatment, as described, supra. Oligomeric compounds targeted to miRNAs were tested in quadruplicate, and ISIS 29848 was tested in triplicate; data is presented as an average of the replicates. Results of these dose response studies are shown in Table 33, where data are expressed as percentage of cells in each phase.

TABLE 33Dose response of oligomeric compounds targetingmiRNAs on cell cycling (48 hours)Doseoligomeric% cells in cell cycle phaseISIS #compoundSubG1G1SG2/MAneuploidUntreatedâ€‚25 nM1.356242012controlâ€‚50 nM1.456242014(UTC)100 nM1.657241911150 nM1.657232015â€‚29848â€‚25 nM2.055252012â€‚50 nM1.556251912100 nM3.252282013150 nM4.248312115327902â€‚25 nM1.657231913â€‚50 nM2.451302014100 nM3.143302711150 nM6.336362812327905â€‚25 nM1.757241812â€‚50 nM2.150302012100 nM2.546302414150 nM4.538382412328383â€‚25 nM1.957251812â€‚50 nM1.356251813100 nM9.336343010150 nM11.829363411328403â€‚25 nM1.558241813â€‚50 nM1.153272014100 nM3.548292311150 nM8.237402413

From these data, it was observed that 48-hours after treatment of HeLa cells with increasing doses of each of these four oligomeric compounds targeting miRNAs, a dose-responsive delay or arrest resulted, exhibited as an increasing percentage of cells in the Sâ€” and G2/M-phases of the cell cycle. Concomittent decreases in the percentage of cells in G1-phase of the cell cycle and increases in the percentage of hypodiploid (subG1) cells were also observed. Likewise, a dose-responsive G2/M delay or arrest was observed in A549 cells treated with 25-, 50-, 100-, or 150 nM of the oligomeric compounds ISIS 327902, ISIS 328383 and ISIS Number 328342.

In a further study, A549 cells were treated with 25-, 50-, 100- or 150 nM of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a, and ISIS 340350 (SEQ ID NO: 855) targeted against mir-95 (Mourelatos). Cells treated with increasing concentrations of oligomeric compounds were compared to untreated cells, to assess the dose-responsiveness of the observed delay or arrest. The random-mer ISIS 29848 was used as a negative control. Cells were prepared for flow cytometry 24-hours after oligonucleotide treatment. Results of these dose response studies are shown in Table 34, where data are expressed as percentage of cells in each phase relative to the untreated control cells in that phase.

TABLE 34Dose response of oligomeric compounds targetingmiRNAs on cell cycling (24 hours)Doseoligomeric% cells in cell cycle phaseISIS #compoundSubG1G1SG2/MAneuploidâ€‚29848â€‚25 nM90121868776â€‚50 nM91116889390100 nM27212574112116150 nM5071267111984338637â€‚25 nM891009910199â€‚50 nM8611089107120100 nM6712673115146150 nM21612366144135338769â€‚25 nM6210194114101â€‚50 nM8211481122132100 nM13012475113157150 nM34111771145184338836â€‚25 nM76971039799â€‚50 nM2321138998111100 nM6811780116153150 nM17811769149114340350â€‚25 nM9110210095120â€‚50 nM1581286712680100 nM26712560155107150 nM40212840211108

From these data, it was observed that 24-hours after treatment of A549 cells with increasing doses of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a, and ISIS 340350 (SEQ ID NO:855) targeted against mir-95 (Mourelatos), a dose-responsive delay or arrest resulted, exhibited as an increasing percentage of cells in the G2/M-phases of the cell cycle. Concomittent decreases in the percentage of cells in S-phase of the cell cycle and increases in the percentage of hypodiploid (subG1) cells were also observed.

In further studies, additional cell lines were treated with oligomeric compounds targeted against miRNAs to assess the effects of each oligomeric compound on cell cycling. BJ, B 16, T47D, and HeLa cells were cultured and transfected as described above. T47D cells are deficient in p53. T47Dp53 cells are T47D cells that have been transfected with and selected for maintenance of a plasmid that expresses a wildtype copy of the p53 gene (for example, pCMV-p53; Clontech, Palo Alto, Calif.), using standard laboratory procedures. BJ cells were treated with 200 nM of each oligomeric compound, and T47D, T47Dp53, HeLa, and B16 cells were treated with 150 nM of each oligomeric compound. The human foreskin fibroblast BJ cell line represents a non-cancer cell line, while HeLa, T47D, T47Dp53 cells and the mouse melanoma cell line B16-F10 represent cancerous cell lines. For comparison, oligomeric compounds ISIS 183891 (SEQ ID NO: 847) and ISIS 285717 (TCGGTTCTTTCCAAGGCTGA; herein incorporated as SEQ ID NO: 857), both targeting the kinesin-like 1/Eg5 mRNA, involved in cell cycling, were used as positive controls. The random-mer ISIS 29848 was used as a negative control. Additionally, the oligomeric compounds ISIS Number 25690 (ATCCCTTTCTTCCGCATGTG; herein incorporated as SEQ ID NO: 858) and ISIS Number 25691 (GCCAAGGCGTGACATGATAT; herein incorporated as SEQ ID NO: 859), targeted to nucleotides 3051-3070 and 3085-3104, respectively, of the mRNA encoding the Drosha RNase III (GenBank Accession NM_â€”013235.2, incorporated herein as SEQ ID NO: 860) were also tested. ISIS Number 25690 and ISIS Number 25691 are 5-10-5 2â€²-MOE gapmer compounds, 20 nucleotides in length, with phosphorothioate internucleoside linkages throughout the oligomeric compound. All cytidine residues are 5-methylcytidines. Transfections were performed using the methods described herein. Cells were prepared for flow cytometry 48-hours after oligonucleotide treatment. Results of these studies are shown in Table 35, where data are expressed as percentage of cells in each phase relative to the untreated control cells in that phase.

TABLE 35Effects of oligomeric compounds targeting miRNAs on cell cycling (48 hours)% cells in cell cycle phaseCellSEQSublineISIS #ID NOtargetG1G1SG2/ManeuploidBJ29848737N/A18710099100105B1629848737N/A143989811099HeLa29848737N/A40383113106155T47D29848737N/A869511398155T47Dp5329848737N/A173121759793BJ183891847kinesin-like 1/eg542258173287158B16285717857kinesin-like 1/eg56277278220178HeLa183891847kinesin-like 1/eg512372295211161T47D183891847kinesin-like 1/eg5855584156161T47Dp53183891847kinesin-like 1/eg5351715318984HeLa25690858Drosha, RNAse III641198987140T47D25690858Drosha, RNAse III979780113140T47Dp5325690858Drosha, RNAse III19397108114144BJ25691859Drosha, RNAse III18394116125209B1625691859Drosha, RNAse III3161168399105HeLa25691859Drosha, RNAse III88153141113203T47D25691859Drosha, RNAse III12594104104203T47Dp5325691859Drosha, RNAse III212130669395HeLa338797624hypothetical14410489115125miRNA-144HeLa338666493hypothetical miRNA2149298130151039HeLa338833660hypothetical miRNA25587100136136181HeLa328707616mir-100-112510387122140BJ328403512mir-106811029592114B16328403512mir-106112111889992HeLa328403512mir-106891258980175T47D328403512mir-1064910411289175T47Dp53328403512mir-106140114879489HeLa341787852MiR-125a, Mouse3248896145177T47D328687596mir-130b14210192115169T47Dp53338769596mir-130b1161038712387B16327933351mir-14510410984116130BJ345395351mir-14513210097104115B16345395351mir-14514710687115150HeLa345395351mir-145871089695139BJ328744653mir-18212594111127158B16328744653mir-18215310887110115HeLa328744653mir-182105753110213178T47D328744653mir-182859087118191T47Dp53328744653mir-1829013059101100BJ327902320mir-192-191998810882B16327902320mir-192-11511128898101HeLa327902320mir-192-1941089693162T47D327902320mir-192-110275120116162T47Dp53327902320mir-192-11551009810297HeLa338665492mir-192-13229292142138HeLa328342451mir-203103968913896BJ327905323mir-20510510077109102B16327905323mir-2051421078910694HeLa327905323mir-205551089990164T47D327905323mir-2058197101103164T47Dp53327905323mir-20510911280104103HeLa338678505mir-20512910394105132BJ328759668mir-21616491117141160B16328759668mir-21613210491110126HeLa328759668mir-2167974082203223T47D328759668mir-2161238687122223T47Dp53328759668mir-2164239993108109HeLa327896314mir-229510394106144HeLa338836660mir-23a3039796121114HeLa328084743mir-30a2868992153125HeLa340350855mir-9513210194112177(Mourelatos)

When treatment of cells with oligomeric compounds resulted in greater than 750% cells in subG1 phase, these oligomeric compounds were deemed to be â€œhits,â€ in that they appear to cause an increase in apoptosis, resulting in hypodiploid DNA contents. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in G1-phase, these oligomeric compounds were deemed â€œhits,â€ as they appeared to cause an arrest or delay in G1-phase and/or blocked entry into S-phase of the cell cycle. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in S-phase, these oligomeric compounds were deemed â€œhits,â€ as they appeared to cause an arrest or delay in DNA synthesis. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in G2/M phase, these oligomeric compounds were deemed â€œhits,â€ as they appeared to cause an arrest or delay in the transition into mitosis, and/or in cell division, itself.

From these data, it was observed that 48-hours after treatment of the various cell lines with the oligomeric compounds, ISIS Number 183891 targeting the kinesin-like 1/Eg5 mRNA results in a delay or arrest in G2/M phase of the cell cycle for all cell lines. Treatment of HeLa cells with ISIS Number 25691, targeted against the Drosha RNase III mRNA, resulted in an increased percentage of cells in S-phase as well as a significant percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with DNA replication and/or maintenance of the integrity of the proper complement of genetic material.

In HeLa cells, ISIS 341787 (SEQ ID NO: 852) targeted against mir-125a (mouse), resulted in an arrest or delay in G2/M as well as an increased percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis.

In HeLa cells treated with ISIS 328744 (SEQ ID NO: 653) targeted against mir-182, an increase in the percentage of cells in the G2/M-phase of the cell cycle as well as in the subG1 category was observed, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis. Notably, genetically normal cells (BJ and T47Dp53 cells) were not affected by ISIS Number 328744, indicating that the oligomeric compound targeting miR-182 may selectively cause a cell cycle delay or arrest in cancer cells and not normal cells, and suggesting that this compound may be particularly useful as a therapeutic agent in the treatment of hyperproliferative disorders such as cancer.

In HeLa cells treated with ISIS 328759 (SEQ ID NO: 668) targeted against mir-216, a delay or arrest resulted in the G2/M-phase of the cell cycle was observed, as well as an increase in the percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis.

Thus, it was observed that treatment of HeLa cells with oligomeric compounds targeting miRNAs is a effective means of identifying compounds that can block progression through various stages of the cell cycle. Notably, a transient increase in G1-phase was observed 24 hours after treatment of HeLa cells with oligomeric compounds targeting miRNAs; for example, oligomeric compounds ISIS Numbers 338769, 338836, 340350, and 338637 caused a transient increase in the percentage of cells delayed or arrested in G1-phase at the 24-hour time point, which, by the 48-hour time point, had shifted to a delay or arrest in S-phase. It was also noted that multiple oligomeric compounds targeting the same miRNA have the same effect on cell cycling. It was also observed that uniform 2â€²-MOE as well as 2â€²-MOE chimeric gapmer oligomeric compounds targeting the mature miRNA, as well as uniform 2â€²-MOE oligomeric compounds targeting the pri-miRNA often have the same effect.

Oligomeric compounds that delay, arrest or prevent cell cycle progression or induce apoptosis may be useful as therapeutic agents for the treatment of hyperproliferative disorders, such as cancer, cancer, as well as diseases associated with a hyperactivated immune response.

It is understood that BJ, B16, HeLa, A549, HMECs, T47D, T47Dp53, MCF7 or other cell lines can be treated with oligomeric compounds designed to mimic miRNAs in studies to examine their effects on progression through the cell cycle. Such oligomeric compounds are within the scope of the present invention.

Example 25A Bioinformatic Approach to Identification of miRNA Targets

Several candidate RNA transcripts identified using the RACE-PCR methods described in Example 20 were the basis for a bioinformatic analysis of predicted targets bound to and/or potentially regulated by miRNAs. The complementarity between the miRNA used as a primer and the 3â€²-UTR of the RNA transcript identified by RACE-PCR was assessed using several methods. Transcripts identified by RACE-PCR were also analyzed using the FASTA sequence alignment program (accessible through the interne at, for example, www.ebi.ac.uk/fasta33) to find the best alignment between complementary sequences of the transcript and the miRNA used as a primer for RACE-PCR. When, using the default parameters, the FASTA alignment program resulted in the identification of the actual primer binding site (PBS) within the 3â€²-UTR of the RNA transcript as the sequence most complementary to the miRNA used as a primer in the RACE-PCR method, the candidate miRNA target transcript was specified by a plus sign (for example, see the â€œmir-143/PBS complementary?â€ column in Table 36 below). When the FASTA program failed to align the actual PBS with the sequence most complementary to the miRNA used in the RACE-PCR, the candidate miRNA target transcript was specified by a minus sign. When the FASTA program could be made to align with the sequence most complementary to the miRNA used in the RACE-PCR by decreasing the stringency of the FASTA program parameters, the candidate miRNA target transcript was specified by â€œÂ±â€ .

A global alignment was also performed to assess whether the sequence of the PBS within the RNA transcript identified by RACE-PCR was conserved between human and mouse orthologs of the RNA target. For example, in Table 36, below, strong conservation of PBS in the human and murine orthologs (homology from 80-100%) was indicated by a plus sign; moderate conservation (homology between 70-80%) was indicated by â€œÂ±â€ , and a minus sign indicates homology below 70%.

A variety of algorithms can be used to predict RNA secondary structures based on thermodynamic parameters and energy calculations. For example, secondary structure prediction can be performed using either M-fold or RNA Structure 2.52. M-fold can be accessed through the Internet at, for example, www.ibc.wustl.edu/-zuker/ma/form2.cgi or can be downloaded for local use on UNIX platforms. M-fold is also available as a part of GCG package. RNA Structure 2.52 is a windows adaptation of the M-fold algorithm and can be accessed through the Internet at, for example, 128.151.176.70/RNAstructure.html. The RNA Structure 2.52 program was used to analyze a series of 30-base fragments spanning the entire length of the human RNA transcript and their potential to hybridize with the miRNA used as a primer in the RACE-PCR, allowing the prediction of the lowest absolute free energy peak representing the most likely site of hybridization (including bulged regions) between the miRNA and the RNA target. If the free energy peak representing the hybridization between the miRNA and the PBS of the RNA transcript identified by RACE-PCR was among the top five peaks predicted by the RNA Structure 2.52 program, the transcript was given a plus sign, â€œ+â€ . If the free energy peak representing the hybridization between the miRNA and the PBS was in the top five to ten peaks predicted by RNA Structure 2.52, the transcript was given a plus/minus sign, â€œÂ±â€ , and if the peak representing the hybridization between the miRNA and the PBS was below the top ten peaks predicted by RNA Structure 2.52, the transcript was given a minus sign, â€œâˆ’â€ .

A list of the RNA transcript targets identified by RACE-PCR employing the mir-143 miRNA as a specific primer is shown in Table 36.

TABLE 36Potential RNA targets of the mir-143 miRNAmir-143/PBSSEQ IDRNA StructurecomplementaryRNA transcript targetNO:PBS conserved?peak??Matrix819+âˆ’+metalloproteinase 2Sec24829âˆ’+/âˆ’+Tripartite motif-828+/âˆ’++/âˆ’containing 32RAN824+/âˆ’++Cystatin B802âˆ’++Glucocorticoid839++/âˆ’+induced transcript 1Protein phosphatase 2809+++Polycystic kidney822âˆ’âˆ’âˆ’disease 2Mannose-6-phosphate801+/âˆ’âˆ’+receptorMitotic control817++âˆ’protein dis3 homologChromosome 14 ORF 103813++/âˆ’âˆ’Rho GDP dissociation823âˆ’âˆ’âˆ’inhibitor betaGlyoxalase I816+++Zinc finger protein818++/âˆ’+36, C3H type-like 1LIM domain only 4804+++

Note that four genes (Sec24, cystatin B, polycystic kidney disease 2, and Rho GDP dissociation inhibitor beta) did not have murine orthologs to compare in a global analysis of the PBS. Because these RNA transcripts were identified as being bound by the mir-143 miRNA used as a primer in the RACE-PCR approach previously described, the mir-143 miRNA is predicted to serve a regulatory role in expression or activity of one or more or all of these RNA transcripts. Of particular note are three targets, protein phosphatase 2, glyoxalase I, and LIM domain only 4 (LMO4) mRNAs, for which all three analyses yielded a positive result. That all three parameters assessed yielded a positive result suggests that these mRNAs are probable targets of mir-143.

The well-studied C. elegans lin-4 miRNA interaction with its lin-28 mRNA target was used as the starting point for a bioinformatics approach to the identification of miRNA binding sites in target nucleic acids. Lin-4 has been experimentally determined to bind at a single site on the lin-28 mRNA. Herein, as a primary determinant of miRNA-target interactions, it was hypothesized that the bimolecular hybridization free energies (Î”G^o₃₇) of the interaction of the miRNA with a true target site would be more negative than the Î”G^o₃₇of other interactions of the miRNA with other binding sites. The nucleotide sequence of the lin-28 mRNA was assessed by computationally deriving 30-nucleotide windows, starting with the first nucleotide of the sequence and defining the first nucleotide in each window by shifting 1 nucleotide in the 3â€² direction. Each window was assessed by hybridizing the 30-nucleotide sequence in the window with the lin-4 miRNA and disallowing unimolecular interactions, thereby spanning the entire length of the lin-28 mRNA, and the resulting Î”G^o₃₇value was plotted against the start position of the window. It was observed that the bimolecular hybridization between the true lin-4 binding site and the lin-28 mRNA had the lowest Î”G^o₃₇value, supporting our hypothesis and our bioinformatic approach to the identification of miRNA binding sites in target nucleic acids.

The mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM_â€”139032.1, incorporated herein as SEQ ID NO: 861) mRNA transcript was previously computationally predicted to be regulated by mir-143 miRNA binding in the 3â€²-UTR regions (Lewis et al., Cell, 2003, 115, 787-798). In order to identify mir-143 binding sites in the ERK5 mRNA, a bimolecular hybridization free energy assessment was performed by performing a hybridization walk to assess possible mir-143 binding sites along the entire length of the ERK5 mRNA. A strong negative Î”G^o₃₇value (âˆ’20.1) was found at the previously predicted mir-143 binding site in the 3â€²-UTR, lending further support to our method. Surprisingly, two additional, and novel, mir-143 binding sites with more negative Î”G^o₃₇values, as well as a third mir-143 binding site with a comparable Î”G^o₃₇value were also identified. Using the ERK5 sequence (GenBank Accession NM_â€”139032.1) as a reference, these binding sites encompass nucleotides 937-966 with a Î”G^o₃₇value of (âˆ’22.8), nucleotides 2041-2070 with a Î”G^o₃₇value of (âˆ’20.6) and nucleotides 2163-2192 with a Î”G^o₃₇value of (âˆ’19.3). See FIG. 1. Thus, three novel mir-143 binding sites (and, thus, a potential regulatory sites) were identified within the coding sequence of the ERK5 gene. Thus, this method of screening for miRNA binding sites by a bimolecular hybridization free energy assessment can be used to confirm previously predicted sites, and further allows the identification of novel miRNA target nucleic acid binding sites. It is believed that this method may more closely mimic the energetic mechanism by which a miRNA scans a target nucleic acid to find its interaction site. In subsequent experiments, the predicted mir-143 binding sites within the ERK5 coding sequence were also tested using the reporter system described below.

Example 26Northern Analysis of miRNA Expression

As described in the adipocyte differentiation assay, the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b, and ISIS Number 327876 (SEQ ID NO: 294), targeted to mir-29b-1, were found to reduce the expression of several hallmark genes of adipocyte differentiation, indicating that mir-23b and mir-29b-1 may play a role in adipocyte differentiation, and that oligomeric compounds targeting these miRNAs may be useful as agents blocking cellular differentiation. Therefore, the expression of mir-23b and mir-29b was assessed by Northern blot of total RNA from multiple tissues. To detect the mir-23b and mir-29b-1 targets, target specific DNA oligonucleotide probes with the sequences GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) and AACACTGATTTCAAA TGGTGCTA (SEQ ID NO: 294), respectively, were synthesized by IDT (Coralville, Iowa). The oligo probes were 5â€² end-labeled with T4 polynucleotide kinase with (Î³-³²P) ATP (Promega). To normalize for variations in loading and transfer efficiency membranes are stripped and probed for U6 RNA. Total RNA from mouse and human tissues as well as total RNA from human adipocytes and HepG2 cells was probed in Northern blot analyses, using methods described in Example 14.

By Northern analyses, the mir-23b miRNA was found to be most highly expressed in human kidney tissue as well as in adipose tissue from ob/ob mice, and was also highly expressed in human liver, adipocytes, preadipocytes and HepG2 cells. Moderate expression of mir-23b was also noted in murine kidney tissue. The mir-29b-1 miRNA was found to be most highly expressed in human and mouse kidney, and was also expressed in human liver, adipocytes, preadipocytes, and HepG2 cells, as well as in murine adipose tissue and liver. Levels of both the mir-23b and mir-29b-1 miRNAs were also found to be upregulated in human differentiated adipocytes.

Similarly, target specific DNA oligonucleotide probes for mir-16, mir-15a, and let-7a were designed and used in Northern blot analyses to assess expression of these miRNAs in human and mouse tissues. The mir-16 and mir-15a miRNAs were each found to be most highly expressed in human spleen, heart, testes, and kidney, and expression was also observed in liver as well as HEK293 and T47D cells. Additionally, low levels of expression of the mir-16 miRNA were observed in NT2 cells. The let-7a miRNA was most highly expressed in human and murine kidney, and expression was also observed in human and murine liver. Additionally, low levels of let-7a expression were found in HepG2 cells.

To detect the mir-21 miRNA in Northern blot analyses, a target specific DNA oligonucleotide probe with the sequences TCAACATCAGTCTGATAAGCTA (SEQ ID NO: 335) was synthesized by IDT (Coralville, Iowa). The oligo probes were 5â€² end-labeled with T4 polynucleotide kinase with (Î³-³²P) ATP (Promega). Twenty micrograms of total RNA from human Promyelocytic Leukemia HL-60 cells, A549, HeLa, HEK293, T47D, HepG2, T-24, MCF7, and Jurkat cells was fractionated by electrophoresis through 15% acrylamide urea gels using a TBE buffer system (Invitrogen). RNA was transferred from the gel to HYBONDâ„¢-N+nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by electroblotting in an Xcell SureLockâ„¢ Minicell (Invitrogen). Membranes were fixed by UV cross-linking using a STRATALINKERâ„¢ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hyb buffer solution (Amersham) using manufacturer's recommendations for oligonucleotide probes. To normalize for variations in loading and transfer efficiency membranes are stripped and probed for U6 RNA. High levels of expression of mir-21 were observed in A549 and HeLa cells; in fact, levels of mir-21 expression were noted to be among the highest of any of the miRNAs observed in HeLa cells.

Example 27Reporter Systems for Assaying Activity of Oligomeric Compounds Targeting or Mimicking miRNAs

Reporter systems have been developed herein to assess the ability of miRNA mimics to provoke a gene silencing response and to assess whether antisense oligomeric compounds targeting miRNAs can inhibit gene silencing activity. The T-RExâ„¢-HeLa cell line (Invitrogen Corp., Carlsbad, Calif.) was used for either stable or transient transfections with plasmids constitutively expressing miRNAs, pre-miRNAs, pri-miRNAs or mimics thereof, and, in some cases, antisense oligomeric compounds targeting the expressed miRNA were also transfected into the cells. It is understood that other mammalian cells lines can also be used in this reporter system. T-RExâ„¢-HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.), supplemented with 10% fetal bovine serum (Invitrogen Corporation). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were harvested when they reached 90% confluence, and on the day before transfection with expression or reporter plasmids (described in detail below), the T-RExâ„¢-HeLa cells were seeded onto 24-well plates at 50,000 cells/well. The following day, cells were transfected according to standard published procedures with various combinations of plasmids using 2 Î¼g Lipofectamineâ„¢ 2000 Reagent (Invitrogen) per Î¼g of plasmid DNA. When transfecting oligomeric compounds, 1-3 Î¼g of Lipofectamineâ„¢ 2000 Reagent was used per 100 nM oligomeric compound.

Plasmids used are as follows: The pcDNA3.1Â©/NT-GFP (Invitrogen) plasmid, containing a CMV promoter controlling expression of a GFP reporter sequence at the N-terminus of the transcription start site was used as a control plasmid. The pcDNA3.1Â©/NT-GFP-mir-143 sensor plasmid contains (in addition to the elements above) three 22-nucleotide sites encoding the mir-143 miRNA binding site, downstream of the GFP coding sequence and upstream of the polyadenylation signal. The pCR3-pri-mir-143 plasmid (â€œpri-mir-143â€ ) is a CMV promoter-driven constitutive expression plasmid which expresses the 110-nucleotide mir-143 pri-miRNA sequence (SEQ ID NO: 38), to act as a mir-143 pri-miRNA mimic. The pCR3-pri-mir control (â€œpri-mir-controlâ€ ) is a CMV promoter-driven constitutive expression plasmid which is designed to express a similar 110-nucleotide pri-miRNA sequence (AGCAGCGCAGCGCCCTGTCTCCCAGCCAAGGTGGAACCTTCTGGGA AGCGGTCAGTTGGGAGTCCCTTCCCTGAAGGTTCCTCCTTGGAAGAGAGAAGTTGTTCTG CAGC; SEQ ID NO: 862) wherein the mature mir-143 sequence has been replaced with an unrelated sequence and the predicted complementary strand opposite it within the pri-miRNA structure is replaced with a nearly complementary sequence in order to preserve the stem loop as well as the bulge structure of the natural mir-143 pri-miRNA. Additionally, in order to test the effect of an oligomeric compound targeting a miRNA, the T-RExâ„¢-HeLa cells were also transfected with the uniform 2â€²-MOE phosphorothioate (PS) antisense oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143 previously described.

Twenty-four hours post-transfection, cells were trypsinized and GFP fluorescence was analyzed by flow cytometry. Results are shown in Table 37.

TABLE 37Mean GFP fluorescence after transfection of T-RExâ€‰â„¢-HeLa cellsTreatmentMeanpri-mirGFP mir-143327901fluores-controlpri-mir-143GFP controlsensoroligocenceâˆ’âˆ’âˆ’âˆ’âˆ’2.2+âˆ’âˆ’âˆ’âˆ’2.7âˆ’+âˆ’âˆ’âˆ’2.6âˆ’âˆ’+âˆ’âˆ’7.9+âˆ’+âˆ’âˆ’22.7âˆ’++âˆ’âˆ’9.6âˆ’âˆ’âˆ’+âˆ’12.4+âˆ’âˆ’+âˆ’21.8âˆ’+âˆ’+âˆ’5.3âˆ’+âˆ’++4.1âˆ’âˆ’âˆ’++4.2âˆ’+âˆ’âˆ’+3.7

Plus signs, â€œ+â€ , indicate the presence of the expression plasmid or oligomeric construct in transfectants; minus signs â€œâˆ’â€ , indicate the absence of same. Mean fluorescence is measured in arbitrary units.

In cells transfected with the sensor plasmid and expressing the mir-143 pri-miRNA mimic from the pCR3-pri-mir-143 plasmid, the mir-143 miRNA is expected to be processed endogenously, allowing it to bind as a mature miRNA to the RNA transcript encoding GFP and containing the mir-143 binding sites expressed from the reporter plasmid, resulting in cleavage of the reporter transcript and a decrease in fluorescence as compared to the control plasmid. From the data shown in Table 37, it was observed that expression of the pCR3-pri-mir-143 plasmid results in an inhibition of expression of GFP indicated by a decrease in fluorescence produced by the pcDNA3.1Â©/NT-GFP-mir-143 sensor plasmid, whereas expression of the pCR3-pri-mir control plasmid had no effect on GFP reporter expression. Neither the pCR3-pri-mir control nor the pCR3-pri-mir-143 plasmid had any inhibitory effect on GFP expression from the pcDNA3.1Â©/NT-GFP control plasmid. Thus, the mir-143 pri-miRNA mimic oligomeric compound silences the expression of RNA transcribed from a reporter plasmid containing mir-143 target sites.

In a further study, T-RExâ„¢-HeLa cells transfected with the pcDNA3.1Â©/NT-GFP-mir-143 sensor plasmid were treated at various dosages with the following oligomeric compounds: 1) a double-stranded RNA oligomeric compound (â€œds-mir-143â€ ) composed of ISIS Number 342199 (TGAGATGAAGCACTGTAGCTCA; SEQ ID NO: 220) representing the mir-143 sense sequence, hybridized to its perfect complement ISIS Number 342200 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319); 2) a negative control dsRNA (â€œds-Controlâ€ ), representing a 10-base mismatched sequence antisense to the unrelated PTP1B mRNA, composed of ISIS Number 342427 (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 863) hybridized to its perfect complement ISIS Number 342430 (GGAGGAACCTTCAGGGAAGG; SEQ ID NO: 864); 3) the pCR3-pri-mir-143 expression plasmid (â€œpCR3-pri-mir-143â€ ) which expresses the 110-nucleotide mir-143 pri-miRNA; 4) the pCR3-pri-mir control (â€œpri-mir-controlâ€ ); 5) an in vitro transcribed RNA oligomeric compound (â€œhairpin mir-143â€ ) representing the 110 bp fragment of the mir-143 pri-miRNA molecule (SEQ ID NO: 38) plus an additional two cytosine nucleobases from the T7 promoter at the 5â€² end; and 6) an in vitro transcribed RNA oligomeric compound (â€œhairpin controlâ€ ) (SEQ ID NO: 862) representing a similar hairpin structure except that the mature mir-143 sequence and its complementary sequence within the pri-miRNA hairpin structure were replaced with sequences unrelated to mir-143. The RNA hairpin oligomeric compounds were in vitro transcribed using the MAXIscript Kit (Ambion Inc., Austin, Tex.) according to the manufacturer's protocol, beginning with a DNA template amplified by PCR. GFP fluorescence of treated cells was assessed using the methods described above, and it was observed that the ds-mir-143 oligomeric compound mimic inhibited expression of GFP from the sensor plasmid in a dose dependent manner. In a further embodiment, pcDNA3.1Â©/NT-GFP-mir-143 sensor-expressing cells treated with 20 nM mir-143 dsRNA oligomeric compound were additionally treated with 4-, 20- or 100 nM uniform 2â€²-MOE oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), or 4-, 20- or 100 nM uniform 2â€²-MOE scrambled mir-143 control ISIS Number 342673 (SEQ ID NO: 758) to assess the ability of compounds to inhibit the inhibitory effect of the mir-143 dsRNA mimic. At all three concentrations, the oligomeric compound ISIS Number 327901 targeting mir-143 blocked the inhibitory effect of the mir-143 dsRNA oligomeric compound, exhibited as a recovery of GFP fluorescence.

In one embodiment, an expression system based on the pGL3-Control (Promega Corp., Madison Wis.) vector containing a CMV promoter controlling expression of a luciferase reporter sequence was used in transient transfections of HeLa cells with plasmids expressing miRNA or pri-miRNA mimics. To assess the ability of miRNA mimics to bind and regulate the expression of the luciferase reporter gene, two reporter plasmids were constructed: 1) a synthetic DNA fragment comprising two sites perfectly complementary to mir-143 were inserted into the pGL3-Control luciferase reporter vector, to create the pGL3-mir-143 sensor plasmid, and 2) a DNA fragment comprising the 3â€²-UTR of the LIM domain only 4 (LMO4) gene (predicted to be regulated by mir-143) was inserted into pGL3-Control to create pGL3-LMO4; this fragment was PCR-amplified using a primer beginning at position 1261 of the LMO4 sequence (GenBank Accession NM_â€”006769.2, incorporated herein as SEQ ID: 809) and the downstream primer hybridizing to the poly-A tail. In each of these plasmids, the target site was placed downstream of the luciferase coding sequence and upstream of the polyadenylation signal in the 3â€²-UTR of the luciferase reporter vector. The unmodified pGL3-Control luciferase reporter vector was used as a control.

HeLa cells were routinely cultured and passaged as described, and on the day before transfection with expression or reporter plasmids, the HeLa cells were seeded onto 24-well plates 50,000 cells/well. Cells were transfected according to standard published procedures with various combinations of plasmids using 2 Î¼g Lipofectamineâ„¢ 2000 Reagent (Invitrogen) per Î¼g of plasmid DNA, or, when transfecting oligomeric compounds, 1.25 Î¼g of Lipofectamineâ„¢ 2000 Reagent per 100 nM oligonucleotide or double-stranded RNA. The luciferase signal in each well was normalized to the Renilla luciferase (RL) activity produced from a co-transfected plasmid, pRL-CMV, which was transfected at 0.5 Î¼g per well. Cells were treated at various dosages (4 nM, 20 nM, and 100 nM) with the following oligomeric compound mimics: 1) â€œds-mir-143,â€ 2) â€œds-Control,â€ 3) â€œpCR3-pri-mir-143,â€ or 4) â€œpri-mir-control,â€ as described supra. In accordance with methods described in Example 12, supra, a luciferase assay was performed 48-hours after transfection. Briefly, cells were lysed in passive lysis buffer (PLB; Promega), and 20 ul of the lysate was then assayed for RL activity using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are an average of three trials and are presented as percent pGL3-Control luciferase expression normalized to pRL-CMV expression (RL). The data are shown in Table 38.

TABLE 38Luciferase assays showing effects of oligomericcompounds mimicking mir-143luciferase expression(% lucif. only control)pGL3-pGL3-pGL3-treatmentControlmir-143 sensorLMO4no luciferase (pRL)0.30.30.4luciferase (pRL) only100.0101.0100.0ds-mir-143 (4 nM)101.514.5151.6ds-mir-143 (20 nM)123.88.0140.1ds-mir-143 (100 nM)131.87.1128.4ds-Control (4 nM)133.6144.5172.4ds-Control (20 nM)126.1169.8151.6ds-Control (100 nM)123.0151.3151.5pCR3-pri-mir-143 (0.25 ug)75.658.6101.9pCR3-pri-mir-14376.650.795.7precursor (0.5 ug)pCR3-pri-mir-143 (1 ug)64.735.082.5pri-mir control (0.25 ug)90.378.3114.8pri-mir control (0.5 ug)57.361.895.4pri-mir control (1 ug)67.964.974.8

From these data, it was observed that the mir-143 dsRNA oligomeric compound and the mir-143 pri-miRNA mimic expressed from the pCR3-pri-mir-143 expression plasmid both inhibited luciferase activity from the pGL3-mir-143 sensor plasmid in a dose-dependent manner.

In another embodiment, HeLa cells were transfected with 0.03 Î¼g pGL3-mir-143 sensor plasmid and 0.01 Î¼g pRL-CMV plasmid, and, in addition, (except those samples described below as â€œwithout mir-143 pri-miRNA,â€ ) were also transfected with 0.01 Î¼g of an expression plasmid designed to express a mir-143 pri-miRNA mimic comprising a larger 430-nt fragment of the mir-143 primary miRNA transcript, referred to as â€œpCR3-pri-mir-143 (430)â€ (AGGTTTGGTCCTGGGTGCTCAAATGGCAGGCCACAGACAGGAAACACAG TTGTGAGGAATTACAACAGCCTCCCGGCCAGAGCTGGAGAGGTGGAGCCCAGGTCCCCT CTAACACCCCTTCTCCTGGCCAGGTTGGAGTCCCGCCACAGGCCACCAGAGCGGAGCAG CGCAGCGCCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGGTCAGTTGGGAGTCT GAGATGAAGCACTGTAGCTCAGGAAGAGAGAAGTTGTTCTGCAGCCATCAGCCTGGAAG TGGTAAGTGCTGGGGGGTTGTGGGGGGCCATAACAGGAAGGACAGAGTGTTTCCAGACT CCATACTATCAGCCACTTGTGATGCTGGGGAAGTTCCTCTACACAAGTTCCCCTGGTGCC ACGATCTGCTTCACGAGTCTGGGCA; SEQ ID NO: 871). It was observed that the mir-143 pri-miRNA mimic expressed by pCR3-pri-mir-143 (430) inhibits luciferase expression from the pGL3-mir-143 sensor plasmid. To further evaluate the ability of the mir-143 pri-miRNA mimic to inhibit luciferase activity from the sensor plasmid, and to assess the ability of oligomeric compounds to interfere with the inhibition of pGL3-mir-143 sensor luciferase expression by the mir-143 pri-miRNA mimic, pGL3-mir-143 sensor-expressing HeLa cells treated with pCR3-pri-mir-143 (430) were additionally treated with varying concentrations (0-, 6.7- or 20 nM) of the following oligomeric compounds: 1) ISIS Number 327901 (SEQ ID NO: 319), a uniform 2â€²-MOE oligomeric compound targeting mir-143; 2) ISIS Number 342673 (SEQ ID NO: 758), a uniform 2â€²-MOE scrambled control; or 3) ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA (mir-129-2). ISIS Numbers 342673 and 327924 were used as negative controls. HeLa cells transfected with the pRL-CMV and pGL3-mir-143 sensor plasmids, but not treated with the pCR3-pri-mir-143 (430) hairpin precursor served as a control. In this experiment, the luciferase assay was performed 24-hours after transfection. The data are presented in Table 39 as relative luciferase activity (normalized to RL expression levels). Where present, â€œN.D.â€ indicates â€œno data.â€

TABLE 39Effects of oligomeric compounds on mir-143 pri-miRNAmimic-mediated inhibition of luciferase expressionRelative luciferase activitySEQ IDDose of oligomeric compoundTreatmentNO0 nM6.7 nM20 nM3279013190.974.06.43426737580.971.31.5negative control3279243420.970.81.2negative controlwithout pCR3-pri-mir-143(430)N/A13.8N.D.N.D.

From these data, it was observed that the oligomeric compound ISIS Number 327901 targeting mir-143 blocked the inhibitory effect of the mir-143 pri-miRNA mimic, exhibited as a 4- to 6.4-fold recovery of luciferase activity in HeLa cells expressing the pGL3-mir-143 sensor plasmid. More than four-hundred target genes have been predicted to be regulated by miRNA binding to the 3â€²-UTR regions of the mRNA transcript (Lewis et al., Cell, 2003, 115, 787-798). For example, at least six genes have been reported to bear regulatory sequences in their 3â€²-UTRs which are predicted to be bound by the mir-143 miRNA; these include the inwardly rectifying potassium channel Kir2.2 (GenBank Accession AB074970, incorporated herein as SEQ ID NO: 872), synaptotagmin III (GenBank Accession BC028379, incorporated herein as SEQ ID NO: 873), mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM_â€”139032.1, SEQ ID NO: 861), protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB) (GenBank Accession NM_â€”004156.1, SEQ ID NO: 814), glyoxalase I (GLO1) (GenBank Accession NM_â€”006708.1, SEQ ID NO: 821), and LIM domain only 4 (LMO4) (GenBank Accession NM_â€”006769.2, SEQ ID NO: 809). It should be noted that one third of miRNA targets predicted in the study by Lewis, et al. are expected to be false positives (Lewis et al., Cell, 2003, 115, 787-798).

Because the present inventors independently identified the PPP2CB and GLO1 genes as potential targets of mir-143 by the RACE-PCR methods as described in Example 20, these targets were selected for further study. In addition, and described in Example 25, a novel mir-143 binding site (and, thus, a potential regulatory site) was identified within the coding sequence of the ERK5 gene; this predicted mir-143 binding site within the ERK5 coding sequence was also tested in these reporter systems.

In some embodiments, an expression system based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising predicted miRNA binding sites was used in stable transfections of HeLa cells, selecting for cells that have integrated the reporter plasmid into their genome. Because pGL3-based reporter vectors have no selectable marker for antibiotic resistance, a neomycin-resistance (Genetecin) gene was cloned into the pCR2 plasmid (Invitrogen Life Technologies, Carlsbad, Calif.) to create the pCR2-neo plasmid, and pCR2-neo was co-transfected into HeLa cells with the pGL3-mir-143-sensor plasmid at a ratio of one pCR2-neo plasmid to ten pGL3-mir-143-sensor plasmids. Co-transfected cells were then selected for the presence of the Genetecin marker and assayed for luciferase activity; Genetecin-resistant cells are very likely to have also integrated the luciferase reporter into their genome.

Establishment of Stably-Transfected Cells:

One day prior to transfection, approximately 750,000 HeLa cells are seeded onto a 10 cm dish or T-75 flask and grown in complete medium overnight at 37Â° C. The next day, 10 Î¼g of pGL3-mir-143-sensor plasmid and 1 Î¼g pCR2-neo are mixed in 2 ml OPTI-MEMâ„¢ (Invitrogen Corporation, Carlsbad, Calif.). (Linearization of circular plasmids by digestion with restriction enzyme may increase the number of stable transfectants per Î¼g transforming DNA, but is not an essential step). 10 Î¼l LIPOFECTINâ„¢ reagent (Invitrogen Corporation, Carlsbad, Calif.) is mixed with 2 ml OPTI-MEMâ„¢. The plasmid/OPTI-MEMâ„¢ and OPTI-MEMâ„¢/LIPOFECTINâ„¢ mixtures are then mixed together, and an additional 11 ml OPTI-MEMâ„¢ is added, and the resulting 15 ml cocktail is added to the cells. Cells are incubated in the plasmid/OPTI-MEMâ„¢/LIPOFECTINâ„¢ cocktail for approximately 4 hours at 37Â° C., after which the cocktail is removed and replaced with fresh complete medium. The following day, cells are trypsinized and transferred to a T-175 flask. Media containing the selection agent, 500 Î¼g/ml G418 (Geneticin; GIBCO/Life Technologies, Gaithersburg, Md.), is added and cells are grown at 37Â° C. Cells are re-fed daily with fresh media containing the selection agent until the majority of the cells appear to have died off and isolated colonies of neomycin-resistant cells appear. In cases where subcloning is desired, selected neomycin-resistant cells are trypsinized and plated at a concentration of 0.5 cells/well in 96-well plates, maintaining the cells in 500 Î¼g/ml G418 selection media.

In one embodiment, five stably-transfected, neomycin-resistant, luciferase-positive, pGL3-mir-143-sensor cell clones were isolated, subcloned and selected for further testing with oligomeric compounds of the present invention. Cells stably expressing the luciferase reporter and comprising one or more miRNA binding sites were then transfected with oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in order to assess the ability of these miRNA mimics to bind and regulate the expression of the luciferase reporter.

An expression system based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising predicted miRNA binding sites was used in transient transfections of HeLa cells with plasmids expressing oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in order to assess the ability of these miRNA mimics to bind and regulate the expression of the luciferase reporter. The effect of increasing the copy number of the miRNA-binding site in the target was also tested by including multiple binding sites in artificial reporter constructs. It is understood that the presence of multiple miRNA-binding sites in a target can include binding sites for different miRNAs.

The following reporter plasmids were constructed by cloning the specified fragment into the XbaI site of the pGL3-control plasmid, placing the potential miRNA-binding site in the 3â€²-UTR of the luciferase reporter: The reporter plasmid pGL3-bulge(Ã—3) contains three contiguous copies of the sequence (TGAGCTACAGCTTCATCTCA; herein incorporated as SEQ ID NO: 874) which represents a sequence complementary to the mir-143 miRNA except that it is missing 2 nucleotides such that the mir-143 miRNA is presumed to adopt a bulged structure when it hybridizes to this target sequence. The pGL3-GLO1 reporter plasmid contains a DNA fragment comprising the 3â€²-UTR of the GLO1 sequence; this fragment was PCR-amplified using a primer beginning at nucleotide number 621 of the GLO1 sequence (GenBank Accession NM_â€”006708.1, SEQ ID NO: 821) and the downstream primer hybridizing to the poly A tail. The pGL3-PP2A reporter plasmid contains a DNA fragment comprising the 3â€²-UTR of the PP2A gene; this fragment was PCR-amplified using a primer beginning at nucleotide number 921 of the PP2A sequence (GenBank Accession NM_â€”004156.1) and the downstream primer hybridizing to the poly A tail. The reporter plasmid pGL3-ERK5-3â€²-UTR(Ã—1) contains one copy of the sequence TATTCTGCAGGTTCATCTCAG (herein incorporated as SEQ ID NO: 875), found in the 3â€²-UTR of ERK5 and predicted by Lewis, et al. to be bound by the mir-143 miRNA, and the reporter plasmid pGL3-ERK5-3â€²UTR(Ã—3) has three contiguous copies of this sequence. The reporter plasmid pGL3-ERK5-3â€²UTR(ext) contains one copy of the sequence CGGCTTGGATTATTCTGCAGGTTCATCTCAGACCCACCTTT (herein incorporated as SEQ ID NO: 876), which includes an additional ten nucleotides at either end of the mir-143 binding site in 3â€²-UTR of ERK5 predicted by Lewis, et al. (Lewis et al., Cell, 2003, 115, 787-798). The plasmids pGL3-ERK5-cds(Ã—1), pGL3-ERK5-cds(Ã—2), pGL3-ERK5-cds(Ã—3), and pGL3-ERK5-cds(Ã—5) contain one, two, three or five contiguous copies, respectively, of the novel mir-143 binding site (TTGAGCCCAGCGCTCGCATCTCA; herein incorporated as SEQ ID NO: 877) we identified within the coding sequence of ERK5. The unmodified pGL3-Control luciferase reporter vector was used as a negative control, and the pGL3-mir-143 sensor reporter plasmid was used as a positive control.

HeLa cells were routinely cultured and passaged as described. In some embodiments, HeLa cells were transfected with 0.05 Î¼g of the relevant pGL3-sensor plasmid and 0.01 Î¼g pRL-CMV plasmid. Additionally, in some embodiments, cells were treated at various dosages (11 nM, 33 nM, and 100 nM) with the following oligomeric compound mimics: 1) ds-mir-143, or 2) ds-Control as described. In accordance with methods described in Example 12, a luciferase assay was performed 24-hours after transfection. The results, shown in Tables 40 and 41, are an average of three trials. Data are presented as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 40Effects of oligomeric compounds mimickingmir-143 on luciferase expressionReporterds-mir-143ds-controlplasmid11 nM33 nM100 nM11 nM33 nM100 nMpGL3-Control90.794.272.5113.479.687.0pGL3-bulge50.735.417.2111.382.684.7(x3)pGL3-ERK5-81.984.762.2103.279.677.63â€²UTR(x1)

From these data, it was observed that, while treatment of HeLa cells expressing the reporter plasmids with the ds-control did not appear to significantly affect luciferase expression, the mir-143 dsRNA mimic compound inhibited luciferase activity from the pGL3-bulge(Ã—3) sensor plasmid in a dose-dependent manner.

TABLE 41Effects of oligomeric compounds mimickingmir-143 on luciferase expressionds-mir-143ds-control11331001133100Reporter plasmidnMnMnMnMnMnMpGL3-Control110.2124.392.3114.195.6103.0pGL3-mir-143 sensor15.015.011.1114.5108.997.1pGL3-bulge(x3)36.133.922.2109.5103.292.4pGL3-ERK5-3â€²UTR(x1)92.2108.181.9106.299.690.1pGL3-ERK5-3â€²UTR(x3)51.751.028.2104.6103.495.7pGL3-ERK5-cds(x1)101.3115.477.4100.6102.196.2pGL3-ERK5-cds(x2)92.7113.863.6111.399.290.4pGL3-ERK5-cds(x3)73.577.949.4105.296.679.9pGL3-ERK5-cds(x5)49.444.523.9103.0113.489.9pGL3-ERK5-3â€²UTR(ext)89.0106.781.496.8108.989.4

From these data it was observed that treatment of HeLa cells expressing the pGL3-bulge(Ã—3) reporter plasmid with the ds-mir-143 miRNA mimic oligomeric compound resulted in a dose-dependent inhibition of luciferase activity while the ds-control oligomeric compound had no effect as described previously. Treatment of HeLa cells expressing the pGL3-ERK5-3â€²UTR(Ã—1) (containing one copy of the mir-143 binding site predicted by Lewis, et al.) with the ds-mir-143 mimic oligomeric compound did not inhibit luciferase activity, although increasing the number of potential mir-143 binding sites in the pGL3-ERK5-3â€²UTR(Ã—3) reporter plasmid to three appeared to favor the binding of the ds-mir-143 mimic and inhibition of luciferase activity. Treatment of cells expressing the pGL3-ERK5-cds(Ã—1) or pGL3-ERK5-cds(Ã—2) reporter plasmids bearing a one or two copies, respectively, of the novel mir-143 binding site identified in the coding sequence of the ERK5 gene with 11- or 33 nM of the ds-mir-143 mimic oligomeric compound did not appear to inhibit luciferase activity, although treatment with 100 nM of the ds-mir-143 mimic did reduce luciferase expression. Treatment of cells expressing the pGL3-ERK5-cds(Ã—3) or pGL3-ERK5-cds(Ã—5) reporter plasmids, bearing three or five of copies, respectively, of the novel mir-143 binding site in the ERK5 coding sequence, with the ds-mir-143 mimic oligomeric compound resulted in a reduction in luciferase activity. The pGL3-ERK5-cds(Ã—5) reporter plasmid exhibited a dose-responsiveness with increasing concentration of the mir-143 mimic oligomeric compound. Taken together, these results support the conclusion that multiple miRNAs and miRNA binding sites may cooperate to silence gene expression.

In order to assess the ability of miRNAs to bind predicted miRNA binding sites and regulate the expression of the luciferase reporter, in some embodiments, expression systems based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising either a mir-15a, mir-21, or a mir-23b miRNA binding site were developed and used in transient transfections of HeLa cells to determine whether the endogenous mir-15a, mir-21, or mir-23b miRNAs, respectively, could repress luciferase reporter gene expression.

The pGL3-mir-15a sensor plasmid was created by cloning the sequence (CACAAACCATTATGTGCTGCTA; SEQ ID NO: 369), complementary to the mir-15a miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3â€²UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-15a miRNA was able to inhibit luciferase expression from the pGL3-mir-15a sensor plasmid. Thus, to further evaluate the ability of the mir-15a miRNA to bind this target site encoded by the pGL3-mir-15a sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-15a-mediated silencing, pGL3-mir-15a sensor-expressing HeLa cells were treated with varying concentrations (3-, 10- or 30 nM) of the following oligomeric compounds: ISIS Number 327951 (SEQ ID NO: 369) is a uniform 2â€²-MOE compound targeting the mature mir-15a-1 miRNA. ISIS Numbers 356213 (SEQ ID NO: 878), 356215 (SEQ ID NO: 879), 356216 (SEQ ID NO: 880), 356218 (SEQ ID NO: 881), 356221 (SEQ ID NO: 882), 356227 (SEQ ID NO: 883) and 356229 (SEQ ID NO: 884) are phosphorothioate, uniform 2â€²-MOE oligomeric compounds designed and synthesized to target the entire length of the mir-15a pri-miRNA molecule (described in detail in Example 28, below). The uniform 2â€²-MOE phosphorothioate oligomeric compounds ISIS Number 327901 (SEQ ID NO: 319), targeting an unrelated miRNA (mir-143) and ISIS Number 342673 (AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), containing 15 mismatches with respect to the mature mir-143 miRNA, were used as negative controls. The data presented in Table 42 are the average of three trials and are presented as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 42Effects of oligomeric compounds on mir-15a miRNA-mediated inhibition of luciferase expressionRelative luciferase activitySEQ IDDose of oligomeric compoundTreatmentNO3 nM10 nM30 nM32790131983.696.688.2negative control342673758104.582.685.7negative control327951369151.0207.6137.1356213878101.280.5109.935621587998.0116.779.6356216880102.884.7113.235621888191.6110.385.7356221882106.874.081.235622788386.1117.8101.5356229884109.7100.397.5

From these data, it was observed that the oligomeric compound ISIS Number 327951 targeting the mature mir-15a miRNA blocked the inhibitory effect of mir-15a, exhibited as a recovery and increase in luciferase activity in HeLa cells expressing the pGL3-mir-15a sensor plasmid.

The pGL3-mir-23b sensor plasmid was created by cloning the sequence (GTGGTAATCCCTGGCAATGTGAT; SEQ ID NO: 307), representing a sequence complementary to the mir-23b miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3â€²UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-23b miRNA was able to inhibit luciferase expression from the pGL3-mir-23b sensor plasmid. Thus, to further evaluate the ability of the mir-23b miRNA to bind this target site encoded by the pGL3-mir-23b sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-23b-mediated silencing, pGL3-mir-23b sensor-expressing HeLa cells were treated with varying concentrations (1.3-, 5- or 20 nM) of the following oligomeric compounds: ISIS Number 327889 (SEQ ID NO: 307), a phosphorothioate uniform 2â€²-MOE oligomeric compound, and ISIS Number 340925 (SEQ ID NO: 307), a 2â€²-MOE 5-10-8 gapmer oligomeric compound, both targeting mir-23b. The uniform 2â€²-MOE phosphorothioate oligomeric compound ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA (mir-129-2) was used as a negative control. The data are the average of three trials, and are presented in Table 43 as relative luciferase activity (normalized to pRL-CMV luciferase plasmid only, not treated with oligomeric compound).

TABLE 43Effects of oligomeric compounds on mir-23b miRNA-mediated inhibition of luciferase expressionFold change luciferaseSEQ IDDose of oligomeric compoundTreatmentNO1.3 nM5 nM20 nM3279243421.150.680.92negative control327889-uniform MOE3073.753.467.40340925-gapmer3070.991.411.19

From these data, it was observed that, at all doses, ISIS Number 327889, the uniform 2â€²-MOE oligomeric compound targeting the mature mir-23b miRNA, de-repressed the expression of the luciferase reporter. Thus, ISIS 327889 reversed the silencing effect of the mir-23b miRNA, apparently by inhibiting the binding of mir-23b to its target site encoded by the pGL3-mir-23b sensor plasmid.

The pGL3-mir-21 sensor plasmid was created by cloning the sequence (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), representing a sequence complementary to the mir-21 miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3â€²UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-21 miRNA was able to inhibit luciferase expression from the pGL3-mir-21 sensor plasmid. Thus, to further evaluate the ability of the mir-21 miRNA to bind this target site encoded by the pGL3-mir-21 sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-21-mediated silencing, pGL3-mir-21 sensor-expressing HeLa cells were treated with varying concentrations (10 nM or 50 nM) of the following oligomeric compounds: ISIS Number 327917 (SEQ ID NO: 335), a phosphorothioate uniform 2â€²-MOE oligomeric compound; ISIS Number 338697 (TGCCATGAGATTCAACAGTC; herein incorporated as SEQ ID NO: 524), a uniform 2â€²-MOE oligomeric compound targeting the mir-21 pri-miRNA molecule; and ISIS Number 328415 (SEQ ID NO: 524), a 2â€²-MOE 5-10-5 gapmer oligomeric compound targeting the mir-21 pri-miRNA. The uniform 2â€²-MOE phosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting an unrelated miRNA (mir-143) was used as a negative control. The data are the average of three trials and are presented in Table 44 as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 44Effects of oligomeric compounds on mir-21 miRNA-mediated inhibition of luciferase expression% UTCSEQ IDDose of oligomeric compoundTreatmentNO10 nM50 nM32790131974.283.1negative control3279173351037.6847.533869752487.084.832841552466.0104.4

From these data, it was observed that, at both doses, treatment of HeLa cells with ISIS Number 327917, the uniform 2â€²-MOE oligomeric compound targeting the mature mir-21 miRNA, de-repressed the expression of the luciferase reporter. Thus, ISIS 327917 reversed the silencing effect of the endogenous mir-21 miRNA, apparently by inhibiting the binding of mir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.

Therefore, oligomeric compounds targeting and/or mimicking the mir-143, mir-15a, mir-23b and mir-21 miRNAs and their corresponding pri-miRNA molecules have been demonstrated to bind to target RNA transcripts and silence reporter gene expression.

Example 28Effects of Oligomeric Compounds on Expression of Pri-miRNAs

As described above in Example 19, pri-miRNAs, often hundreds of nucleotides in length, are processed by a nuclear enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs or foldback miRNA precursors), and pre-miRNAs are subsequently exported from the nucleus to the cytoplasm, where they are processed by human Dicer into double-stranded miRNAs, which are subsequently processed by the Dicer RNase into mature miRNAs. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3â€² overhang (Lee, et al., Nature, 2003, 425, 415-419). The 3â€² two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9).

The oligomeric compounds of the present invention are believed to disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder Drosha and/or Dicer cleavage, respectively. Additionally, oligomeric compounds capable of binding to the mature miRNA are believed to prevent the RISC-mediated binding of a miRNA to its mRNA target, either by cleavage or steric occlusion of the miRNA.

Using the real-time RT-PCR methods described in Example 19, the expression levels of the mir-15a pri-miRNA were compared in HepG2 cells treated with a nested series of chimeric gapmer oligomeric compounds, targeting and spanning the entire length of the mir-15a pri-miRNA; these compounds are shown in Table 45, below. Each gapmer is 20 nucleotides in length, composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five-nucleotide â€œwings.â€ The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. Using the transfection methods described herein, HepG2 cells were treated with 100 nM of each of these gapmer oligomeric compounds. Total RNA was isolated from HepG2 cells by lysing cells in 1 mL TRIZOLâ„¢ (Invitrogen) using the manufacturer's recommended protocols. Real-time RT-PCR analysis was performed using a primer/probe set specific for the mir-15a pri-miRNA molecule to assess the effects of these compounds on expression of the mir-15a pri-miRNA molecule. ISIS 339317 (GTGTGTTTAAAAAAAATAAAACCTTGGA; SEQ ID NO.: 885) was used as the forward primer, ISIS 339318 (TGGCCTGCACCTTTTCAAA; SEQ ID NO.: 886) was used as the reverse primer, and ISIS 339319 (AAAGTAGCAGCACATAATGGTTTGTGG; SEQ ID NO.: 887) was used as the probe. Total RNA was quantified using RiboGreenâ„¢ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target are normalized to 5.8S rRNA, and values are expressed relative to the untreated control. Inhibition of expression of the mir15a pri-miRNA by these gapmer oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are described in Table 45 below:

TABLE 45Effects of chimeric oligomeric compoundson expression of the mir-15a pri-miRNAExpression ofSEQmir-15aIDpri-miRNAISIS NumberNOSequence(% UTC)347964878TATAACATTGATGTAATATG13.7 347965888GCTACTTTACTCCAAGGTTT86.0 347966879TGCTACTTTACTCCAAGGTT39.2 347967880GCACCTTTTCAAAATCCACA152.3 347968889CCTGCACCTTTTCAAAATCC8.4 347969881TGGCCTGCACCTTTTCAAAA39.5 347970890ATATGGCCTGCACCTTTTCA2.2 347971891ACAATATGGCCTGCACCTTT92.8 347972882AGCACAATATGGCCTGCACC98.6 347973892GGCAGCACAATATGGCCTGC143.3 347974893TGAGGCAGCACAATATGGCC98.1 347975894TTTTGAGGCAGCACAATATG9.2 347976895TATTTTTGAGGCAGCACAAT73.0 347977896TTGTATTTTTGAGGCAGCAC111.3 347978883TCCTTGTATTTTTGAGGCAG51.1 347979897AGATCCTTGTATTTTTGAGG74.9 347980884AGATCAGATCCTTGTATTTT3.6 347981898AGAAGATCAGATCCTTGTATN/D 347982899TTCAGAAGATCAGATCCTTG82.2 347983900AAATATATTTTCTTCAGAAG13.0

From these data, it was observed that oligomeric compounds ISIS Numbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 show significant inhibition of expression of the mir-15a pri-miRNA molecule. Thus, it is believed that the antisense oligomeric compounds ISIS Numbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 bind to the mir-15a pri-miRNA and/or pre-miRNA molecules and cause their degradation and cleavage.

From these data, it was observed that oligomeric compounds ISIS Numbers 347967, 347977 and 347973 stimulate an increase in expression levels of the mir-15a pri-miRNA. It is believed that the oligomeric compounds ISIS Numbers 347967, 347977 and 347973 bind to the mir-15a pri-miRNA and inhibit its processing into the mature mir-15a miRNA. It is believed that, in addition to the increase in the levels of the mir-15a pri-miRNA observed upon treatment of cells with the oligomeric compounds ISIS Numbers 347977, 347967 and 347973, a drop in expression levels of the fully processed mature mir-15a miRNA may also trigger a feedback mechanism signaling these cells to increase production of the mir-15a pri-miRNA.

The gapmer oligomeric compounds targeting the mir-15b and mir-15-a-1 mature miRNAs described above were also transfected into T47D cells according to standard procedures. In addition, uniform 2â€²-MOE and 2â€²-MOE gapmer oligomeric compounds targeting the mature mir-15a-1 and mir-15b miRNAs were also transfected into T47D cells, for analysis of their effects on mir-15a-1 and mir-15b pri-miRNA levels. The oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345), a uniform 2â€²-MOE compound and ISIS Number 345391 (SEQ ID NO: 345), a 2â€²-MOE 5-10-7 gapmer compound, both target mir-15b. The oligomeric compounds ISIS Number 327951 (SEQ ID NO: 369), a uniform 2â€²-MOE compound, and ISIS Number 345411 (SEQ ID NO: 369), a 2â€²-MOE 5-10-7 gapmer compound, both target mir-15a-1. Oligomeric compounds ISIS Number 129686 (CGTTATTAACCTCCGTTGAA; SEQ ID NO: 901), and ISIS Number 129691 (ATGCATACTACGAAAGGCCG; SEQ ID NO:902), both universal scrambled controls, as well as ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844) targeting an unrelated gene, PTEN, were used as negative controls. ISIS Numbers 129686, 129691, and 116847 are phosphorothiated 2â€²-MOE 5-10-5 gapmers, and all cytosines are 5-methylcytosines. T47D cells (seeded in 12-well plates) were treated with these oligomeric compounds, and RNA was isolated from the treated cells by lysing in 1 mL TRIZOLâ„¢ (Invitrogen) and total RNA was prepared using the manufacturer's recommended protocols. To assess the effects of these compounds on expression of the mir-15a or mir-15b pri-miRNA molecules, real-time RT-PCR analysis was performed using either the primer/probe set specific for the mir-15a pri-miRNA molecule described above, or a primer probe set specific for the mir-15b pri-miRNA molecule: ISIS 339320 (CCTACATTTTTGAGGCCTTAAAGTACTG; SEQ ID NO: 903) was used as the forward primer for the mir-15b pri-miRNA, ISIS 339321 (CAAATAATGATTCGCATCTTG ACTGT; SEQ ID NO: 904) was used as the reverse primer for the mir-15b pri-miRNA, and ISIS 339322 (AGCAGCACATCATGGTTTACATGC; SEQ ID NO: 905) was used as the probe. Total RNA was quantified using RiboGreenâ„¢ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values are expressed relative to the untreated control Inhibition of expression of the mir15a or mir-15b pri-miRNA molecules upon treatment with these oligomeric compounds is was assessed and expressed as a percentage of RNA levels in untreated control cells.

On multiple repeats of these experiments, it was observed that the uniform 2â€²-MOE oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345) and ISIS Number 327951 (SEQ ID NO: 369), targeted to the mature mir-15b and mir-15a-1 miRNAs, respectively, each stimulate an approximately 2.5- to 3.5-fold increase in expression of the mir-15a pri-miRNA molecule and an approximately 1.5- to 2.5-fold increase in the expression of the mir-15b pri-miRNA molecule. Therefore, it is believed that ISIS Numbers 327927 and 327951 can bind to the mir-15a and/or mir-15b pri-miRNA or pre-miRNA molecules and interfere with their processing into the mature mir-15a or mir-15b miRNAs. It is also recognized that a decrease in levels of the mature, processed forms of the mir-15a or mir-15b miRNAs in T47D cells treated with ISIS Number 345411 (SEQ ID NO: 369), ISIS Number 327927 (SEQ ID NO: 345) or ISIS Number 327951 (SEQ ID NO: 369) may also trigger a feedback mechanism that signals these cells to increase production of the mir-15a and/or mir-15b pri-miRNA molecules.

In accordance with the present invention, a nested series of uniform 2â€²-MOE oligomeric compounds were designed and synthesized to target the entire length of the mir-15a pri-miRNA molecule. Each compound is 19 nucleotides in length, composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides and phosphorothioate (Pâ• S) internucleoside linkages throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds are shown in Table 46. The compounds can be analyzed for their effect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.

TABLE 46Uniform 2â€²-MOE PS Compounds targeting themir-15a pri-miRNAISISSEQ IDNumberNOSequence356213878TATAACATTGATGTAATATG 356214879GCTACTTTACTCCAAGGTTT 356215880TGCTACTTTACTCCAAGGTT 356216881GCACCTTTTCAAAATCCACA 356217882CCTGCACCTTTTCAAAATCC 356218883TGGCCTGCACCTTTTCAAAA 356219884ATATGGCCTGCACCTTTTCA 356220888ACAATATGGCCTGCACCTTT 356221889AGCACAATATGGCCTGCACC 356222890GGCAGCACAATATGGCCTGC 356223891TGAGGCAGCACAATATGGCC 356224892TTTTGAGGCAGCACAATATG 356225893TATTTTTGAGGCAGCACAAT 356226894TTGTATTTTTGAGGCAGCAC 356227895TCCTTGTATTTTTGAGGCAG 356228896AGATCCTTGTATTTTTGAGG 356229897AGATCAGATCCTTGTATTTT 356230898AGAAGATCAGATCCTTGTAT 356231899TTCAGAAGATCAGATCCTTG 356232900AAATATATTTTCTTCAGAAG

Using the real-time RT-PCR methods described, the expression levels of the mir-15a pri-miRNA were compared in T47D cells treated with the nested series of uniform 2â€²-MOE oligomeric compounds, targeting and spanning the entire length of the mir-15a pri-miRNA. The region encompassing the mir-15a primary transcript (the complement of nucleotides 31603159 to 31603468 of GenBank Accession number NT_â€”024524.13; AAATAATTATG CATATTACATCAATGTTATAATGTTTAAACATAGATTTTTTTACATGCATTCTTTTTTTCCT GAAAGAAAATATTTTTTATATTCTTTAGGCGCGAATGTGTGTTTAAAAAAAATAAAACCT TGGAGTAAAGTAGCAGCACATAATGGTTTGTGGATTTTGAAAAGGTGCAGGCCATATTG TGCTGCCTCAAAAATACAAGGATCTGATCTTCTGAAGAAAATATATTTCTTTTTATTCATA GCTCTTATGATAGCAATGTCAGCAGTGCCTTAGCAGCACGTAAATATTGGCGTTAAG) is incorporated herein as SEQ ID NO: 906. ISIS Number 356215 (SEQ ID NO: 879) targets a region flanking and immediately 5â€² to the predicted 5â€² Drosha cleavage site in the mir-15a pri-miRNA. ISIS Number 356218 (SEQ ID NO: 881) targets a region in the loop of the mir-15a pri-miRNA. ISIS 356227 (SEQ ID NO: 883) targets a region flanking and immediately 3â€² to the predicted 3â€² Drosha cleavage site in the mir-15a pri-miRNA. Additionally, oligomeric compound ISIS 327951 (SEQ ID NO: 369), a uniform 2â€²-MOE compound targeting the mature mir-15a-1 miRNA, was tested for comparison. Oligomeric compounds ISIS 327901 (SEQ ID NO: 319) targeting the mature mir-143 miRNA; ISIS 129690, (TTAGAATACGTCGCGTTATG; SEQ ID NO: 907), a phosphorothioate 5-10-5 MOE gapmer used as a universal scrambled control; and ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), a uniform 5-10-5 2â€²-MOE gapmer targeting an unrelated gene, PTEN, were used as negative controls. Using the transfection methods previously described, T47D cells were treated with 100 nM of each of these oligomeric compounds. Total RNA was isolated by lysing cells in 1 mL TRIZOLâ„¢ (Invitrogen) using the manufacturer's recommended protocols. real-time RT-PCR analysis was performed using a primer/probe set specific for the mir-15a pri-miRNA molecule [forward primer=ISIS 339317 (SEQ ID NO.: 885), reverse primer=ISIS 339318 (SEQ ID NO.: 886), and probe=ISIS 339319 (SEQ ID NO.: 887)]. Total RNA was quantified using RiboGreenâ„¢ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values were expressed relative to the untreated control (UTC). Effects on expression of the mir-15a pri-miRNA molecule resulting from treatment of T47D cells with these uniform 2â€²-MOE oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are described in Table 47 below:

TABLE 47Effects of uniform 2â€²-MOE oligomericcompounds on mir-15a pri-miRNA expressionISIS #SEQ ID NO:target% UTCUTCN/AN/A100129690XXXN/A121scrambled control327901319mir-143132116847844PTEN mRNA132327951369mature mir-15a-1713356213878>100 bp upstream of171mature mir-15a356215879flanking 5â€² Drosha1005cleavage siteof mir-15a-1 pri-miRNA356216880mir-15a-1 pri-miRNA503356218881loop of mir-15a-1392pri-miRNA356221882mir-15a-1 pri-miRNA444356224894mir-15a-1 pri-miRNA592356227883flanking 3â€² Drosha879cleavage siteof mir-15a-1 pri-miRNA356229884mir-15a-1 pri-miRNA818356231899mir-15a-1 pri-miRNA811356232900mir-15a-1 pri-miRNA631

From these data, it was observed that the uniform 2â€²-MOE oligomeric compounds ISIS Numbers 327927, 327951, 356215, 356216, 356218, 356221, 356224, 356227, 356229, 356231 and 356232 stimulate an increase in levels of the mir-15a pri-miRNA molecule as detected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers 356215 and 356227 which target the regions immediately flanking the predicted 5â€² and 3â€² Drosha cleavage sites in the mir-15a pri-miRNA, respectively, were observed to stimulate the greatest increases in expression of the mir-15a pri-miRNA. It is believed that these oligomeric compounds bind to the mir-15a pri-miRNA and/or pre-miRNA molecules and interfere with their processing into the mature mir-15a miRNA, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer and/or Drosha. The resultant decrease in levels of the processed mature mir-15a miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-15a pri-miRNA molecule. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-15a pri-miRNA or pre-miRNA molecules. It is understood that such oligomeric compound-triggered mechanisms may be operating not only upon regulation of mir-15a production and processing, but may also be found to regulate the production and processing of other miRNAs.

The expression levels of mir-24-2, let-71, and let-7d were assessed in HeLa or T-24 cells treated with various uniform 2â€²-MOE oligomeric compounds targeting mature miRNAs. For example, using the transfection methods previously described, HeLa cells were treated with 100 nM of the oligomeric compound ISIS Number 327945 (SEQ ID NO: 363) targeting the mir-24-2 mature miRNA. Total RNA was isolated and expression levels of the mir-24-2 pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the mir-24-2 pri-miRNA molecule [forward primer=ISIS 359358 (CCCTGGGCTCTGCCT; herein incorporated as SEQ ID NO.: 908), reverse primer=ISIS 359359 (TGTACACAAACCAACTGTGTTTC; herein incorporated as SEQ ID NO.: 909), and probe=ISIS 359360 (CGTGCCTACTGAGC; herein incorporated as SEQ ID NO.: 910)]. An approximately 35-fold increase in expression levels of the mir-24-2 pri-miRNA molecule was observed in HeLa cells treated with the oligomeric compound ISIS 327945 as detected by real-time RT-PCR.

Using the transfection methods previously described, HeLa cells were treated with 100 nM of the oligomeric compound ISIS Number 327890 (SEQ ID NO: 308) targeting the let-71 mature miRNA. Total RNA was isolated and expression levels of the let-71 pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the let-71 pri-miRNA molecule [forward primer=ISIS 341684 (TGAGGTAGTAGTTTGTGCTGTTGGT; herein incorporated as SEQ ID NO.: 777), reverse primer=ISIS 341685 (AGGCAGTAGCTTGCGCAGTTA; herein incorporated as SEQ ID NO.: 778), and probe=ISIS 341686 (TTGTGACATTGCCCGCTGTGGAG; herein incorporated as SEQ ID NO.: 779)]. An approximately 4-fold increase in expression levels of the let-7i pri-miRNA molecule was observed in HeLa cells treated with the oligomeric compound ISIS 327890 as detected by real-time RT-PCR.

Using the transfection methods previously described, supra, T-24 cells were treated with 100 nM of the oligomeric compound ISIS Number 327926 (SEQ ID NO: 344) targeting the let-7d mature miRNA. Total RNA was isolated and expression levels of the let-7d pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the let-7d pri-miRNA molecule (forward primer=ISIS 341678 (CCTAGGAAGAGGTAG TAGGTTGCA; herein incorporated as SEQ ID NO.: 771), reverse primer=ISIS 341679 (CAGCAGGTCGTATAGTTACCTCCTT; herein incorporated as SEQ ID NO.: 772), and probe=ISIS 341680 (AGTTTTAGGGCAGGGATTTTGCCCA; herein incorporated as SEQ ID NO.: 773)). An approximately 1.7-fold increase in expression levels of the let-7d pri-miRNA molecule was observed in T-24 cells treated with the oligomeric compound ISIS 327926 as detected by real-time RT-PCR.

Thus, treatment with uniform 2â€²-MOE oligomeric compounds targeting mature miRNAs appears to result in an induction of expression of the corresponding pri-miRNA molecule.

In one embodiment, the expression of mir-21 (noted to be expressed at high levels in HeLa cells) was assessed in cells treated with oligomeric compounds. Using the transfection methods previously described, HeLa cells were treated with 100 nM of the uniform 2â€²-MOE oligomeric compound ISIS Number 327917 (SEQ ID NO: 335) targeting the mir-21 mature miRNA. Total RNA was isolated by lysing cells in 1 mL TRIZOLâ„¢ (Invitrogen) using the manufacturer's recommended protocols. By Northern blot analysis of total RNA from HeLa cells treated with ISIS 327917, expression levels of the mir-21 mature miRNA were observed to be reduced to 50% of those of untreated control cells. Furthermore, expression levels of the mir-21 pri-miRNA were found to increase in these HeLa cells treated with the oligomeric compound ISIS 327917. Real-time RT-PCR analysis was also performed on HeLa cells treated with ISIS 327917 using a primer/probe set specific for the mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (GCTGTACCACCTTGTCGGGT; herein incorporated as SEQ ID NO.: 911), reverse primer=ISIS 339333 (TCGACTGGTGTTGCCATGA; herein incorporated as SEQ ID NO.: 912), and probe=ISIS 339334 (CTTATCAGACTGATGTTGACTGTTGAAT; herein incorporated as SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreenâ„¢ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for the target were normalized to 5.8S rRNA, and values were expressed relative to an untreated control (UTC). ISIS Number 327917 was observed to stimulate an approximately 2-fold increase in levels of the mir-21 pri-miRNA molecule as detected by real-time RT-PCR.

Thus, it is believed that, in addition to binding the mir-21 mature miRNA and interfering with the RISC-mediated binding of mir-21 to its mRNA target, the oligomeric compound, ISIS 327917, binds to the mir-21 pri-miRNA and/or pre-miRNA molecules and interferes with their processing into the mature mir-21 miRNA, inhibiting expression of the mature mir-21 miRNA in HeLa cells, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer or Drosha. The resultant decrease in levels of mature mir-21 miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-21 pri-miRNA molecule. Treatment with this oligomeric compound could also stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNA or pre-miRNA molecules.

In accordance with the present invention, a nested series of uniform 2â€²-MOE oligomeric compounds were designed and synthesized to target the entire length of the mir-21 pri-miRNA molecule. The region encompassing the mir-21 primary transcript (nucleotides 16571584 to 16571864 of GenBank Accession number NT_â€”010783.14; CTGGGTTTTTTTGGTTTGTTTTTGTTTTTGTTTTTTTATCAAATCCTGCCTGACTGTCTGCTT GTTTTGCCTACCATCGTGACATCTCCATGGCTGTACCACCTTGTCGGGTAGCTTATCAGAC TGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGACATTTTGGTA TCTTTCATCTGACCATCCATATCCAATGTTCTCATTTAAACATTACCCAGCATCATTGTTT ATAATCAGAAACTCTGGTCCTTCTGTCTGGTGGCAC) is incorporated herein as SEQ ID NO: 914. Each compound is 20 nucleotides in length, composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides and phosphorothioate (Pâ• S) internucleoside linkages throughout the compound. All cytidine residues are 5-methylcytidines. The compounds are shown in Table 48. The compounds can be analyzed for their effect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.

TABLE 48Uniform 2â€²-MOE PS Compoundstargeting the mir-21 pri-miRNAISISSEQ IDNumberNOSequence358765915ACAAGCAGACAGTCAGGCAG 358766916GGTAGGCAAAACAAGCAGAC 358767917GGAGATGTCACGATGGTAGG 358768918AGGTGGTACAGCCATGGAGA 358769919GATAAGCTACCCGACAAGGT 358770920AGTCTGATAAGCTACCCGAC 358771921CAACAGTCAACATCAGTCTG 358772922GAGATTCAACAGTCAACATC 358773923CTGGTGTTGCCATGAGATTC 358774924CATCGACTGGTGTTGCCATG 358775925ACAGCCCATCGACTGGTGTT 358776926TGTCAGACAGCCCATCGACT 358777927CCAAAATGTCAGACAGCCCA 358778928GATACCAAAATGTCAGACAG 358779929GGTCAGATGAAAGATACCAA 358780930AACATTGGATATGGATGGTC 358781931TAATGTTTAAATGAGAACAT 358782932AACAATGATGCTGGGTAATG 358783933GAGTTTCTGATTATAAACAA 358784934CGACAAGGTGGTACAGCCAT 358785935GAAAGATACCAAAATGTCAG

Using the real-time RT-PCR methods, the expression levels of the mir-21 pri-miRNA were compared in HeLa cells treated with this nested series of uniform 2â€²-MOE oligomeric compounds, targeting and spanning the entire length of the mir-21 pri-miRNA. ISIS Number 358768 (SEQ ID NO: 918) targets a region flanking the predicted 5â€² Drosha cleavage site in the mir-21 pri-miRNA. ISIS Number 358777 (SEQ ID NO: 927) targets a region spanning the 3â€² Drosha cleavage site in the mir-21 pri-miRNA. ISIS 358779 (SEQ ID NO: 929) targets a region flanking the predicted 3â€² Drosha cleavage site in the mir-21 pri-miRNA. Additionally, oligomeric compounds ISIS 327917 (SEQ ID NO: 335), a uniform 2â€²-MOE compound targeting the mature mir-21 miRNA, and ISIS 345382 (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), a 5-10-7 phosphorothioate 2â€²-MOE gapmer targeting mir-21, were tested for comparison. Oligomeric compound ISIS 327863 (ACGCTAGCCTAATAGCGAGG; herein incorporated as SEQ ID NO: 936), a phosphorothioate 5-10-5 2â€²-MOE gapmer, was used as scrambled control. Using the transfection methods previously described, HeLa cells were treated with 100 nM of each of these oligomeric compounds. Total RNA was isolated by lysing cells in 1 mL TRIZOLâ„¢ (Invitrogen) using the manufacturer's recommended protocols. real-time RT-PCR analysis was performed using the primer/probe set specific for the mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (SEQ ID NO.: 911), reverse primer=ISIS 339333 (SEQ ID NO.: 912), and probe=ISIS 339334 (SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreenâ„¢ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values were expressed relative to the untreated control (UTC). Effects on expression of the mir-21 pri-miRNA molecule resulting from treatment of HeLa cells with these uniform 2â€²-MOE oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are shown in Table 49 below:

TABLE 49Effects of oligomeric compounds on mir-21 pri-miRNA expressionISIS #SEQ ID NO:target% UTCUTCN/AN/A100327863936N/A107gapmer control327917335mature mir-21249uniform 2â€²-MOE345382335mature mir-211195-10-7 2â€²-MOEgapmer358765915mir-21 pri-miRNA133358766916mir-21 pri-miRNA142358767917mir-21 pri-miRNA248358768918flanking 5â€² Drosha987cleavage siteof mir-21 pri-miRNA358769919mir-21 pri-miRNA265358770920mir-21 pri-miRNA250358771921mir-21 pri-miRNA181358772922mir-21 pri-miRNA245358773923mir-21 pri-miRNA148358774924mir-21 pri-miRNA104358775925mir-21 pri-miRNA222358776926mir-21 pri-miRNA367358777927spanning 3â€² Drosha536cleavage siteof mir-21 pri-miRNA358778928mir-21 pri-miRNA503358779929flanking 3â€² Drosha646cleavage siteof mir-21 pri-miRNA358780930mir-21 pri-miRNA269358781931mir-21 pri-miRNA122358782932mir-21 pri-miRNA155358783933mir-21 pri-miRNA133358784934mir-21 pri-miRNA358358785935mir-21 pri-miRNA257

From these data, it was observed that the uniform 2â€²-MOE oligomeric compounds ISIS Numbers 327917, 358767, 358768, 358769, 358770, 358772, 358775, 358776, 358777, 358778, 358779, 358780, 358784 and 358785 stimulate an increase in levels of the mir-21 pri-miRNA molecule as detected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers 358768 and 358779 which target the regions flanking the predicted 5â€² and 3â€² Drosha cleavage sites, respectively, and ISIS Number 358777, which targets a region spanning the 3â€² Drosha cleavage site in the mir-21 pri-miRNA were observed to stimulate the greatest increases in expression of the mir-21 pri-miRNA. Furthermore, treatment of HeLa cells with increasing concentrations (25, 50, 100, and 200 nM) of ISIS Numbers 358768, 358779, and 327917 was observed to result in a dose-responsive induction of mir-21 pri-miRNA levels. Thus, it is believed that these oligomeric compounds bind to the mir-21 pri-miRNA and/or pre-miRNA molecules and interfere with their processing into the mature mir-21 miRNA, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer and/or Drosha. The resultant decrease in levels of the processed mature mir-21 miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-21 pri-miRNA molecule. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNA or pre-miRNA molecules. It is understood that such oligomeric compound-triggered mechanisms may be operating not only upon regulation of mir-21 production and processing, but may also be found to regulate the production and processing of other miRNAs or target nucleic acids.

In one embodiment, the oligomeric compounds ISIS Number 327917 (SEQ ID NO: 335), the phosphorothioate uniform 2â€²-MOE targeting mature mir-21; ISIS Number 358768 (SEQ ID NO: 918), the uniform 2â€²-MOE targeting the mir-21 pri-miRNA which stimulated the largest increase in pri-miRNA expression levels by real time quantitative RT-PCR; and ISIS Number 345382 (SEQ ID NO: 335), the 5-10-7 phosphorothioate 2â€²-MOE gapmer targeting mature mir-21 were selected for dose response studies in HeLa cells using the luciferase reporter system described in Example 27. ISIS Number 342683 (SEQ ID NO: 790), representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide, was used as a negative control. HeLa cells expressing the pGL3-mir-21 sensor plasmid (described in Example 27) were treated with 1.9, 5.5, 16.7, and 50 nM of these oligomeric compounds, to assess the ability of oligomeric compounds to interfere with endogenous mir-21-mediated silencing of the pGL3-mir-21 sensor plasmid. The data are presented in Table 50 as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.

TABLE 50Effects of oligomeric compounds on mir-21 miRNA-mediated inhibition of luciferase expression% UTCDose of oligomeric compoundTreatment1.9 nM5.5 nM16.7 nM50 nM342683127171104108negative control32791752212932470453435876810316314611834538210113511795

From these data, it was observed that, at all doses, treatment of HeLa cells with ISIS Number 327917, the uniform 2â€²-MOE oligomeric compound targeting the mature mir-21 miRNA, de-repressed the expression of the luciferase reporter, in a dose-dependent fashion. Thus, ISIS 327917 reversed the silencing effect of the endogenous mir-21 miRNA, possibly by inhibiting the binding of mir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.

Example 29Diseases Associated with miRNA-Containing Loci

Using the public databases Online Mendelian Inheritance in Man (OMIM) (accessible through the Internet at, for example, ftp.ncbi.nih.gov/repository/OMIM/) and LocusLink (accessible at, for example, ftp.ncbi.nlm.nih.gov/refseq/LocusLink/), a bioinformatic analysis was performed which allowed the prediction of miRNAs associated with several human diseases. First, miRNAs encoded within genes having LocusLink identification numbers were identified, and these were compared to tables (for example, â€œmim2loc,â€ which connects LocusLink identification numbers with OMIM identification numbers, as well as â€œgenemap,â€ â€œgenemap.key,â€ â€œmim-title,â€ and â€œmorbidmapâ€ tables) for the construction of a new database called â€œdbl.mdbâ€ linking miRNAs to LocusLink and OMIM identification numbers and linking these to human diseases.

It was observed that, beginning with 95 pri-miRNAs, a subset of 49 had OMIM identification numbers, 48 of which were linked to OMIM names. Six of these miRNAs were associated with specific diseased patients (some in each category were duplicates). Thus, the majority of miRNAs with OMIM identification numbers are not directly linked to observed diseases, but are likely to be important in pathways (such as cholesterol homeostasis) associated with diseases. Tables 51 and 52 summarize information retrieved from these studies.

TABLE 51miRNA genes associated with specific diseasesOMIMID:locus containing miRNADisease association:120150collagen, type I, alpha 1/Osteogenesishypothetical miRNA-144imperfecta,type I, 166200114131calcitonin receptor containingOsteoporosis,hypothetical miRNA 30postmenopausalsusceptibility,166710605317forkhead box P2/Speech-languagehypothetical miRNA 169disorder-1, 602081600700LIM domain-containing preferredLipoma; Leukemia,translocation partner in lipomamyeloidcontaining miR-28160710myosin, heavy polypeptide 6,Cardiomyopathy,cardiac muscle, alphafamilial(cardiomyopathy, hypertrophic 1)hypertrophic, 192600containing miR-208606157hypothetical protein FLJ11729Neurodegeneration,containing mir-103-2pantothenate kinase-associated, 234200

The previous table shows miRNAs associated with an OMIM record that were also associated with diseased patients.

The following table, Table 52, describes diseases or disease-related phenotypes found to be associated with genetic loci associated with a miRNA.

TABLE 52miRNAs associated with disease phenotypesOMIMID:Locus containing miRNADisease association:114131calcitonin receptor containingOsteoporosis, postmenopausal,hypothetical miRNA-30susceptibility, 166710120150collagen, type I, alpha 1/Osteogenesis imperfecta,hypothetical miRNA-144type I, 166200138247glutamate receptor, ionotropic,cerebellar long-term depressionAMPA 2/hypothetical miRNA-171160710myosin, heavy polypeptide 6,Cardiomyopathy, familialcardiac muscle, alphahypertrophic, 192600(cardiomyopathy, hypertrophic 1)containing miR-208184756sterol regulatory element-bindingEmery-Dreifuss muscularprotein-1/mir-33bdystrophy, 310300; dilatedcardiomyopathy (CMD1A),115200; familial partiallipodystrophy (FPLD), 151660300093gamma-aminobutyric acid (GABA) Aearly-onset parkinsonism, orreceptor, epsilonWaisman syndrome, 311510;and MRX3 X-linked mentalretardation, 309541305660gamma-aminobutyric acid (GABA) Amanic depressive illness,receptor, alpha 3 containing miR-colorblindness, and G6PD105 (Mourelatos) and miR-105-2305915glutamate receptor, ionotrophic,complex bipolar disorder;AMPA 3/hypothetical miRNA-033drug addiction600150potassium large conductancecardiovascular diseasecalcium-activated channel,subfamily M, alpha member 1containing hypothetical miRNA-172600395glypican 1 containing miR-149angiogenesis600481Sterol regulatory element bindingLDL and cholesterol homeostasistranscription factor 2 containingmir-33a600592Minichromosome maintenanceincreased chromosomal loss,deficient (S. cerevisiae) 7DNA replication and recombinationcontaining miR-93 (Mourelatos)and miR-25 and miR-94600700LIM domain-containing preferredLipoma; Leukemia, myeloidtranslocation partner in lipomacontaining miR-28600758Focal adhesion kinase, p125/oncogenesismir-151601009tight junction protein 1 (zonapeptic ulcer disease andoccludens 1)/hypothetical miRNA-gastric carcinoma183601029mesoderm specific transcriptintrauterine and postnatal(mouse) homolog containing mir-growth retardation240* (Kosik)601698protein tyrosine phosphatase,insulin-dependent diabetes mellitus (IDDM)receptor type, N polypeptide 2containing mir-153-2601773protein tyrosine phosphatase,insulin-dependent diabetesreceptor type, N containing mir-mellitus (IDDM), 222100153-1603576melastatin 1 containing mir-211metastatic human melanoma603634ribosomal protein L5/colorectal cancershypothetical miRNA 168-2603745slit (Drosophila) homolog 3congenital diaphragmaticcontaining mir-218-2hernia603746slit (Drosophila) homolog 2retinal ganglion cell axoncontaining mir-218-1guidance603803dachshund (Drosophila) homologcell proliferation duringcontaining hypothetical miRNA-083mammalian retinogenesis andpituitary development605317forkhead box P2/hypotheticalautism & speech-languagemiRNA 169disorder-1, 602081605547follistatin-like 1 containingsystemic rheumatic diseasesmir-198605575SMC4 (structural maintenance ofcell proliferationchromosomes 4, yeast)-like 1containing mir-16-3 and mir-15b605766deleted in lymphocytic leukemia,B-cell chronic lymphocytic leukemia2 containing mir-16-1 and mir-15a-1606157hypothetical protein FLJ11729Neurodegeneration, pantothenate kinase-containing mir-103-2associated, 234200 (3);606160pantothenate kinase containingpantothenate kinase-mir-107associated neurodegeneration606161hypothetical protein FLJ12899pantothenate kinase-containing mir-103-1associated neurodegeneration

From these data, it was observed that several miRNAs are predicted to be associated with human disease states. For example, several studies of autistic disorder have demonstrated linkage to a similar region of 7q (the AUTS1 locus), leading to the proposal that a single genetic factor on 7q31 contributes to both autism and language disorders, and it has been reported that the FOXP2 gene, located on human 7q31, encoding a transcription factor containing a polyglutamine tract and a forkhead domain, is mutated in a severe monogenic form of speech and language impairment, segregating within a single large pedigree, and is also disrupted by a translocation. In one recent study, association and mutation screening analysis of the FOXP2 gene was performed to assess the impact of this gene on complex language impairments and autism, and it was concluded that coding-region variants in FOXP2 do not underlie the AUTS1 linkage and that the gene is unlikely to play a role in autism or more common forms of language impairment (Newbury, et al., Am. J. Hum. Genet. 2002, 70, 1318-27). However, hypothetical mir-169 is also encoded by this same genetic locus, and it is possible that mutations affecting the hypothetical mir-169 miRNA could underlie the AUTS1 linkage and play a role in language impairment. To this end, oligomeric compounds targeting or mimicking the mir-169 miRNA may prove useful in the study, diagnosis, treatment or amelioration of this disease.

Example 30Effects of Oligomeric Compounds Targeting miRNAs on Insulin Signaling and Hallmark Gene Expression in HepG2 Cells

Additional oligomeric compounds were screened in the assays described in Example 18. As stated above, insulin inhibits the expression of IGFBP-1, PEPCK-c and follistatin mRNAs.

Protocols for treatment of HepG2 cells and transfection of oligomeric compounds are as described in Example 18. Also as described in Example 18, forty-four hours post-transfection, the cells in the transfected wells were treated with either no insulin (â€œbasalâ€ Experiment 3 (below), for identification of insulin-mimetic compounds) or with 1 nM insulin (â€œinsulin treatedâ€ Experiment 4 (below), for identification of insulin sensitizers) for four hours. At the same time, in both plates, cells in some of the un-transfected control wells are treated with 100 nM insulin to determine maximal insulin response. At the end of the insulin or no-insulin treatment (forty-eight hours post-transfection), total RNA is isolated from both the basal and insulin treated (1 nM) 96-well plates, and the amount of total RNA from each sample is determined using a Ribogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR is performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and valuesÂ±standard deviation are expressed relative to the transfectant only and negative control oligonucleotides. The compound ISIS Number 186515 (AGGTAGCTTTGATTATGTAA; SEQ ID NO: 939) is targeted to IGFBP-1 and is a phosphorothioate 5-10-5 MOE gapmer where all cytosines are 5-methylcytosines, as is used as a transfection control. The oligomeric compound ISIS Number 340341 (TAGCTTATCAGACTGATGTTGA; SEQ ID NO: 236) is a uniform 2â€²-MOE phosphorothioate compound targeted to mir-104 (Mourelatos), ISIS 340362 (GACTGTTGAATCTCATGGCA; SEQ ID NO: 937) is a 5-10-5 gapmer compound also targeted to mir-104 (Mourelatos), and ISIS Number 341813 (AGACACGTGCACTGTAGA; SEQ ID NO: 938) is a uniform 2â€²-MOE phosphorothioate compound targeted to mir-139. Results of these experiments are shown in Tables 53 and 54.

TABLE 53Experiment 3: Effects of oligomeric compounds targeting miRNAson insulin-repressed gene expression in HepG2 cellsSEQ IDIGFBP-1PEPCK-cFollistatinISIS NO:NOPri-miRNA(% UTC)(% UTC)(% UTC)UTCN/AN/A10010010029848737N/A10410090n-mer186515939IGFBP-11937067328384493hypothetical139142110miRNA-039328677586hypothetical208145130miRNA-120328685594mir-219157219100328691600mir-14510510893328759668mir-21635698266328761670hypothetical1184891miRNA-138328765674mir-215889387328773682mir-15a-2148138131328779688hypothetical135123109mir-177340341236mir-10411012994(Mourelatos)340362937mir-104157168123(Mourelatos)341813938mir-139137121100

From these data, it is evident that the oligomeric compound ISIS Number 328761 targeting hypothetical mir-138, for example, results in a 52% decrease in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, this oligomeric compound may be useful as a pharmaceutical agent with insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

TABLE 54Experiment 4: Effects of oligomeric compounds targeting miRNAson insulin-sensitization of gene expression in HepG2 cellsSEQIGFBP-1PEPCK-cFollistatinISIS NO:ID NOPri-miRNA(% UTC)(% UTC)(% UTC)UTC(1 nmN/AN/A100100100insulin)29848737N/A929095n-mer186515939IGFBP-11054039328384493hypothetical102114121miRNA-039328677586hypothetical159117118miRNA-120328685594mir-219143184157328691600mir-14510197104328759668mir-21621292224328761670hypothetical935598miRNA-138328765674mir-215947397328773682mir-15a-213693148328779688hypothetical12878119mir-177340341236mir-104113115120(Mourelatos)340362937mir-104129104119(Mourelatos)341813938mir-13911788102

In HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.

From these data, it is evident that the oligomeric compounds, ISIS Number 328761 targeting hypothetical mir-138 and ISIS Number 328765 targeting mir-215, for example, were observed to result in a 45% and a 27% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Furthermore, mRNA levels of the IGFBP-1 and follistatin genes were also reduced. Thus, these oligomeric compounds may be useful as pharmaceutical agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.

Example 31Adipocyte Assay of Oligomeric Compounds

The effect of several oligomeric compounds of the present invention targeting miRNA target nucleic acids on the expression of markers of cellular differentiation was examined in differentiating adipocytes.

As described in Example 13, some genes known to be upregulated during adipocyte differentiation include HSL, aP2, Glut4 and PPARÎ³. These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. An increase in triglyceride content is another well-established marker for adipocyte differentiation.

For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARÎ³, as well as triglyceride (TG) accumulation were measured as previously described in adipocytes transfected with uniform 2â€²-MOE or chimeric gapmer phosphorothioate (PS) oligomeric compounds. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of untreated control (UTC) levels. Results are shown in Table 55.

TABLE 55Effects of oligomeric compounds targeting miRNAs onexpression of adipocyte differentiation markersISISSEQPPARNumberID NOTGHSLAP2Glut4gammaUTCN/A100100100100100ISIS-2984873789848996100n-mer327877295109827711985327888306132134102841033279043225642654054327909327132130881329632792734512512011412010832792834645527739573279333511271328212710032793735581777663923279513697610091818432795337194949211290327956374809010269913279603784752523476328093395598997739932811241492897397793281144161101341231161063281324341208981679432834044976130851121103283624717383598078328400509604034186732841752683988768943284345439196858379328651560931098478106328677586346861448932868559450100736991328691600130156166144105328759668871051086695

For these data, values for triglyceride accumulation above 100 are considered to indicate that the compound has the ability to stimulate triglyceride accumulation, whereas values at or below 100 indicate that the compound inhibits triglyceride accumulation. With respect to leptin secretion, values above 100 are considered to indicate that the compound has the ability to stimulate secretion of the leptin hormone, and values at or below 100 indicate that the compound has the ability to inhibit secretion of leptin. With respect to the four adipocyte differentiation hallmark genes, values above 100 are considered to indicate induction of cell differentiation, whereas values at or below 100 indicate that the compound inhibits differentiation.

Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 327904 (SEQ ID NO: 322), targeted to mir-181a-1, ISIS Number 327928 (SEQ ID NO: 346), targeted to mir-29a, ISIS Number 327960 (SEQ ID NO: 378), targeted to mir-215, ISIS Number 328400 (SEQ ID NO: 509), targeted to mir-196-2, and ISIS Number 328677 (SEQ ID NO: 586), targeted to hypothetical miRNA-120 were shown to reduce the expression levels of all five markers of adipocyte differentiation, indicating that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as in the maintenance of the pluripotent phenotype of stem or precursor cells.

The oligomeric compounds ISIS Number 328691 (SEQ ID NO: 600) targeted to mir-145, ISIS Number 328114 (SEQ ID NO: 416) targeted to hypothetical miRNA-138, and ISIS Number 327927 (SEQ ID NO: 345) targeted to mir-15b are examples of compounds which exhibit an increase in all five markers of adipocyte differentiation. Additionally, the oligomeric compound ISIS Number 327909 (SEQ ID NO: 327) targeted to mir-196-2 exhibited an increase in three of the five markers of adipocyte differentiation. Thus, these oligomeric compounds may be useful as pharmaceutical agents in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue would be beneficial. Furthermore, these oligomeric compounds may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.

Example 32Effects of Oligomeric Compounds on Endothelial Tube Formation Assay

Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J Mol. Cell. Cardiol., 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol. Histopathol., 1999, 14, 1287-1294).

Endothelial Tube Formation Assay as a Measure of Angiogenesis:

Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluates the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. In this assay, human umbilical vein endothelial cells (HuVECs) are plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HuVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.

In one embodiment, primary human umbilical vein endothelial cells (HuVECs) were used to measure the effects of oligomeric compounds targeted to miRNAs on tube formation activity. HuVECs were routinely cultured in EBM (Clonetics Corporation, Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence and were maintained for up to 15 passages. HuVECs are plated at 3000 cells/well in 96-well plates. One day later, cells are transfected with oligomeric compounds. The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.).

A scrambled control compound, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; where N is A, T, C or G; herein incorporated as SEQ ID NO: 737) served as a negative control. ISIS 196103 (AGCCCATTGCTGGACATGCA; incorporated herein as SEQ ID NO: 940) targets integrin beta 3 and was used as a positive control to inhibit endothelial tube formation. ISIS 29248 and ISIS 196103 are chimeric 5-10-5 2â€²-MOE gapmer oligonucleotides. The internucleoside (backbone) linkages are phosphorothioate (Pâ• S) throughout the oligonucleotides. All cytidine residues are 5-methylcytidines. ISIS 342672 (SEQ ID NO: 789) contains 13 mismatches with respect to the mature mir-143 miRNA, and was also used as a negative control. ISIS 342672 is a uniform 2â€²-MOE phosphorothioate oligomeric compound 22 nucleotides in length. All cytidine residues are 5-methylcytidines.

Oligomeric compound was mixed with LIPOFECTINâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEMâ„¢ (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 75 nM of oligomeric compound and 2.25 Î¼g/mL LIPOFECTINâ„¢. Before adding to cells, the oligomeric compound, LIPOFECTINâ„¢ and OPTI-MEMâ„¢ were mixed thoroughly and incubated for 0.5 hrs. Untreated control cells received LIPOFECTINâ„¢ only. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 Î¼l of phosphate-buffered saline. The wash buffer in each well was replaced with 100 Î¼L of the oligomeric compound/OPTI-MEMâ„¢/LIPOFECTINâ„¢ cocktail. Compounds targeted to miRNAs were tested in triplicate, and ISIS 29848 was tested in up to six replicates. The plates were incubated for 4 hours at 37Â° C., after which the medium was removed and the plate was tapped on sterile gauze. 100 Î¼l of full growth medium was added to each well. Fifty hours after transfection, cells are transferred to 96-well plates coated with ECMa-trixâ„¢ (Chemicon Inter-national). Under these conditions, untreated HuVECs form tube-like structures. After an overnight incubation at 37Â° C., treated and untreated cells are inspected by light microscopy. Individual wells are assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network. Results are expressed as a percentage of the level of the tube formation observed in cultures not treated with oligonucleotide, and are shown in Tables 56-59.

TABLE 56Effect of compounds targeting miRNAs on Tube FormationActivity in HuVECsSEQ ID% Activity RelativeISIS NO:NO:Pri-miRNAto UTCUTCN/AN/A100196103940Integrin beta 335.7positive control342672789N/A46.4negative control327873291mir-140100.0327875293mir-3471.4327876294mir-29b-150.0327877295mir-16-378.6327878296mir-20357.1327879297mir-7-171.4327880298mir-10b57.1327881299mir-128a50.0327882300mir-153-1107.1327883301mir-27b92.9327884302mir-9678.6327885303mir-17as/mir-9150.0327886304mir-123/mir-126as42.9327887305mir-13257.1327888306mir-108-1100.0327889307mir-23b50.0327890308let-7i92.9327891309mir-21250.0327892310mir-131-2/mir-957.1327893311let-7b100.0327894312mir-1d100.0327895313mir-122a100.0327896314mir-2264.3327898316mir-142100.0

From these data, it was observed that ISIS Number 327886 targeted to mir-123/mir126 as suppressed tube formation, indicating that this compound may be useful as an angiogenesis inhibitor and/or anti-tumor agent, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

TABLE 57Effect of compounds targeting miRNAs on Tube FormationActivity in HuVECsSEQ ID% ActivityISIS NO:NO:Pri-miRNARelative to UTCUTCN/AN/A100196103940Integrin beta 324.1positive control342672789N/A58.6negative control327899317mir-18334.5327900318mir-21455.2327901319mir-14348.3327902320mir-192-141.4327903321let-7a-3103.5327904322mir-181a89.7327905323mir-20548.3327906324mir-103-169.0327907325mir-26a62.1327908326mir-33a103.5327909327mir-196-296.6327910328mir-10755.2327911329mir-10675.9327913331mir-29c69.0327914332mir-130a82.8327915333mir-218-169.0327916334mir-124a-296.6327917335mir-2182.8327918336mir-14496.6327919337mir-221103.5327920338mir-22241.4327921339mir-30d96.6327922340mir-19b-289.7327923341mir-128b48.3

From these data, it was observed that ISIS Number 327899 targeted to mir-183, ISIS Number 327902 targeted to mir-192-1, and ISIS Number 327920 targeted to mir-222 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

TABLE 58Effect of compounds targeting miRNAs on TubeFormation Activity in HuVECsSEQ ID% Activity RelativeISIS NO:NO:Pri-miRNAto UTCUTCN/AN/A100196103940Integrin beta 329.6positive control342672789N/A55.6negative control327924342mir-129-288.9327925343mir-133b44.4327926344let-7d96.3327927345mir-15b59.3327928346mir-29a-137.0327929347mir-199b51.9327930348let-7e88.9327931349let-7c103.7327932350mir-20451.9327933351mir-14559.3327934352mir-213/mir-181a51.9327935353mir-2074.1327936354mir-133a-151.9327937355mir-138-288.9327938356mir-9896.3327939357mir-125b-166.7327940358mir-199a-259.3327941359mir-181b74.1327942360mir-14174.1327943361mir-1881.5327944362mir-22037.0327945363mir-24-259.3327946364mir-21151.9327947365mir-101-381.5

From these data, it was observed that ISIS Number 327925 targeted to mir-133b, ISIS Number 327928 targeted to mir-29a-1, and ISIS Number 327944 targeted to mir-220 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

TABLE 59Effect of compounds targeting miRNAs on TubeFormation Activity in HuVECsSEQ ID% Activity RelativeISIS NumberNO:Pri-miRNAto UTCUTCN/AN/A100196103940Integrin beta 326.7positive control342672789N/A60.0negative control327874292mir-30a46.7327897315mir-92-140.0327901319mir-143100.0327948366mir-30b33.3327949367mir-10a66.7327950368mir-19a73.3327951369mir-15a-173.3327952370mir-13753.3327953371mir-21953.3327954372mir-148b53.3327955373mir-130b46.7327956374mir-21646.7327957375mir-100-166.7327958376mir-18740.0327959377mir-21040.0327960378mir-21553.3327961379mir-22353.3327962380mir-30c53.3327963381mir-26b93.3327964382mir-15286.7327965383mir-135-1100.0327966384mir-21740.0327967385let-7g93.3327968386mir-33b93.3

From these data, it was observed that ISIS Number 327948 targeted to mir-30b, ISIS Number 327958 targeted to mir-187, ISIS Number 327959 targeted to mir-210, and ISIS Number 327966 targeted to mir-217 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.

Example 33Effect of Oligomeric Compounds on miRNA Target Protein Expression

Several mRNA transcripts have been predicted to be regulated by miRNAs (Lewis et al., Cell, 2003, 115, 787-798). For example, the mRNAs encoded by six genes, 1) inwardly rectifying potassium channel Kir2.2 (GenBank Accession AB074970, SEQ ID NO: 872); 2) synaptotagmin III (GenBank Accession BC028379, SEQ ID NO: 873); 3) mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM_â€”139032.1, SEQ ID NO: 861); 4) protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB) (GenBank Accession NM_â€”004156.1, SEQ ID NO: 814); 5) glyoxalase I (GenBank Accession NM_â€”006708.1, SEQ ID NO: 821); and 6) LIM domain only 4 (LMO4) (GenBank Accession NM_â€”006769.2, SEQ ID NO: 865), are believed to have mir-143 binding sites within their 3â€²-UTRs. The latter three genes encode mRNAs that were identified as potential targets of mir-143 by the RACE-PCR experiments described, supra. Thus, the mir-143 miRNA is predicted to regulate some or all of these genes.

When miRNAs have effects on the expression of downstream genes or proteins encoded by these genes, it is advantageous to measure the protein levels of those gene products, and to do this, western blot (immunoblot) analysis is employed Immunoblot analysis is carried out using standard methods. Briefly, preadipocytes and differentiating adipocytes were cultured as described previously, and differentiating adipocytes are sampled at several timepoints after stimulation of differentiation. Cells were treated with 250 nM oligomeric compounds and harvested 16-20 h after oligomeric compound treatment. Cells were washed, lysed in RIPA buffer with protease inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, Ind.), suspended in Laemmli buffer (20 ul/well), boiled for 5 minutes and loaded onto either an 8% SDS-PAGE or a 4-20% gradient SDS-PAGE gel. Gels are run for approximately 1.5 hours at 150 V, and transferred to PVDF membrane for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Because expression levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein remain constant, an antibody recognizing the GAPDH protein (Abcam, Cambridge, Mass.) can be used in a re-probing of the membrane to verify equal protein loading. It is also understood that antisense oligomeric compounds specifically targeting and known to inhibit the expression of the mRNA and protein endproducts of the gene of interest can be used as controls in these experiments. Bands are visualized and quantitated using a PHOSPHORIMAGERâ„¢ (Molecular Dynamics, Sunnyvale Calif.) or the ChemiDocâ„¢ system (Bio-Rad Laboratories, Inc., Hercules, Calif.). Thus, the effects of treatment of many cell types (including, but not limited to, preadipocytes, differentiating adipocytes, HeLa, T-24 and A549 cells) with oligomeric compounds of the present invention on the levels of gene expression products can be assessed. It is understood that one of ordinary skill in the art can use immunoblot analysis to examine the expression of any protein predicted to be the downstream expression product of a target of a miRNA. Similarly, using methods described above, real-time RT-PCR methods can also be used to examine the mRNA expression levels of any of these predicted targets of the mir-143 miRNA. More specifically, immunoblot analysis and/or real-time RT-PCR methods can be used to examine the effects of treatment with oligomeric compounds on the protein or mRNA levels, respectively, produced by the Kir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LMO4 genes in a variety of cell types.

In one embodiment of the invention, immunoblot analysis was used to assess the effects of the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319) targeting mir-143, on expression levels of the ERK5 protein in differentiating adipocytes. It was observed that, upon treatment of cells with ISIS 327901, ERK5 protein levels were approximately 2-2.5-fold higher in differentiating adipocytes both 7- and 10-days post-differentiation than in pre-adipocytes or in untreated differentiating adipocytes from the same timepoints. Thus, mir-143 appears to negatively regulate the expression of the ERK5 gene presumably by inhibiting translation of the ERK5 mRNA into protein, either directly (by mir-143 binding an ERK5 cis-regulatory sequence) or indirectly (by mir-143 regulating another target gene that regulates ERK5); upon treatment with the oligomeric compound ISIS 327901, this mir-143-dependent inhibition of ERK5 expression was relieved. It is known that ERK5 promotes cell growth and proliferation in response to tyrosine kinase signaling. In light of the involvement of mir-143 in adipocyte differentiation disclosed in several examples in the present invention, as well as the role of mir-143 in regulating ERK5, it is predicted that ERK5 and mir-143 are together involved regulating the balance between cellular proliferation and differentiation.

It is understood that the oligomeric compounds of the present invention, including miRNA mimics, can also be tested for their effects on the expression of the protein endproducts of targets of miRNAs. For example, an oligomeric compound such as a mir-143 mimic can be used to treat differentiating adipocytes, and is predicted to result in a reduction of Kir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LMO4 protein expression levels.

The phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) tumor suppressor mRNA (GenBank Accession NM_â€”000314, incorporated herein as SEQ ID NO: 941) has been predicted to be a potential target of the mir-19a miRNA (Lewis et al., Cell, 2003, 115, 787-798). Oligomeric compounds that target or mimic the mir-19a miRNA or mir-19a pri-miRNA can be used to treat cells and, using the methods described above, the effects of these oligomeric compounds on the expression of the PTEN protein and mRNA levels can be assessed. It is predicted that the mir-19a miRNA, or an oligomeric compound acting as a mir-19a mimic, would inhibit expression of the PTEN tumor suppressor mRNA and protein, and that treatment with oligomeric compounds targeting mir-19a would reverse this inhibition. It is also understood that other antisense oligomeric compounds specifically targeting and known to inhibit the expression of the mRNA and protein endproducts of the gene interest can be used as controls in these experiments.

Example 34Additional Oligomeric Compounds Targeting miRNAs

In accordance with the present invention, oligomeric compounds were designed and synthesized to target or mimic one or more miRNA genes or gene products. Pri-miRNAs, pre-miRNAs and mature miRNAs represent target nucleic acids to which the oligomeric compounds of the present invention were designed and synthesized. Oligomeric compounds of the present invention can also be designed and synthesized to mimic the pri-miRNA, pre-miRNA or mature miRNA structure while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior properties as compared to the endogenous precursor or mature miRNA.

In accordance with the present invention, oligomeric compounds were designed to target or mimic one or more human, mouse, rat, or Drosophila pri-miRNAs, pre-miRNAs or mature miRNAs.

A list of human pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 60. â€œPri-miRNA nameâ€ indicates the gene name for each of the pri-miRNAs. Also given in table 60 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5â€² to 3â€² direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 60Human pri-miRNA sequences and the corresponding mature miRNAsSEQSEQ IDIDPri-miRNA nameNOMature miRNA nameMature miRNA sequenceNOmir-27b17mir-27bTTCACAGTGGCTAAGTTCTG202 mir-27b17miR-27* (MichaelTTCACAGTGGCTAAGTTCTGC1059et al) mir-23b23mir-23bATCACATTGCCAGGGATTACCAC208 glutamate36hypotheticalTGTTATAGTATTCCACCTACC1060receptor,miRNA-033ionotrophic, AMPA3/hypotheticalmiRNA-033 LOC 11461474hypotheticalTGCTAATCGTGATAGGGGTTT1061containing miR-miRNA-071155/hypotheticalmiRNA-071 LOC 11461474mir-155 (RFAM)TTAATGCTAATCGTGATAGGGG1062containing miR-155/hypotheticalmiRNA-071 collagen, type I,147hypotheticalAGACATGTTCAGCTTTGTGGA1063alpha 1/miRNA-144hypotheticalmiRNA-144 sterol regulatory168mir-33bGTGCATTGCTGTTGCATTG286element-bindingprotein-1/mir-33b tight junction186hypotheticalAGCCTGTGGAGCTGCGCTTAC1064protein 1 (zonamiRNA-183occludens 1)/hypotheticalmiRNA-183 mir-1404mir-140AGTGGTTTTACCCTATGGTAG192 mir-1404miR-140-asTACCACAGGGTAGAACCACGGA1065 mir-1404mir-239* (Kosik)TACCACAGGGTAGAACCACGGACA1066 mir-346mir-34TGGCAGTGTCTTAGCTGGTTGT194 mir-346miR-172 (RFAM-M. mu.)TGGCAGTGTCTTAGCTGGTTGTT1067 mir-20310mir-203GTGAAATGTTTAGGACCACTAG197 mir-20310miR-203 (RFAM-M. mu.)TGAAATGTTTAGGACCACTAG1068 mir-20310miR-203 (Tuschl)TGAAATGTTTAGGACCACTAGA1069 mir-7_1/mir-7_1*11mir-7_1*_RuvkunCAACAAATCACAGTCTGCCATA1070 mir-7_1/mir-7_1*11mir-7TGGAAGACTAGTGATTTTGTT198 mir-10b12miR-10b (Tuschl)CCCTGTAGAACCGAATTTGTGT1071 mir-10b12mir-10bTACCCTGTAGAACCGAATTTGT199 mir-10b12miR-10b (MichaelTACCCTGTAGAACCGAATTTGTG1072et al) mir-128a13mir-128 (Kosik)TCACAGTGAACCGGTCTCTTT1073 mir-128a13mir-128aTCACAGTGAACCGGTCTCTTTT200 mir-153_114mir-153TTGCATAGTCACAAAAGTGA201 mir-153_215mir-153TTGCATAGTCACAAAAGTGA201 hypothetical miR-16hypotheticalTATCAAACATATTCCTACAGT107413/miR-190miRNA-013 hypothetical miR-16miR-190TGATATGTTTGATATATTAGGT107513/miR-190 mir-123/mir-12620mir-123/mir-126asCATTATTACTTTTGGTACGCG205 mir-123/mir-12620mir-126TCGTACCGTGAGTAATAATGC1076 mir-13221miR-132 (RFAM-TAACAGTCTACAGCCATGGTCG1077Human) mir-13221mir-132TAACAGTCTACAGCCATGGTCGC206 mir-108_122mir-108ATAAGGATTTTTAGGGGCATT207 let-7i24let-7iTGAGGTAGTAGTTTGTGCT209 let-7i24let-7i_RuvkunTGAGGTAGTAGTTTGTGCTGTT1078 mir-21225mir-212TAACAGTCTCCAGTCACGGCC210 hypothetical miRNA26hypotheticalTGGGCAAGAGGACTTTTTAAT1079023miRNA-023 mir-131_2/mir-927mir-131TAAAGCTAGATAACCGAAAGT211 mir-131_2/mir-927mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA1080 mir-131_2/mir-927miR-9TCTTTGGTTATCTAGCTGTATGA1081 let-7b28let-7bTGAGGTAGTAGGTTGTGTGGTT212 let-7b28let-7b_RuvkunTGAGGTAGTAGGTTGTGTGGTTT1082 mir-1d_129miR-1 (RFAM)TGGAATGTAAAGAAGTATGTA1083 mir-1d_129mir-1dTGGAATGTAAAGAAGTATGTAT213 mir-122a30miR-122a,bTGGAGTGTGACAATGGTGTTTG1084(Tuschl) mir-122a30mir-122aTGGAGTGTGACAATGGTGTTTGT214 mir-2231mir-22AAGCTGCCAGTTGAAGAACTGT215 hypothetical miRNA33hypotheticalTGACATCACATATACGGCAGC108530miRNA-030 mir-14234mir-142CATAAAGTAGAAAGCACTAC217 mir-14234miR-142-asTGTAGTGTTTCCTACTTTATGG1086 mir-14234miR-142asTGTAGTGTTTCCTACTTTATGGA1087(Michael et al) mir-18335mir-183TATGGCACTGGTAGAATTCACTG218 mir-21437mir-214ACAGCAGGCACAGACAGGCAG219 mir-14338miR-143 (MichaelTGAGATGAAGCACTGTAGCTC1088et al) mir-14338mir-143TGAGATGAAGCACTGTAGCTCA220 mir-192_139miR-192 (Tuschl)CTGACCTATGAATTGACA1089 mir-192_139mir-192CTGACCTATGAATTGACAGCC221 mir-192_139miR-192 (MichaelTGACCTATGAATTGACAGCCAG1090et al) hypothetical miRNA42hypotheticalTAAGACTTGCAGTGATGTTTA1091039miRNA-039 hypothetical miRNA43hypotheticalTGTCAACAAAACTGCTTACAA1092040miRNA-040 hypothetical miRNA44hypotheticalTACCAGTTGTTTTCTCTGTGA1093041miRNA-041 let-7a_345let-7aTGAGGTAGTAGGTTGTATAGTT222 hypothetical miRNA46hypotheticalTGACAGGAAATCTTTGAGAGG1094043miRNA-043 hypothetical miRNA47hypotheticalTTCCACTCTGTTTATCTGACA1095044miRNA-044 mir-181a_148mir-178 (Kosik)AACATTCAACGCTGTCGGTGAG1096 mir-181a_148mir-181aAACATTCAACGCTGTCGGTGAGT223 let-7a_149let-7aTGAGGTAGTAGGTTGTATAGTT222 mir-20550mir-205TCCTTCATTCCACCGGAGTCTG224 mir-33a53mir-33aGTGCATTGTAGTTGCATTG227 mir-196_254miR-196 (Tuschl)TAGGTAGTTTCATGTTGTTGG1097 mir-196_254mir-196TAGGTAGTTTCATGTTGTTGGG228 let-7f_157let-7f (MichaelTGAGGTAGTAGATTGTATAGT1098et al) let-7f_157let-7fTGAGGTAGTAGATTGTATAGTT231 hypothetical miRNA58hypotheticalTTGCATGCCCTATTGATTCTC1099055miRNA-055 mir-29c59mir-29cCTAGCACCATTTGAAATCGGTT232 mir-29c59miR-29c (Tuschl)TAGCACCATTTGAAATCGGTTA1100 mir-130a60mir-130aCAGTGCAATGTTAAAAGGGC233 mir-130a60mir-130 (Kosik)CAGTGCAATGTTAAAAGGGCAT1101 hypothetical miRNA61hypotheticalTGTCAGATGCTTAATGTTCTT1102058miRNA-058 mir-218_162mir-218TTGTGCTTGATCTAACCATGT234 mir-218_162mir-253* (Kosik)TTGTGCTTGATCTAACCATGTG1103 mir-124a_263mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA1104 mir-124a_263mir-124aTTAAGGCACGCGGTGAATGCCA235 mir-124a_263mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA1105 mir-14466mir-144TACAGTATAGATGATGTACTAG237 mir-22167mir-221 (RFAM-AGCTACATTGTCTGCTGGGTTT1106mmu) mir-22167mir-221AGCTACATTGTCTGCTGGGTTTC238 mir-22268mir-222 (RFAM-AGCTACATCTGGCTACTGGGTCT1107mmu) mir-22268mir-222AGCTACATCTGGCTACTGGGTCTC239 mir-30d69mir-30dTGTAAACATCCCCGACTGGAAG240 mir-30d69mir-30d_RuvkunTGTAAACATCCCCGACTGGAAGCT1108 mir-128b71mir-128 (Kosik)TCACAGTGAACCGGTCTCTTT1073 mir-128b71mir-128bTCACAGTGAACCGGTCTCTTTC242 mir-219_272mir-219TGATTGTCCAAACGCAATTCT271 hypothetical miRNA73hypotheticalTCACATTTGCCTGCAGAGATT1109070miRNA-070 mir-129_276mir-129as/mir-AAGCCCTTACCCCAAAAAGCAT1110258* (Kosik) mir-129_276mir-129CTTTTTGCGGTCTGGGCTTGC243 mir-129_276miR-129b (RFAM-CTTTTTGCGGTCTGGGCTTGCT1111Human) mir-133b77mir-133bTTGGTCCCCTTCAACCAGCTA244 hypothetical miRNA78hypotheticalTGGTTAAAATATTAATGGGGC1112075miRNA-075 let-7d79let-7dAGAGGTAGTAGGTTGCATAGT245 let-7d79let-7d_RuvkunAGAGGTAGTAGGTTGCATAGTT1113 let-7d79let-7d* (RFAM-M. mu.)CTATACGACCTGCTGCCTTTCT1114 mir-15b80miR-15b (MichaelTAGCAGCACATCATGGTTTAC1115et al) mir-15b80mir-15bTAGCAGCACATCATGGTTTACA246 mir-29a81mir-29aCTAGCACCATCTGAAATCGGTT247 mir-29a81mir-29a_RuvkunTAGCACCATCTGAAATCGGTTA1116 hypothetical miRNA82hypotheticalTGATATGTTTGATATTGGG1117079miRNA-079 mir-199b83mir-199b (human)CCCAGTGTTTAGACTATCTGTTC248 mir-199b83miR-199-asTACAGTAGTCTGCACATTGGTT1118 mir-129_184mir-129CTTTTTGCGGTCTGGGCTTGC243 mir-129_184miR-129b (RFAM-CTTTTTGCGGTCTGGGCTTGCT1111Human) let-7e85let-7eTGAGGTAGGAGGTTGTATAGT249 hypothetical miRNA86hypotheticalTTACATGGGGAAGCTATCATA1119083miRNA-083 let-7c_187let-7cTGAGGTAGTAGGTTGTATGGTT250 let-7c_187let-7c_RuvkunTGAGGTAGTAGGTTGTATGGTTT1120 mir-20488mir-204TTCCCTTTGTCATCCTATGCCT251 mir-20488miR-204 (Tuschl)TTCCCTTTGTCATCCTATGCCTG1121 mir-14589miR-145 (MichaelGTCCAGTTTTCCCAGGAATCC1122et al) mir-14589mir-145GTCCAGTTTTCCCAGGAATCCCTT252 mir-124a_190mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA1104 mir-124a_190mir-124aTTAAGGCACGCGGTGAATGCCA235 mir-124a_190mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA1105 DiGeorge syndrome91hypotheticalTGTGATTTCCAATAATTGAGG1123critical regionmiRNA-088gene 8/hypotheticalmiRNA-088 mir-213/mir-92mir-178 (Kosik)AACATTCAACGCTGTCGGTGAG1096181a_2 mir-213/mir-92mir-181aAACATTCAACGCTGTCGGTGAGT223181a_2 mir-213/mir-92mir-213ACCATCGACCGTTGATTGTACC253181a_2 hypothetical miRNA93hypotheticalTAGGCCAAATGGCGCATCAAT1124090miRNA-090 mir-2094miR-20* (human)ACTGCATTATGAGCACTTAAA1125 mir-2094miR-20 (RFAM-TAAAGTGCTTATAGTGCAGGTA1126Human) mir-2094mir-20TAAAGTGCTTATAGTGCAGGTAG254 mir-133a_195mir-133aTTGGTCCCCTTCAACCAGCTGT255 mir-138_296mir-138AGCTGGTGTTGTGAATC256 mir-138_296mir-138_RuvkunAGCTGGTGTTGTGAATCAGGCCG1127 mir-196_198miR-196 (Tuschl)TAGGTAGTTTCATGTTGTTGG1097 mir-196_198mir-196TAGGTAGTTTCATGTTGTTGGG228 mir-125b_199mir-125bTCCCTGAGACCCTAACTTGTGA258 mir-199a_2100miR-199-sCCCAGTGTTCAGACTACCTGTT1128 mir-199a_2100mir-199aCCCAGTGTTCAGACTACCTGTTC259 mir-199a_2100miR-199-asTACAGTAGTCTGCACATTGGTT1118 hypothetical miRNA102hypotheticalAGGCAGATAGAGAAGTCACAG1272099miRNA-099 mir-181b_1103mir-181bAACATTCATTGCTGTCGGTGGGTT260 hypothetical miRNA104hypotheticalTGACAGTCAATTAACAAGTTT1130101miRNA-101 mir-141105mir-141AACACTGTCTGGTAAAGATGG261 mir-131_1/mir-9106mir-131TAAAGCTAGATAACCGAAAGT211 mir-131_1/mir-9106mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA1080 mir-131_1/mir-9106miR-9TCTTTGGTTATCTAGCTGTATGA1081 mir-133a_2107mir-133aTTGGTCCCCTTCAACCAGCTGT255 hypothetical miRNA108miR-202 (human)AGAGGTATAGGGCATGGGAAAA1131105 hypothetical miRNA108hypotheticalTTCCTATGCATATACTTCTTT1132105miRNA-105 hypothetical miRNA110hypotheticalTGACAGTTTATTGGCTTTATC1133107miRNA-107 mir-1d_2111miR-1 (RFAM)TGGAATGTAAAGAAGTATGTA1083 mir-1d_2111mir-1dTGGAATGTAAAGAAGTATGTAT213 mir-1d_2111miR-1d (Tuschl)TGGAATGTAAAGAAGTATGTATT1134 mir-220113mir-220CCACACCGTATCTGACACTTT263 hypothetical miRNA114hypotheticalTTCCTCCTCCTCCGACTCGGA1135111miRNA-111 mir-7_3115mir-7TGGAAGACTAGTGATTTTGTT198 mir-218_2116mir-218TTGTGCTTGATCTAACCATGT234 mir-218_2116mir-253* (Kosik)TTGTGCTTGATCTAACCATGTG1103 mir-211120mir-211 (human)TTCCCTTTGTCATCCTTCGCCT1136 mir-30b122mir-30bTGTAAACATCCTACACTCAGC266 mir-30b122mir-30b_RuvkunTGTAAACATCCTACACTCAGCT1137 hypothetical miRNA123hypotheticalTTACAGCAATCCAGTAATGAT1138120miRNA-120 mir-10a125mir-10a (Tuschl)TACCCTGTAGATCCGAATTTGT1139 mir-10a125mir-10aTACCCTGTAGATCCGAATTTGTG267 let-7f_2127let-7f (MichaelTGAGGTAGTAGATTGTATAGT1098et al) let-7f_2127let-7fTGAGGTAGTAGATTGTATAGTT231 mir-108_2129mir-108ATAAGGATTTTTAGGGGCATT207 mir-137130mir-137TATTGCTTAAGAATACGCGTAG270 mir-148b132mir-148bTCAGTGCATCACAGAACTTTGT272 mir-130b133mir-130bCAGTGCAATGATGAAAGGGC273 mir-130b133mir-266* (Kosik)CAGTGCAATGATGAAAGGGCAT1140 let-7a_4135let-7aTGAGGTAGTAGGTTGTATAGTT222 mir-216136mir-216TAATCTCAGCTGGCAACTGTG274 hypothetical miRNA140hypotheticalTAAACTGGCTGATAATTTTTG1141137miRNA-137 hypothetical miRNA141hypotheticalTGCAAGTATGAAAATGAGATT1142138miRNA-138 mir-124a_3143mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA1104 mir-124a_3143mir-124aTTAAGGCACGCGGTGAATGCCA235 mir-124a_3143mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA1105 mir-7_2144mir-7TGGAAGACTAGTGATTTTGTT198 hypothetical miRNA145hypotheticalTGACGCTGCTCCCCACCTTCT1143142miRNA-142 hypothetical miRNA146hypotheticalTGCAATTTGCTTGCAATTTTG1144143miRNA-143 mir-210148mir-210CTGTGCGTGTGACAGCGGCTG277 mir-215149mir-215ATGACCTATGAATTGACAGAC278 mir-223150mir-223TGTCAGTTTGTCAAATACCCC279 mir-131_3/mir-9151mir-131TAAAGCTAGATAACCGAAAGT211 mir-131_3/mir-9151mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA1080 mir-131_3/mir-9151miR-9TCTTTGGTTATCTAGCTGTATGA1081 mir-199a_1152miR-199-sCCCAGTGTTCAGACTACCTGTT1128 mir-199a_1152mir-199aCCCAGTGTTCAGACTACCTGTTC259 mir-199a_1152miR-199-asTACAGTAGTCTGCACATTGGTT1118 mir-30c_1153mir-30cTGTAAACATCCTACACTCTCAGC280 mir-30c_1153mir-30c_RuvkunTGTAAACATCCTACACTCTCAGCT1129 hypothetical miRNA156hypotheticalTGCAAGCAGATGCTGATAATA1145153miRNA-153 hypothetical miRNA157hypotheticalTTAAAGTGGATGTGTGTTATT1146154miRNA-154 mir-26b158miR-26b (RFAM-TTCAAGTAATTCAGGATAGGT1147Human) mir-26b158mir-26bTTCAAGTAATTCAGGATAGGTT281 hypothetical miRNA159hypotheticalTGCTTTCCCTCCTTCCTTCTT1148156miRNA-156 mir-152160mir-152TCAGTGCATGACAGAACTTGG282 mir-135_1161miR-135 (RFAM-TATGGCTTTTTATTCCTATGTGA1149Human) mir-135_1161mir-135TATGGCTTTTTATTCCTATGTGAT283 non-coding RNA in162miR-135 (RFAM-TATGGCTTTTTATTCCTATGTGA1149rhabdomyosarcoma/Human)mir-135_2 non-coding RNA in162mir-135TATGGCTTTTTATTCCTATGTGAT283rhabdomyosarcoma/mir-135_2 mir-217163mir-217 (human)TACTGCATCAGGAACTGATTGGAT284 hypothetical miRNA164hypotheticalTGGCCATAAACTTGTAGTCAT1150161miRNA-161 mir-15a165mir-15_RuvkunTAGCAGCACATAATGGTTTGT1151 mir-15a165mir-15aTAGCAGCACATAATGGTTTGTG269 let-7g166let-7gTGAGGTAGTAGTTTGTACAGT285 let-7g166let-7gL_RuvkunTGAGGTAGTAGTTTGTACAGTT1152 hypothetical miRNA167hypotheticalTGCAAGGATTTTTATGTTTTG1153164miRNA-164 hypothetical miRNA169hypotheticalTTCCAGTTGCAGCACCTGTAA1154166miRNA-166 hypothetical miRNA171hypotheticalAGCCAGGTGCCTTCACCTGCT1155168_1/similar tomiRNA-168ribosomal proteinL5 forkhead box172hypotheticalTGGCAGCTCTGGCATTTCATA1156P2/hypotheticalmiRNA-169miRNA-169 hypothetical miRNA173hypotheticalTGATCTTGCTCTAACACTTGG1157170miRNA-170 glutamate174hypotheticalTGACAAGTATGTTTTATCGTT1158receptor,miRNA-171ionotropic, AMPA 2/hypotheticalmiRNA-171 hypothetical miRNA175hypotheticalTCCAACTGCAAGAAGTTACT1159172miRNA-172 hypothetical miRNA176hypotheticalTAGTACGAGAAGAAGGAGGCT1160173miRNA-173 mir-182177miR-182* (RFAM-TGGTTCTAGACTTGCCAACTA1161Human) mir-182177mir-182TTTGGCAATGGTAGAACTCACA287 hypothetical miRNA178hypotheticalTCTCCTTCAACCACCTGAGGT1162175miRNA-175 hypothetical miRNA179hypotheticalTAGGAGTTTGATATGACATAT1163176miRNA-176 hypothetical180hypotheticalAGACAAACATGCTACTCTCAC1164miRNA-177_1miRNA-177 hypothetical miRNA181hypotheticalTAGCCTATCTCCGAACCTTCA1165178miRNA-178 hypothetical miRNA182hypotheticalTGAAAGGCACTTTGTCCAATT1166179miRNA-179 hypothetical miRNA184hypotheticalTCACCTGCTCTGGAAGTAGTT1167181miRNA-181 mir-148a185mir-148aTCAGTGCACTACAGAACTTTGT288 hypothetical miRNA188hypotheticalTGATGGCCAGCTGAGCAGCTC1168185miRNA-185 hypothetical189hypotheticalAGACAAACATGCTACTCTCAC1164miRNA-177_2/miRNA-177hypothetical miRNA186 mir-181c190mir-181cAACATTCAACCTGTCGGTGAGT290 hypothetical miRNA191hypotheticalTGGTGAGGGGAATGAAAAGTA1169188miRNA-188 mir-100_1945mir-100AACCCGTAGATCCGAACTTGTG275 mir-101_1946mir-101TACAGTACTGTGATAACTGA265 mir-101_1946miR-101 (RFAM-TACAGTACTGTGATAACTGAAG1170Human) mir-101_3947mir-101TACAGTACTGTGATAACTGA265 mir-101_3947miR-101 (RFAM-TACAGTACTGTGATAACTGAAG1170Human) mir-29b_2948miR-29b (RFAM-TAGCACCATTTGAAATCAGT1172Human) mir-29b_2948miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT1173 mir-29b_2948mir-29bTAGCACCATTTGAAATCAGTGTT195 mir-29b_1949miR-29b (RFAM-TAGCACCATTTGAAATCAGT1172Human) mir-29b_1949miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT1173 mir-29b_1949mir-29bTAGCACCATTTGAAATCAGTGTT195 mir-103_1950mir-103AGCAGCATTGTACAGGGCTATGA225 mir-106951mir-106 (human)AAAAGTGCTTACAGTGCAGGTAGC230 mir-107952mir-107AGCAGCATTGTACAGGGCTATCA229 mir-16_1953mir-16TAGCAGCACGTAAATATTGGCG196 mir-16_1953mir-16_RuvkunTAGCAGCACGTAAATATTGGCGT1176 mir-16_3954mir-16TAGCAGCACGTAAATATTGGCG196 mir-16_3954mir-16_RuvkunTAGCAGCACGTAAATATTGGCGT1176 mir-18955mir-18TAAGGTGCATCTAGTGCAGATA262 mir-18955mir-18_RuvkunTAAGGTGCATCTAGTGCAGATAG1177 mir-19a956mir-19aTGTGCAAATCTATGCAAAACTGA268 mir-19b_1957mir-19b* (MichaelAGTTTTGCAGGTTTGCATCCAGC1179et al) mir-19b_1957mir-19bTGTGCAAATCCATGCAAAACTGA241 mir-19b_2958mir-19bTGTGCAAATCCATGCAAAACTGA241 mir-21959mir-21TAGCTTATCAGACTGATGTTGA236 mir-23a960mir-23aATCACATTGCCAGGGATTTCC289 mir-24_2961mir-24TGGCTCAGTTCAGCAGGAACAG264 mir-17/mir-91962mir-17 (human,ACTGCAGTGAAGGCACTTGT1180rat) mir-17/mir-91962mir-91_RuvkunCAAAGTGCTTACAGTGCAGGTAG1181 mir-17/mir-91962mir-17as/mir-91CAAAGTGCTTACAGTGCAGGTAGT204 mir-92_1963miR-92 (RFAM-M. mu.)TATTGCACTTGTCCCGGCCTG1182 mir-92_1963mir-92TATTGCACTTGTCCCGGCCTGT216 mir-96964mir-96TTTGGCACTAGCACATTTTTGC203 mir-96964miR-96 (RFAM-M. mu.)TTTGGCACTAGCACATTTTTGCT1183 mir-30a965mir-30aCTTTCAGTCGGATGTTTGCAGC193 mir-30a965miR-30a-sTGTAAACATCCTCGACTGGAAGC1184 mir-98966mir-98TGAGGTAGTAAGTTGTATTGTT257 mir-104967miR-104TCAACATCAGTCTGATAAGCTA335(Mourelatos)(Mourelatos) mir-105968miR-105TCAAATGCTCAGACTCCTGT1185(Mourelatos)(Mourelatos) mir-27969miR-27TTCACAGTGGCTAAGTTCC1186(Mourelatos)(Mourelatos) mir-27969miR-27a (RFAM-M. mu.)TTCACAGTGGCTAAGTTCCGC1187(Mourelatos) mir-27969miR-27a (RFAM-TTCACAGTGGCTAAGTTCCGCC1188(Mourelatos)Human) mir-92_2970miR-92 (RFAM-M. mu.)TATTGCACTTGTCCCGGCCTG1182 mir-92_2970mir-92TATTGCACTTGTCCCGGCCTGT216 mir-93971miR-93AAAGTGCTGTTCGTGCAGGTAG1189(Mourelatos)(Mourelatos) mir-93971miR-93 (Tuschl)CAAAGTGCTGTTCGTGC1190(Mourelatos) mir-93971miR-93 (RFAM-M. mu.)CAAAGTGCTGTTCGTGCAGGTAG1191(Mourelatos) mir-95972miR-95TTCAACGGGTATTTATTGAGCA1192(Mourelatos)(Mourelatos) mir-99973miR-99AACCCGTAGATCCGATCTTGTG1193(Mourelatos)(Mourelatos) mir-99973miR-99a (Tuschl)ACCCGTAGATCCGATCTTGT1194(Mourelatos) mir-25974miR-25 (Tuschl)CATTGCACTTGTCTCGGTCTGA1195 mir-28975miR-28 (Tuschl)AAGGAGCTCACAGTCTATTGAG1196 mir-31976miR-31 (RFAM-M. mu.)AGGCAAGATGCTGGCATAGCTG1197 mir-31976miR-31 (Tuschl)GGCAAGATGCTGGCATAGCTG1198 mir-32977miR-32 (Tuschl)TATTGCACATTACTAAGTTGC1199 mir-149978miR-149TCTGGCTCCGTGTCTTCACTCC1200 mir-30c_2979mir-30cTGTAAACATCCTACACTCTCAGC280 mir-30c_2979mir-30c_RuvkunTGTAAACATCCTACACTCTCAGCT1129 mir-99b980miR-99bCACCCGTAGAACCGACCTTGCG1201 MiR-125a981miR-125aTCCCTGAGACCCTTTAACCTGTG1202 MiR-125b_2982mir-125bTCCCTGAGACCCTAACTTGTGA258 mir-26a_2983miR-26a (MichaelTTCAAGTAATCCAGGATAGGC1203et al) mir-26a_2983mir-26aTTCAAGTAATCCAGGATAGGCT226 mir-127984mir-127_RuvkunTCGGATCCGTCTGAGCTTGG1204 mir-127984miR-127TCGGATCCGTCTGAGCTTGGCT1205 mir-136985miR-136ACTCCATTTGTTTTGATGATGGA1206 mir-154986miR-154TAGGTTATCCGTGTTGCCTTCG1207 mir-26a_1987miR-26a (MichaelTTCAAGTAATCCAGGATAGGC1203et al) mir-26a_1987mir-26aTTCAAGTAATCCAGGATAGGCT226 mir_186988miR-186CAAAGAATTCTCCTTTTGGGCTT1208 mir_198989mir-198GGTCCAGAGGGGAGATAGG1209 mir_191990mir-191CAACGGAATCCCAAAAGCAGCT1210 mir_191990mir-191_RuvkunCAACGGAATCCCAAAAGCAGCTGT1211 mir_206991mir-206TGGAATGTAAGGAAGTGTGTGG1212 mir-94/mir-106b992miR-94AAAGTGCTGACAGTGCAGAT1213 mir-94/mir-106b992miR-106b (RFAM-M. mu.)TAAAGTGCTGACAGTGCAGAT1214 mir_184993miR-184TGGACGGAGAACTGATAAGGGT1215 mir_195994miR-195TAGCAGCACAGAAATATTGGC1216 mir_193995miR-193AACTGGCCTACAAAGTCCCAG1217 mir_185996miR-185TGGAGAGAAAGGCAGTTC1218 mir_188997miR-188CATCCCTTGCATGGTGGAGGGT1219 mir_197998miR-197aTTCACCACCTTCTCCACCCAGC1220 mir_194_1999miR-194TGTAACAGCAACTCCATGTGGA1221 mir_2081000miR-208ATAAGACGAGCAAAAAGCTTGT1222 mir_194_21001miR-194TGTAACAGCAACTCCATGTGGA1221 mir_1391002miR-139TCTACAGTGCACGTGTCT1223 mir-200b1003miR-200a (RFAM-CTCTAATACTGCCTGGTAATGATG1224Human) mir-200b1003miR-200b (MichaelTAATACTGCCTGGTAATGATGA1225et al) mir-200b1003miR-200bTAATACTGCCTGGTAATGATGAC1226 mir-200a1004miR-200aTAACACTGTCTGGTAACGATG1227 mir-200a1004miR-200a (RFAM-M. mu.)TAACACTGTCTGGTAACGATGT1228 mir-240* (Kosik)1005mir-240* (Kosik)TCAAGAGCAATAACGAAAAATGT1229 mir-232* (Kosik)1006mir-232* (Kosik)CTGGCCCTCTCTGCCCTTCCGT1230 mir-227*1007mir-226* (Kosik)ACTGCCCCAGGTGCTGCTGG1231(Kosik)/mir-226*(Kosik) mir-227*1007mir-324-3p_RuvkunCCACTGCCCCAGGTGCTGCTGG1232(Kosik)/mir-226*(Kosik) mir-227*1007mir-227* (Kosik)CGCATCCCCTAGGGCATTGGTGT1233(Kosik)/mir-226*(Kosik) mir-244* (Kosik)1008mir-244* (Kosik)TCCAGCATCAGTGATTTTGTTGA1234 mir-224* (Kosik)1009mir-224* (Kosik)GCACATTACACGGTCGACCTCT1235 mir-248* (Kosik)1010mir-248* (Kosik)TCTCACACAGAAATCGCACCCGTC1236 ribosomal protein1011hypotheticalAGCCAGGTGCCTTCACCTGCT1155L5/hypotheticalmiRNA-168miRNA 168_2 hypothetical1012hypotheticalAGACAAACATGCTACTCTCAC1164miRNA-177_3miRNA-177 mir-138_31013mir-138AGCTGGTGTTGTGAATC256 mir-138_31013mir-138_RuvkunAGCTGGTGTTGTGAATCAGGCCG1127 mir-138_41014mir-138AGCTGGTGTTGTGAATC256 mir-181b_21015mir-181bAACATTCATTGCTGTCGGTGGGTT260 mir-219_11016mir-219TGATTGTCCAAACGCAATTCT271 mir-105_21017miR-105TCAAATGCTCAGACTCCTGT1185(Mourelatos) hypothetical miRNA1018hypotheticalTTACAGCAATCCAGTAATGAT1138120_2miRNA-120 cezanne 2/1019hypotheticalTCCTGTCAGACTTTGTTCGGT1237hypotheticalmiRNA-180miRNA-180 mir-103_21020mir-103AGCAGCATTGTACAGGGCTATGA225 mir-147 (Sanger)1021miR-147 (RFAM-GTGTGTGGAAATGCTTCTGC1238Human) mir-224 (Sanger)1022miR-224 (RFAM-CAAGTCACTAGTGGTTCCGTTTA1239Human) mir-134 (Sanger)1023miR-134 (RFAM-TGTGACTGGTTGACCAGAGGG1240Human) mir-146 (Sanger)1024miR-146 (RFAM-TGAGAACTGAATTCCATGGGTT1241Human) mir-150 (Sanger)1025miR-150 (RFAM-TCTCCCAACCCTTGTACCAGTG1242Human) mir-30e (RFAM/mmu)1026miR-30e (RFAM-M. mu.)TGTAAACATCCTTGACTGGA1243 mir-30e (RFAM/mmu)1026miR-97 (MichaelTGTAAACATCCTTGACTGGAAG1244et al) mir-296 (RFAM/mmu)1027miR-296 (RFAM-M. mu.)AGGGCCCCCCCTCAATCCTGT1245 mir-299 (RFAM/mmu)1028miR-299 (RFAM-M. mu.)TGGTTTACCGTCCCACATACAT1246 mir-301 (RFAM/mmu)1029miR-301 (RFAM-M. mu.)CAGTGCAATAGTATTGTCAAAGC1247 mir-301 (RFAM/mmu)1029mir-301_RuvkunCAGTGCAATAGTATTGTCAAAGCAT1248 mir-302 (RFAM/mmu)1030miR-302 (RFAM-M. mu.)TAAGTGCTTCCATGTTTTGGTGA1249 mir-34a (RFAM/mmu)1031mir-34c (RFAM)AGGCAGTGTAGTTAGCTGATTG1250 mir-34a (RFAM/mmu)1031miR-34a (RFAM-M. mu.)AGGCAGTGTAGTTAGCTGATTGC1251 mir_3201032miR-320AAAAGCTGGGTTGAGAGGGCGAA1252 mir-321_11033miR-321-1TAAGCCAGGGATTGTGGGTTC1253 mir-135b (Ruvkun)1034mir-135b (Ruvkun)TATGGCTTTTCATTCCTATGTG1254 mir-151* (Ruvkun)1035mir-151 (human)ACTAGACTGAAGCTCCTTGAGG1255 mir-151* (Ruvkun)1035mir-151* (Ruvkun)TCGAGGAGCTCACAGTCTAGTA1256 mir-340 (Ruvkun)1036mir-340 (Ruvkun)TCCGTCTCAGTTACTTTATAGCC1257 mir-331 (Ruvkun)1037mir-331 (Ruvkun)GCCCCTGGGCCTATCCTAGAA1258 mir_200c (RFAM)1038mir-200c (RFAM)AATACTGCCGGGTAATGATGGA1259 mir_34b (RFAM)1039mir-34b (RFAM)AGGCAGTGTCATTAGCTGATTG1260 mir_339_1 (RFAM)1040mir-339 (RFAM)TCCCTGTCCTCCAGGAGCTCA1261 mir_339_2 (RFAM)1041mir-339 (RFAM)TCCCTGTCCTCCAGGAGCTCA1261 mir-325 (Ruvkun)1042mir-325 (human)CCTAGTAGGTGTCCAGTAAGTGT1262 mir-326 (Ruvkun)1043miR-326 (Ruvkun)CCTCTGGGCCCTTCCTCCAG1263 mir-326 (Ruvkun)1044mir-326 (human)CCTCTGGGCCCTTCCTCCAGC1264 mir-329-1 (Ruvkun)1045mir-329 (human)AACACACCTGGTTAACCTCTTT1265 mir-329-2 (Ruvkun)1046mir-329 (human)AACACACCTGGTTAACCTCTTT1265 mir-330 (Ruvkun)1047mir-330 (human)GCAAAGCACACGGCCTGCAGAGA1266 mir-337 (Ruvkun)1048mir-337 (human)TCCAGCTCCTATATGATGCCTTT1267 mir-345 (Ruvkun)1049mir-345 (human)TGCTGACTCCTAGTCCAGGGC1268 mir-346 (Ruvkun)1050mir-346 (human)TGTCTGCCCGCATGCCTGCCTCT1269 mir-1871051miR-187 (RFAM-TCGTGTCTTGTGTTGCAGCCG1270Human) mir-1871051mir-187TCGTGTCTTGTGTTGCAGCCGG276 miR-24-11052miR-189 (RFAM-GTGCCTACTGAGCTGATATCAGT1271Human) miR-24-11052mir-24TGGCTCAGTTCAGCAGGAACAG264 mir-2151053mir-215ATGACCTATGAATTGACAGAC278

A list of mouse pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 61. â€œPri-miRNA nameâ€ indicates the gene name for each of the pri-miRNAs. Also given in table 61 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5â€² to 3â€² direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 61Mouse pri-miRNA sequences and the corresponding mature miRNAsSEQSEQIDIDPri-miRNA nameNOMature miRNA nameMature miRNA sequenceNOmir-26b1273miR-99TTCAAGTAATTCAGGATAGGT1147(Mourelatos) mir-26b1273miR-199-asTTCAAGTAATTCAGGATAGGTT281 mir-30a1274miR-199-asCTTTCAGTCGGATGTTTGCAGC193 mir-30a1274miR-26b (RFAM-TGTAAACATCCTCGACTGGAAGC1184Human) mir-30c_11275miR-32 (Tuschl)TGTAAACATCCTACACTCTCAGC280 mir-30c_11275let-7c_RuvkunTGTAAACATCCTACACTCTCAGCT1129 mir-128a1276mir-214TCACAGTGAACCGGTCTCTTT1073 mir-128a1276miR-29b (RFAM-TCACAGTGAACCGGTCTCTTTT200Human) mir-29b_11277mir-196TAGCACCATTTGAAATCAGT1172 mir-29b_11277hypotheticalTAGCACCATTTGAAATCAGTGT1173miRNA-079 mir-29b_11277mir-30cTAGCACCATTTGAAATCAGTGTT195 mir-29c1278mir-131_RuvkunCTAGCACCATTTGAAATCGGTT232 mir-29c1278hypotheticalTAGCACCATTTGAAATCGGTTA1100miRNA-033 mir-123/mir-1261279mir-326 (rodent)CATTATTACTTTTGGTACGCG205 mir-123/mir-1261279mir-126TCGTACCGTGAGTAATAATGC1076 mir-130a1280mir-23aCAGTGCAATGTTAAAAGGGC233 mir-130a1280hypotheticalCAGTGCAATGTTAAAAGGGCAT1101miRNA-040 mir-1d_11281mir-132TGGAATGTAAAGAAGTATGTA1083 mir-1d_11281mir-124a (Kosik)TGGAATGTAAAGAAGTATGTAT213 mir-1d_11281miR-200bTGGAATGTAAAGAAGTATGTATT1134 mir-124a_31282mir-100TAAGGCACGCGGTGAATGCCA1104 mir-124a_31282mir-212TTAAGGCACGCGGTGAATGCCA235 mir-124a_31282let-7aTTAAGGCACGCGGTGAATGCCAA1105 mir-133a_21283miR-189 (RFAM-TTGGTCCCCTTCAACCAGCTGT255Human) mir-124a_21284mir-181cTAAGGCACGCGGTGAATGCCA1104 mir-124a_21284mir-108TTAAGGCACGCGGTGAATGCCA235 mir-124a_21284mir-239* (Kosik)TTAAGGCACGCGGTGAATGCCAA1105 mir-131_1/mir-91285mir-325 (rodent)TAAAGCTAGATAACCGAAAGT211 mir-131_1/mir-91285mir-19bTAAAGCTAGATAACCGAAAGTA1080 mir-131_1/mir-91285mir-124a_RuvkunTCTTTGGTTATCTAGCTGTATGA1081 mir-15b1286mir-152TAGCAGCACATCATGGTTTAC1115 mir-15b1286hypotheticalTAGCAGCACATCATGGTTTACA246miRNA-111 mir-16_31287miR-104TAGCAGCACGTAAATATTGGCG196(Mourelatos) mir-16_31287mir-128aTAGCAGCACGTAAATATTGGCGT1176 mir-1371288mir-30bTATTGCTTAAGAATACGCGTAG270 mir-101_11289mir-18TACAGTACTGTGATAACTGA265 mir-101_11289mir-128bTACAGTACTGTGATAACTGAAG1170 mir-29a1291miR-27a (RFAM-M. mu.)CTAGCACCATCTGAAATCGGTT247 mir-29a1291mir-153TAGCACCATCTGAAATCGGTTA1116 mir-29b_21292mir-138_RuvkunTAGCACCATTTGAAATCAGT1172 mir-29b_21292hypotheticalTAGCACCATTTGAAATCAGTGT1173miRNA-075 mir-29b_21292miR-30a-sTAGCACCATTTGAAATCAGTGTT195 mir-148a1293miR-1d (Tuschl)TCAGTGCACTACAGAACTTTGT288 mir-1411294mir-16_RuvkunAACACTGTCTGGTAAAGATGG261 mir-131_3/mir-91295mir-124a (Kosik)TAAAGCTAGATAACCGAAAGT211 mir-131_3/mir-91295mir-7b (rodent)TAAAGCTAGATAACCGAAAGTA1080 mir-131_3/mir-91295mir-19aTCTTTGGTTATCTAGCTGTATGA1081 mir-23a1296miR-1 (RFAM)ATCACATTGCCAGGGATTTCC289 mir-24_21297mir-124a_RuvkunTGGCTCAGTTCAGCAGGAACAG264 mir-1401298miR-199b (mouse)AGTGGTTTTACCCTATGGTAG192 mir-1401298mir-205TACCACAGGGTAGAACCACGGA1065 mir-1401298mir-26bTACCACAGGGTAGAACCACGGACA1066 let-7a_41299mir-16_RuvkunTGAGGTAGTAGGTTGTATAGTT222 mir-125b_11300mir-131_RuvkunTCCCTGAGACCCTAACTTGTGA258 mir-26a_11301mir-29bTTCAAGTAATCCAGGATAGGC1203 mir-26a_11301hypotheticalTTCAAGTAATCCAGGATAGGCT226miRNA-154 let-7i1302hypotheticalTGAGGTAGTAGTTTGTGCT209miRNA-179 let-7i1302miR-1d (Tuschl)TGAGGTAGTAGTTTGTGCTGTT1078 mir-211303mir-125bTAGCTTATCAGACTGATGTTGA236 mir-221304mir-131AAGCTGCCAGTTGAAGAACTGT215 mir-1421305mir-131_RuvkunCATAAAGTAGAAAGCACTAC217 mir-1421305hypotheticalTGTAGTGTTTCCTACTTTATGG1086miRNA-105 mir-1421305mir-218TGTAGTGTTTCCTACTTTATGGA1087 mir-1441306mir-26aTACAGTATAGATGATGTACTAG237 mir-1521307miR-99a (Tuschl)TCAGTGCATGACAGAACTTGG282 mir-153_21308mir-29cTTGCATAGTCACAAAAGTGA201 let-7a_11309mir-16TGAGGTAGTAGGTTGTATAGTT222 let-7d1310mir-144AGAGGTAGTAGGTTGCATAGT245 let-7d1310hypotheticalAGAGGTAGTAGGTTGCATAGTT1113miRNA-171 let-7d1310miR-204 (Tuschl)CTATACGACCTGCTGCCTTTCT1114 let-7f_11311miR-9TGAGGTAGTAGATTGTATAGT1098 let-7f_11311hypotheticalTGAGGTAGTAGATTGTATAGTT231miRNA-138 mir-23b1312mir-1dATCACATTGCCAGGGATTACCAC208 miR-24-11313mir-124a (Kosik)GTGCCTACTGAGCTGATATCAGT1271 miR-24-11313hypotheticalTGGCTCAGTTCAGCAGGAACAG264miRNA-070 mir-27b1314miR-29c (Tuschl)TTCACAGTGGCTAAGTTCTG202 mir-27b1314mir-135TTCACAGTGGCTAAGTTCTGC1059 mir-131_2/mir-91315mir-107TAAAGCTAGATAACCGAAAGT211 mir-131_2/mir-91315miR-224 (RFAM-TAAAGCTAGATAACCGAAAGTA1080mouse) mir-131_2/mir-91315mir-124aTCTTTGGTTATCTAGCTGTATGA1081 mir-15a1316miR-20 (RFAM-TAGCAGCACATAATGGTTTGT1151Human) mir-15a1316miR-92 (RFAM-TAGCAGCACATAATGGTTTGTG269M. mu.) mir-16_11317mir-98TAGCAGCACGTAAATATTGGCG196 mir-16_11317mir-30c_RuvkunTAGCAGCACGTAAATATTGGCGT1176 mir-124a_11318miR-132 (RFAM-TAAGGCACGCGGTGAATGCCA1104Human) mir-124a_11318miR-140-asTTAAGGCACGCGGTGAATGCCA235 mir-124a_11318hypotheticalTTAAGGCACGCGGTGAATGCCAA1105miRNA-181 mir-181319mir-124aTAAGGTGCATCTAGTGCAGATA262 mir-181319miR-27TAAGGTGCATCTAGTGCAGATAG1177(Mourelatos) mir-201320mir-23bTAAAGTGCTTATAGTGCAGGTA1126 mir-201320mir-199aTAAAGTGCTTATAGTGCAGGTAG254 mir-30b1321miR-31 (Tuschl)TGTAAACATCCTACACTCAGC266 mir-30b1321mir-18_RuvkunTGTAAACATCCTACACTCAGCT1137 mir-30d1322miR-186TGTAAACATCCCCGACTGGAAG240 mir-30d1322let-7i_RuvkunTGTAAACATCCCCGACTGGAAGCT1108 let-7b1323mir-135TGAGGTAGTAGGTTGTGTGGTT212 let-7b1323mir-133aTGAGGTAGTAGGTTGTGTGGTTT1082 let7c_21324let-7d* (RFAM-M. mu.)TGAGGTAGTAGGTTGTATGGTT250 let7c_21324hypotheticalTGAGGTAGTAGGTTGTATGGTTT1120miRNA-170 let-7c_11325let-7dTGAGGTAGTAGGTTGTATGGTT250 let-7c_11325miR-135 (RFAM-TGAGGTAGTAGGTTGTATGGTTT1120Human) mir-991326miR-203 (Tuschl)AACCCGTAGATCCGATCTTGTG1193(Mourelatos) mir-991326mir-34ACCCGTAGATCCGATCTTGT1194(Mourelatos) LOC 1146141327mir-187TTAATGCTAATTGTGATAGGGG1459containing miR-155/hypotheticalmiRNA-071 let-7e1328let-7aTGAGGTAGGAGGTTGTATAGT249 mir-1d_21329miR-10b (MichaelTGGAATGTAAAGAAGTATGTA1083et al) mir-1d_21329miR-139TGGAATGTAAAGAAGTATGTAT213 mir-1d_21329mir-124aTGGAATGTAAAGAAGTATGTATT1134 mir-133a_11330mir-24TTGGTCCCCTTCAACCAGCTGT255 mir-1431331miR-15b (MichaelTGAGATGAAGCACTGTAGCTC1088et al) mir-1431331mir-253* (Kosik)TGAGATGAAGCACTGTAGCTCA220 mir-1451332mir-148bGTCCAGTTTTCCCAGGAATCC1122 mir-1451332let-7fGTCCAGTTTTCCCAGGAATCCCTT252 mir-122a1333miR-172 (RFAM-M. mu.)TGGAGTGTGACAATGGTGTTTG1084 mir-122a1333mir-124a_RuvkunTGGAGTGTGACAATGGTGTTTGT214 mir-19b_21334mir-22TGTGCAAATCCATGCAAAACTGA241 let-7f_21335hypotheticalTGAGGTAGTAGATTGTATAGT1098miRNA-137 let-7f_21335mir-131TGAGGTAGTAGATTGTATAGTT231 mir-26a_21336mir-29a_RuvkunTTCAAGTAATCCAGGATAGGC1203 mir-26a_21336hypotheticalTTCAAGTAATCCAGGATAGGCT226miRNA-153 mir-1271337mir-103TCGGATCCGTCTGAGCTTGG1204 mir-1271337mir-17as/mir-91TCGGATCCGTCTGAGCTTGGCT1205 mir-1361338mir-91_RuvkunACTCCATTTGTTTTGATGATGGA1206 mir-1541339mir-17-3p (mouse)TAGGTTATCCGTGTTGCCTTCG1207 mir-1491340let-7gL_RuvkunTCTGGCTCCGTGTCTTCACTCC1200 mir-30c_21341miR-31 (RFAM-TGTAAACATCCTACACTCTCAGC280M. mu.) mir-30c_21341let-7cTGTAAACATCCTACACTCTCAGCT1129 mir-99b1342mir-101b (rodent)CACCCGTAGAACCGACCTTGCG1201 MiR-125a1343mir-106 (mouse)TCCCTGAGACCCTTTAACCTGTG1202 MiR-125b_21344miR-9TCCCTGAGACCCTAACTTGTGA258 mir-2211345miR-200a (RFAM-AGCTACATTGTCTGCTGGGTTT1106Human) mir-2211345miR-26a (MichaelAGCTACATTGTCTGCTGGGTTTC238et al) mir-2031346mir-10bGTGAAATGTTTAGGACCACTAG197 mir-2031346mir-128 (Kosik)TGAAATGTTTAGGACCACTAG1068 mir-2031346mir-204TGAAATGTTTAGGACCACTAGA1069 let-7g1347hypotheticalTGAGGTAGTAGTTTGTACAGT285miRNA-176 let-7g1347mir-1dTGAGGTAGTAGTTTGTACAGTT1152 mir-101_31348miR-200aTACAGTACTGTGATAGCTGAAG1460 mir-1061349miR-200a (RFAM-CAAAGTGCTAACAGTGCAGGTA1461M. mu.) mir-17/mir-911350mir-123/mir-126asACTGCAGTGAGGGCACTTGT1462 mir-17/mir-911350mir-227* (Kosik)CAAAGTGCTTACAGTGCAGGTAG1181 mir-17/mir-911350miR-195CAAAGTGCTTACAGTGCAGGTAGT204 mir-199b1351mir-226* (Kosik)CCCAGTGTTTAGACTACCTGTTC1463 mir-199b1351mir-217 (rodent)TACAGTAGTCTGCACATTGGTT1118 hypothetical1352mir-324-3p_RuvkunAGAGGTATAGCGCATGGGAAGA1464miRNA 105 hypothetical1352miR-127TTCCTATGCATATACTTCTTT1132miRNA 105 mir-2111353mir-244* (Kosik)TTCCCTTTGTCATCCTTTGCCT1465 mir-2171354mir-224* (Kosik)TACTGCATCAGGAACTGACTGGAT1466 mir-224 (Sanger)1355mir-248* (Kosik)TAAGTCACTAGTGGTTCCGTTTA1467 mir-7_31356mir-138TGGAAGACTTGTGATTTTGTT1468 mir-325 (Ruvkun)1357mir-138_RuvkunCCTAGTAGGTGCTCAGTAAGTGT1469 mir-326 (Ruvkun)1358mir-181bCCTCTGGGCCCTTCCTCCAG1263 mir-326 (Ruvkun)1358miR-298CCTCTGGGCCCTTCCTCCAGT1470 mir-329-11359mir-103AACACACCCAGCTAACCTTTTT1471(Ruvkun) mir-330 (Ruvkun)1360miR-134 (RFAM-GCAAAGCACAGGGCCTGCAGAGA1472Human) mir-337 (Ruvkun)1361miR-146 (RFAM-TTCAGCTCCTATATGATGCCTTT1473Human) mir-345 (Ruvkun)1362miR-30e (RFAM-TGCTGACCCCTAGTCCAGTGC1474M. mu.) mir-346 (Ruvkun)1363miR-97 (MichaelTGTCTGCCCGAGTGCCTGCCTCT1475et al) mir-151* (Ruvkun)1364miR-193ACTAGACTGAGGCTCCTTGAGG1476 mir-151* (Ruvkun)1364mir-340 (Ruvkun)CTAGACTGAGGCTCCTTGAGG1477 mir-151* (Ruvkun)1364miR-299 (RFAM-TCGAGGAGCTCACAGTCTAGTA1256M. mu.) mir_34b (RFAM)1365mir-331 (Ruvkun)TAGGCAGTGTAATTAGCTGATTG1478 glutamate1366miR-143 (MichaelTGTTATAGTATTCCACCTACC1060receptor,et al)ionotrophic, AMPA3/hypotheticalmiRNA-033 mir-341367mir-138TGGCAGTGTCTTAGCTGGTTGT194 mir-341367mir-30aTGGCAGTGTCTTAGCTGGTTGTT1067 mir-7_1/mir-7_1*1368mir-191_RuvkunCAACAAATCACAGTCTGCCATA1070 mir-7_1/mir-7_1*1368mir-29bTGGAAGACTAGTGATTTTGTT198 mir-10b1369mir-210CCCTGTAGAACCGAATTTGTGT1071 mir-10b1369miR-29b (RFAM-TACCCTGTAGAACCGAATTTGT199M. mu.) mir-10b1369mir-34b (mouse)TACCCTGTAGAACCGAATTTGTG1072 mir-1321370mir-130aTAACAGTCTACAGCCATGGTCG1077 mir-1321370miR-196 (Tuschl)TAACAGTCTACAGCCATGGTCGC206 mir-108_11371mir-130 (Kosik)ATAAGGATTTTTAGGGGCATT207 mir-2121372miR-1 (RFAM)TAACAGTCTCCAGTCACGGCC210 hypothetical26mir-143TGGGCAAGAGGACTTTTTAAT1079miRNA 023 mir-21437mir-15bACAGCAGGCACAGACAGGCAG219 hypothetical43mir-145TGTCAACAAAACTGCTTACAA1092miRNA 040 hypothetical1373miR-145 (MichaelTGACAGGAAATCTTTGAGAGG1094miRNA 043et al) mir-2051374mir-101TCCTTCATTCCACCGGAGTCTG224 mir-33a1375miR-29b (RFAM-GTGCATTGTAGTTGCATTG227M. mu.) mir-196_21376mir-7-1*_RuvkunTAGGTAGTTTCATGTTGTTGG1097 mir-196_21376mir-148aTAGGTAGTTTCATGTTGTTGGG228 hypothetical1377mir-122aTTGCATGCCCTATTGATTCTC1099miRNA 055 hypothetical1378miR-122a,bTGTCAGATGCTTAATGTTCTT1102miRNA 058(Tuschl) mir-218_11379mir-140TTGTGCTTGATCTAACCATGT234 mir-218_11379mir-196TTGTGCTTGATCTAACCATGTG1103 mir-2221380miR-200b (MichaelAGCTACATCTGGCTACTGGGTCT1107et al) mir-2221380let-7iAGCTACATCTGGCTACTGGGTCTC239 mir-128b1381mir-142TCACAGTGAACCGGTCTCTTT1073 mir-128b1381hypotheticalTCACAGTGAACCGGTCTCTTTC242miRNA-023 mir-219_21382mir-30b_RuvkunTGATTGTCCAAACGCAATTCT271 hypothetical1383mir-19bTCACATTTGCCTGCAGAGATT1109miRNA 070 mir-129_21384miR-196 (Tuschl)AAGCCCTTACCCCAAAAAGCAT1110 mir-129_21384mir-128 (Kosik)CTTTTTGCGGTCTGGGCTTGC243 mir-129_21384miR-142-asCTTTTTGCGGTCTGGGCTTGCT1111 mir-133b1385miR-142asTTGGTCCCCTTCAACCAGCTA244(Michael et al) hypothetical78let-7fTGGTTAAAATATTAATGGGGC1112miRNA 075 hypothetical1386let-7f (MichaelTGATATGTTTGATATTGGG1117miRNA 079et al) mir-2041387let-7d_RuvkunTTCCCTTTGTCATCCTATGCCT251 mir-2041387miR-10b (Tuschl)TTCCCTTTGTCATCCTATGCCTG1121 mir-213/mir-1388mir-137AACATTCAACGCTGTCGGTGAG1096181a_2 mir-213/mir-1388hypotheticalAACATTCAACGCTGTCGGTGAGT223181a_2miRNA-043 mir-213/mir-1388let-7f (MichaelACCATCGACCGTTGATTGTACC253181a_2et al) hypothetical1389mir-26aTAGGCCAAATGGCGCATCAAT1124miRNA 090 mir-138_21390mir-92AGCTGGTGTTGTGAATC256 mir-138_21390miR-27* (MichaelAGCTGGTGTTGTGAATCAGGCCG1127et al) mir-196_11391miR-29b (RFAM-TAGGTAGTTTCATGTTGTTGG1097Human) mir-196_11391mir-7TAGGTAGTTTCATGTTGTTGGG228 mir-199a_21392miR-202 (mouse)CCCAGTGTTCAGACTACCTGTT1128 mir-199a_21392mir-15aCCCAGTGTTCAGACTACCTGTTC259 mir-199a_21392mir-211 (rodent)TACAGTAGTCTGCACATTGGTT1118 mir-181b_11393mir-16AACATTCATTGCTGTCGGTGGGTT260 hypothetical1394miR-26a (MichaelTGACAGTCAATTAACAAGTTT1130miRNA 101et al) hypothetical1395mir-127_RuvkunTTCCTCCTCCTCCGACTCGGA1135miRNA 111 mir-218_21396mir-33aTTGTGCTTGATCTAACCATGT234 mir-218_21396mir-24TTGTGCTTGATCTAACCATGTG1103 mir-148b1397mir-30dTCAGTGCATCACAGAACTTTGT272 mir-2161398mir-30d_RuvkunTAATCTCAGCTGGCAACTGTG274 hypothetical1399miR-136TAAACTGGCTGATAATTTTTG1141miRNA 137 hypothetical1400miR-154TGCAAGTATGAAAATGAGATT1142miRNA 138 mir-2101401let-7cCTGTGCGTGTGACAGCGGCTG277 mir-2231402let-7c_RuvkunTGTCAGTTTGTCAAATACCCC279 hypothetical1403miR-149TGCAAGCAGATGCTGATAATA1145miRNA 153 hypothetical1404mir-30cTTAAAGTGGATGTGTGTTATT1146miRNA 154 mir-135_11405hypotheticalTATGGCTTTTTATTCCTATGTGA1149miRNA-101 mir-135_11405let-7eTATGGCTTTTTATTCCTATGTGAT283 non-coding RNA in1406mir-181bTATGGCTTTTTATTCCTATGTGA1149rhabdomyosarcoma/mir-135_2 non-coding RNA in1406miR-155/TATGGCTTTTTATTCCTATGTGAT283rhabdomyosarcoma/hypotheticalmir-135_2miRNA-071 hypothetical1407mir-30c_RuvkunTGATCTTGCTCTAACACTTGG1157miRNA 170 glutamate174miR-99bTGACAAGTATGTTTTATCGTT1158receptor,ionotropic, AMPA2/hypotheticalmiRNA-171 hypothetical179miR-125aTAGGAGTTTGATATGACATAT1163miRNA 176 hypothetical1408mir-125bTGAAAGGCACTTTGTCCAATT1166miRNA 179 hypothetical1409mir-221TCACCTGCTCTGGAAGTAGTT1167miRNA 181 mir-181c1410mir-133aAACATTCAACCTGTCGGTGAGT290 mir-100_11411let-7bAACCCGTAGATCCGAACTTGTG275 mir-103_1950mir-29aAGCAGCATTGTACAGGGCTATGA225 mir-1071412mir-141AGCAGCATTGTACAGGGCTATCA229 mir-19a1413mir-20TGTGCAAATCTATGCAAAACTGA268 mir-19b_11414mir-21AGTTTTGCAGGTTTGCATCCAGC1179 mir-19b_11414mir-223TGTGCAAATCCATGCAAAACTGA241 mir-92_11415hypotheticalTATTGCACTTGTCCCGGCCTG1182miRNA-090 mir-92_11415miR-9TATTGCACTTGTCCCGGCCTGT216 mir-981416mir-131TGAGGTAGTAAGTTGTATTGTT257 mir-1041417mir-221 (RFAM-TCAACATCAGTCTGATAAGCTA335(Mourelatos)mmu) mir-271418mir-213TTCACAGTGGCTAAGTTCC1186(Mourelatos) mir-271418mir-222 (RFAM-TTCACAGTGGCTAAGTTCCGC1187(Mourelatos)mmu) mir-271418mir-203TTCACAGTGGCTAAGTTCCGCC1188(Mourelatos) mir-311419mir-178 (Kosik)AGGCAAGATGCTGGCATAGCTG1197 mir-311419miR-203 (RFAM-GGCAAGATGCTGGCATAGCTG1198M. mu.) mir-321420let-7gTATTGCACATTACTAAGTTGC1199 mir_1861421miR-326 (Ruvkun)CAAAGAATTCTCCTTTTGGGCTT1208 mir_1911422mir-329 (mouse)CAACGGAATCCCAAAAGCAGCT1210 mir_1911422miR-27a (RFAM-CAACGGAATCCCAAAAGCAGCTGT1211Human) mir_1951423mir-330 (rodent)TAGCAGCACAGAAATATTGGC1216 mir_1931424mir-337 (rodent)AACTGGCCTACAAAGTCCCAG1217 mir_1881425mir-345 (rodent)CATCCCTTGCATGGTGGAGGGT1219 mir_2081426mir-346 (mouse)ATAAGACGAGCAAAAAGCTTGT1222 mir_1391427mir-151* (Ruvkun)TCTACAGTGCACGTGTCT1223 mir-200b1428mir-151 (rodent)CTCTAATACTGCCTGGTAATGATG1224 mir-200b1428mir-216TAATACTGCCTGGTAATGATGA1225 mir-200b1428mir-219TAATACTGCCTGGTAATGATGAC1226 mir-200a1429mir-181aTAACACTGTCTGGTAACGATG1227 mir-200a1429mir-151L (rodent)TAACACTGTCTGGTAACGATGT1228 mir-227*1430mir-191ACTGCCCCAGGTGCTGCTGG1231(Kosik)/mir-226*(Kosik) mir-227*1430hypotheticalCCACTGCCCCAGGTGCTGCTGG1232(Kosik)/mir-226*miRNA-058(Kosik) mir-227*1430hypotheticalCGCATCCCCTAGGGCATTGGTGT1233(Kosik)/mir-226*miRNA-055(Kosik) mir-244* (Kosik)1431mir-218TCCAGCATCAGTGATTTTGTTGA1234 mir-224* (Kosik)1432mir-253* (Kosik)GCACATTACACGGTCGACCTCT1235 mir-248* (Kosik)1433mir-222TCTCACACAGAAATCGCACCCGTC1236 mir-138_31434mir-19b* (MichaelAGCTGGTGTTGTGAATC256et al) mir-138_31434mir-27bAGCTGGTGTTGTGAATCAGGCCG1127 mir-181b_21435mir-15_RuvkunAACATTCATTGCTGTCGGTGGGTT260 mir-103_21436miR-101 (RFAM-AGCAGCATTGTACAGGGCTATGA225Human) mir-134 (Sanger)1437mir-129TGTGACTGGTTGACCAGAGGG1240 mir-146 (Sanger)1438mir-129as/mir-TGAGAACTGAATTCCATGGGTT1241258* (Kosik) mir-30e1439miR-129b (RFAM-TGTAAACATCCTTGACTGGA1243(RFAM/mmu)Human) mir-30e1439miR-135 (RFAM-TGTAAACATCCTTGACTGGAAG1244(RFAM/mmu)Human) mir-2991440mir-133bTGGTTTACCGTCCCACATACAT1246(RFAM/mmu) mir-340 (Ruvkun)1441miR-188TCCGTCTCAGTTACTTTATAGCC1257 mir-331 (Ruvkun)1442miR-208GCCCCTGGGCCTATCCTAGAA1258 mir-1871443miR-199-sTCGTGTCTTGTGTTGCAGCCG1270 mir-1871443let-7b_RuvkunTCGTGTCTTGTGTTGCAGCCGG276 miR-2011444miR-187 (RFAM-TACTCAGTAAGGCATTGTTCT1479Human) miR-2071445miR-201GCTTCTCCTGGCTCTCCTCCCTC1480 miR-2911446miR-291AAAGTGCTTCCACTTTGTGTGCC1481 miR-2911446miR-207CATCAAAGTGGAGGCCCTCTCT1482 miR-2921447miR-291AAGTGCCGCCAGGTTTTGAGTGT1483 miR-2921447miR-292ACTCAAACTGGGGGCTCTTTTG1484 miR-2931448miR-292AGTGCCGCAGAGTTTGTAGTGT1485 miR-2941449miR-293AAAGTGCTTCCCTTTTGTGTGT1486 miR-2951450miR-294AAAGTGCTACTACTTTTGAGTCT1487 miR-3001451miR-295TATGCAAGGGCAAGCTCTCTTC1488 miR-3221452miR-300AAACATGAAGCGCTGCAACA1489 miR-3441453miR-322TGATCTAGCCAAAGCCTGACTGT1490 miR-3501454miR-344TTCACAAAGCCCATACACTTTCAC1491 miR-2901455miR-350CTCAAACTATGGGGGCACTTTTT1492 miR-3511456miR-290TCCCTGAGGAGCCCTTTGAGCCTG1493 miR-3411457miR-351TCGATCGGTCGGTCGGTCAGT1494 miR-2981458miR-341GGCAGAGGAGGGCTGTTCTTCC1495

A list of rat pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 62. â€œPri-miRNA nameâ€ indicates the gene name for each of the pri-miRNAs. Also given in table 62 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5â€² to 3â€² direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 62Rat pri-miRNA sequences and the corresponding mature miRNAsSEQSEQIDIDPri-miRNA nameNOMature miRNA nameMature miRNA sequenceNOmir-201496miR-20* (mouse)ACTGCATTACGAGCACTTACA1608 mir-201496miR-20 (RFAM-TAAAGTGCTTATAGTGCAGGTA1126Human) mir-201496mir-20TAAAGTGCTTATAGTGCAGGTAG254 mir-151* (Ruvkun)1497mir-151L (rodent)ACTAGACTGAGGCTCCTTGAGG1476 mir-151* (Ruvkun)1497mir-151 (rodent)CTAGACTGAGGCTCCTTGAGG1477 mir-151* (Ruvkun)1497mir-151* (Ruvkun)TCGAGGAGCTCACAGTCTAGTA1256 mir-346 (Ruvkun)1498miR-346 (rat)TGTCTGCCTGAGTGCCTGCCTCT1609 mir-1431499miR-143 (MichaelTGAGATGAAGCACTGTAGCTC1088et al) mir-1431499mir-143TGAGATGAAGCACTGTAGCTCA220 mir-2031500mir-203GTGAAATGTTTAGGACCACTAG197 mir-2031500miR-203 (RFAM-M. mu.)TGAAATGTTTAGGACCACTAG1068 mir-2031500miR-203 (Tuschl)TGAAATGTTTAGGACCACTAGA1069 mir-26b1501miR-26b (RFAM-TTCAAGTAATTCAGGATAGGT1147Human) mir-26b1501mir-26bTTCAAGTAATTCAGGATAGGTT281 mir-128a1276mir-128 (Kosik)TCACAGTGAACCGGTCTCTTT1073 mir-128a1276mir-128aTCACAGTGAACCGGTCTCTTTT200 mir-29b_11277miR-29b (RFAM-TAGCACCATTTGAAATCAGT1172Human) mir-29b_11277miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT1173 mir-29b_11502mir-29bTAGCACCATTTGAAATCAGTGTT195 mir-29c1278mir-29cCTAGCACCATTTGAAATCGGTT232 mir-29c1278miR-29c (Tuschl)TAGCACCATTTGAAATCGGTTA1100 mir-123/mir-1261503mir-123/mir-126asCATTATTACTTTTGGTACGCG205 mir-123/mir-1261503mir-126TCGTACCGTGAGTAATAATGC1076 mir-130a1504mir-130aCAGTGCAATGTTAAAAGGGC233 mir-130a1504mir-130 (Kosik)CAGTGCAATGTTAAAAGGGCAT1101 mir-124a_31282mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA1104 mir-124a_31282mir-124aTTAAGGCACGCGGTGAATGCCA235 mir-124a_31282mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA1105 mir-15b1286miR-15b (MichaelTAGCAGCACATCATGGTTTAC1115et al) mir-15b1286mir-15bTAGCAGCACATCATGGTTTACA246 mir-16_31505mir-16TAGCAGCACGTAAATATTGGCG196 mir-16_31505mir-16_RuvkunTAGCAGCACGTAAATATTGGCGT1176 mir-1371288mir-137TATTGCTTAAGAATACGCGTAG270 mir-101_11289mir-101TACAGTACTGTGATAACTGA265 mir-101_11289miR-101 (RFAM-TACAGTACTGTGATAACTGAAG1170Human) mir-29a1291mir-29aCTAGCACCATCTGAAATCGGTT247 mir-29a1291mir-29a_RuvkunTAGCACCATCTGAAATCGGTTA1116 mir-29b_21292miR-29b (RFAM-TAGCACCATTTGAAATCAGT1172Human) mir-29b_21292miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT1173 mir-29b_21292mir-29bTAGCACCATTTGAAATCAGTGTT195 mir-131_3/mir-91506mir-131TAAAGCTAGATAACCGAAAGT211 mir-131_3/mir-91506mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA1080 mir-131_3/mir-91506miR-9TCTTTGGTTATCTAGCTGTATGA1081 mir-23a1507mir-23aATCACATTGCCAGGGATTTCC289 mir-1401508mir-140AGTGGTTTTACCCTATGGTAG192 mir-1401508miR-140-asTACCACAGGGTAGAACCACGGA1065 mir-1401508mir-239* (Kosik)TACCACAGGGTAGAACCACGGACA1066 mir-125b_11509mir-125bTCCCTGAGACCCTAACTTGTGA258 mir-26a_11510miR-26a (MichaelTTCAAGTAATCCAGGATAGGC1203et al) mir-26a_11510mir-26aTTCAAGTAATCCAGGATAGGCT226 let-7i1302let-7iTGAGGTAGTAGTTTGTGCT209 let-7i1302let-7i_RuvkunTGAGGTAGTAGTTTGTGCTGTT1078 mir-211511mir-21TAGCTTATCAGACTGATGTTGA236 mir-221512mir-22AAGCTGCCAGTTGAAGAACTGT215 mir-1421513mir-142CATAAAGTAGAAAGCACTAC217 mir-1421513miR-142-asTGTAGTGTTTCCTACTTTATGG1086 mir-1421513miR-142asTGTAGTGTTTCCTACTTTATGGA1087(Michael et al) mir-1441514mir-144TACAGTATAGATGATGTACTAG237 mir-1521515mir-152TCAGTGCATGACAGAACTTGG282 mir-153_21516mir-153TTGCATAGTCACAAAAGTGA201 let-7a_11517let-7aTGAGGTAGTAGGTTGTATAGTT222 let-7d1518let-7dAGAGGTAGTAGGTTGCATAGT245 let-7d1518let-7d_RuvkunAGAGGTAGTAGGTTGCATAGTT1113 let-7d1518let-7d* (RFAM-M. mu.)CTATACGACCTGCTGCCTTTCT1114 let-7f_11519let-7f (MichaelTGAGGTAGTAGATTGTATAGT1098et al) let-7f_11519let-7fTGAGGTAGTAGATTGTATAGTT231 miR-24-11313miR-189 (RFAM-GTGCCTACTGAGCTGATATCAGT1271Human) miR-24-11313mir-24TGGCTCAGTTCAGCAGGAACAG264 mir-124a_11318mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA1104 mir-124a_11318mir-124aTTAAGGCACGCGGTGAATGCCA235 mir-124a_11318mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA1105 mir-181319mir-18TAAGGTGCATCTAGTGCAGATA262 mir-181319mir-18_RuvkunTAAGGTGCATCTAGTGCAGATAG1177 mir-30b1520mir-30bTGTAAACATCCTACACTCAGC266 mir-30b1520mir-30b_RuvkunTGTAAACATCCTACACTCAGCT1137 mir-30d1521mir-30dTGTAAACATCCCCGACTGGAAG240 mir-30d1521mir-30d_RuvkunTGTAAACATCCCCGACTGGAAGCT1108 let-7b1522let-7bTGAGGTAGTAGGTTGTGTGGTT212 let-7b1522let-7b_RuvkunTGAGGTAGTAGGTTGTGTGGTTT1082 let-7e1328let-7eTGAGGTAGGAGGTTGTATAGT249 mir-133a_11330mir-133aTTGGTCCCCTTCAACCAGCTGT255 mir-1451332miR-145 (MichaelGTCCAGTTTTCCCAGGAATCC1122et al) mir-1451332mir-145GTCCAGTTTTCCCAGGAATCCCTT252 mir-122a1523miR-122a,bTGGAGTGTGACAATGGTGTTTG1084(Tuschl) mir-122a1523mir-122aTGGAGTGTGACAATGGTGTTTGT214 let-7f_21335let-7f (MichaelTGAGGTAGTAGATTGTATAGT1098et al) let-7f_21335let-7fTGAGGTAGTAGATTGTATAGTT231 mir-1271337mir-127_RuvkunTCGGATCCGTCTGAGCTTGG1204 mir-1271337miR-127TCGGATCCGTCTGAGCTTGGCT1205 mir-1361338miR-136ACTCCATTTGTTTTGATGATGGA1206 mir-1541339miR-154TAGGTTATCCGTGTTGCCTTCG1207 mir-30c_21341mir-30cTGTAAACATCCTACACTCTCAGC280 mir-30c_21341mir-30c_RuvkunTGTAAACATCCTACACTCTCAGCT1129 mir-99b1342miR-99bCACCCGTAGAACCGACCTTGCG1201 MiR-125a1524miR-125aTCCCTGAGACCCTTTAACCTGTG1202 mir-2211525mir-221 (RFAM-AGCTACATTGTCTGCTGGGTTT1106mmu) mir-2211525mir-221AGCTACATTGTCTGCTGGGTTTC238 mir-101_31526mir-101b (rodent)TACAGTACTGTGATAGCTGAAG1460 mir-17/mir-911527mir-17 (human,ACTGCAGTGAAGGCACTTGT1180rat) mir-17/mir-911527mir-91_RuvkunCAAAGTGCTTACAGTGCAGGTAG1181 mir-17/mir-911527mir-17as/mir-91CAAAGTGCTTACAGTGCAGGTAGT204 hypothetical1528hypotheticalTTCCTATGCATATACTTCTTT1132miRNA 105miRNA-105 mir-2111529mir-211 (rodent)TTCCCTTTGTCATCCTTTGCCT1465 mir-2171530mir-217 (rodent)TACTGCATCAGGAACTGACTGGAT1466 mir-7_31531mir-7b (rodent)TGGAAGACTTGTGATTTTGTT1468 mir-325 (Ruvkun)1357mir-325 (rodent)CCTAGTAGGTGCTCAGTAAGTGT1469 mir-326 (Ruvkun)1532miR-326 (Ruvkun)CCTCTGGGCCCTTCCTCCAG1263 mir-326 (Ruvkun)1532mir-326 (rodent)CCTCTGGGCCCTTCCTCCAGT1470 mir-330 (Ruvkun)1533mir-330 (rodent)GCAAAGCACAGGGCCTGCAGAGA1472 mir-337 (Ruvkun)1361mir-337 (rodent)TTCAGCTCCTATATGATGCCTTT1473 mir-345 (Ruvkun)1362mir-345 (rodent)TGCTGACCCCTAGTCCAGTGC1474 mir_34b (RFAM)1365mir-34b (mouse)TAGGCAGTGTAATTAGCTGATTG1478 mir-341534mir-34TGGCAGTGTCTTAGCTGGTTGT194 mir-341534miR-172 (RFAM-M. mu.)TGGCAGTGTCTTAGCTGGTTGTT1067 mir-7_1/mir-7_1*1535mir-7_1*_RuvkunCAACAAATCACAGTCTGCCATA1070 mir-7_1/mir-7_1*1535mir-7TGGAAGACTAGTGATTTTGTT198 mir-10b1536miR-10b (Tuschl)CCCTGTAGAACCGAATTTGTGT1071 mir-10b1536mir-10bTACCCTGTAGAACCGAATTTGT199 mir-10b1536miR-10b (MichaelTACCCTGTAGAACCGAATTTGTG1072et al) mir-1321370miR-132 (RFAM-TAACAGTCTACAGCCATGGTCG1077Human) mir-1321370mir-132TAACAGTCTACAGCCATGGTCGC206 mir-2121537mir-212TAACAGTCTCCAGTCACGGCC210 mir-108_11538mir-108ATAAGGATTTTTAGGGGCATT207 hypothetical26hypotheticalTGGGCAAGAGGACTTTTTAAT1079miRNA 023miRNA-023 mir-2141539mir-214ACAGCAGGCACAGACAGGCAG219 hypothetical43hypotheticalTGTCAACAAAACTGCTTACAA1092miRNA 040miRNA-040 hypothetical1540hypotheticalTGACAGGAAATCTTTGAGAGG1094miRNA 043miRNA-043 mir-2051541mir-205TCCTTCATTCCACCGGAGTCTG224 mir-33a1542mir-33aGTGCATTGTAGTTGCATTG227 mir-196_21543miR-196 (Tuschl)TAGGTAGTTTCATGTTGTTGG1097 mir-196_21543mir-196TAGGTAGTTTCATGTTGTTGGG228 mir-218_11544mir-218TTGTGCTTGATCTAACCATGT234 mir-218_11544mir-253* (Kosik)TTGTGCTTGATCTAACCATGTG1103 mir-2221545mir-222 (RFAM-AGCTACATCTGGCTACTGGGTCT1107mmu) mir-2221545mir-222AGCTACATCTGGCTACTGGGTCTC239 mir-128b1381mir-128 (Kosik)TCACAGTGAACCGGTCTCTTT1073 mir-128b1381mir-128bTCACAGTGAACCGGTCTCTTTC242 mir-219_21546mir-219TGATTGTCCAAACGCAATTCT271 hypothetical1547hypotheticalTCACATTTGCCTGCAGAGATT1109miRNA 070miRNA-070 mir-129_21548mir-129as/mir-AAGCCCTTACCCCAAAAAGCAT1110258* (Kosik) mir-129_21548mir-129CTTTTTGCGGTCTGGGCTTGC243 mir-129_21548miR-129b (RFAM-CTTTTTGCGGTCTGGGCTTGCT1111Human) mir-133b1385mir-133bTTGGTCCCCTTCAACCAGCTA244 hypothetical78hypotheticalTGGTTAAAATATTAATGGGGC1112miRNA 075miRNA-075 mir-2041549mir-204TTCCCTTTGTCATCCTATGCCT251 mir-2041549miR-204 (Tuschl)TTCCCTTTGTCATCCTATGCCTG1121 mir-213/mir-1550mir-178 (Kosik)AACATTCAACGCTGTCGGTGAG1096181a_2 mir-213/mir-1550mir-181aAACATTCAACGCTGTCGGTGAGT223181a_2 mir-213/mir-1550mir-213ACCATCGACCGTTGATTGTACC253181a_2 hypothetical1551hypotheticalTAGGCCAAATGGCGCATCAAT1124miRNA 090miRNA-090 mir-138_21552mir-138AGCTGGTGTTGTGAATC256 mir-138_21552mir-138_RuvkunAGCTGGTGTTGTGAATCAGGCCG1127 mir-199a_21553miR-199-sCCCAGTGTTCAGACTACCTGTT1128 mir-199a_21553mir-199aCCCAGTGTTCAGACTACCTGTTC259 mir-199a_21553miR-199-asTACAGTAGTCTGCACATTGGTT1118 hypothetical1554hypotheticalTGACAGTCAATTAACAAGTTT1130miRNA 101miRNA-101 mir-148b1397mir-148bTCAGTGCATCACAGAACTTTGT272 mir-2161555mir-216TAATCTCAGCTGGCAACTGTG274 hypothetical1399hypotheticalTAAACTGGCTGATAATTTTTG1141miRNA 137miRNA-137 hypothetical1556hypotheticalTGCAAGTATGAAAATGAGATT1142miRNA 138miRNA-138 mir-2101557mir-210CTGTGCGTGTGACAGCGGCTG277 mir-2231558mir-223TGTCAGTTTGTCAAATACCCC279 hypothetical1404hypotheticalTTAAAGTGGATGTGTGTTATT1146miRNA 154miRNA-154 non-coding RNA in13miR-135 (RFAM-TATGGCTTTTTATTCCTATGTGA1149rhabdomyosarcoma/Human)mir-135_2 non-coding RNA in13mir-135TATGGCTTTTTATTCCTATGTGAT283rhabdomyosarcoma/mir-135_2 hypothetical1559hypotheticalTGATCTTGCTCTAACACTTGG1157miRNA 170miRNA-170 glutamate174hypotheticalTGACAAGTATGTTTTATCGTT1158receptor,miRNA-171ionotropic, AMPA2/hypotheticalmiRNA-171 hypothetical179hypotheticalTAGGAGTTTGATATGACATAT1163miRNA 176miRNA-176 hypothetical1560hypotheticalTGAAAGGCACTTTGTCCAATT1166miRNA 179miRNA-179 hypothetical1409hypotheticalTCACCTGCTCTGGAAGTAGTT1167miRNA 181miRNA-181 mir-181c1410mir-181cAACATTCAACCTGTCGGTGAGT290 mir-100_11561mir-100AACCCGTAGATCCGAACTTGTG275 mir-103_1950mir-103AGCAGCATTGTACAGGGCTATGA225 mir-1071562mir-107AGCAGCATTGTACAGGGCTATCA229 mir-19a1563mir-19aTGTGCAAATCTATGCAAAACTGA268 mir-19b_11414mir-19b* (MichaelAGTTTTGCAGGTTTGCATCCAGC1179et al) mir-19b_11414mir-19bTGTGCAAATCCATGCAAAACTGA241 mir-92_11564miR-92 (RFAM-M. mu.)TATTGCACTTGTCCCGGCCTG1182 mir-92_11564mir-92TATTGCACTTGTCCCGGCCTGT216 mir-981565mir-98TGAGGTAGTAAGTTGTATTGTT257 mir-1041566miR-104TCAACATCAGTCTGATAAGCTA335(Mourelatos)(Mourelatos) mir-271567miR-27TTCACAGTGGCTAAGTTCC1186(Mourelatos)(Mourelatos) mir-271567miR-27a (RFAM-M. mu.)TTCACAGTGGCTAAGTTCCGC1187(Mourelatos) mir-271567miR-27a (RFAM-TTCACAGTGGCTAAGTTCCGCC1188(Mourelatos)Human) mir-311568miR-31 (RFAM-M. mu.)AGGCAAGATGCTGGCATAGCTG1197 mir-311568miR-31 (Tuschl)GGCAAGATGCTGGCATAGCTG1198 mir-321569miR-32 (Tuschl)TATTGCACATTACTAAGTTGC1199 mir_1861570miR-186CAAAGAATTCTCCTTTTGGGCTT1208 mir_1911571mir-191CAACGGAATCCCAAAAGCAGCT1210 mir_1911422mir-191_RuvkunCAACGGAATCCCAAAAGCAGCTGT1211 mir_1951572miR-195TAGCAGCACAGAAATATTGGC1216 mir_1931573miR-193AACTGGCCTACAAAGTCCCAG1217 mir_2081574miR-208ATAAGACGAGCAAAAAGCTTGT1222 mir_1391427miR-139TCTACAGTGCACGTGTCT1223 mir-200b1428miR-200a (RFAM-CTCTAATACTGCCTGGTAATGATG1224Human) mir-200b1428miR-200b (MichaelTAATACTGCCTGGTAATGATGA1225et al) mir-200b1428miR-200bTAATACTGCCTGGTAATGATGAC1226 mir-200a1429miR-200aTAACACTGTCTGGTAACGATG1227 mir-200a1429miR-200a (RFAM-M. mu.)TAACACTGTCTGGTAACGATGT1228 mir-227*1430mir-226* (Kosik)ACTGCCCCAGGTGCTGCTGG1231(Kosik)/mir-226*(Kosik) mir-227*1430mir-324-3p_RuvkunCCACTGCCCCAGGTGCTGCTGG1232(Kosik)/mir-226*(Kosik) mir-227*1430mir-227* (Kosik)CGCATCCCCTAGGGCATTGGTGT1233(Kosik)/mir-226*(Kosik) mir-244* (Kosik)1431mir-244* (Kosik)TCCAGCATCAGTGATTTTGTTGA1234 mir-224* (Kosik)1432mir-224* (Kosik)GCACATTACACGGTCGACCTCT1235 mir-248* (Kosik)1433mir-248* (Kosik)TCTCACACAGAAATCGCACCCGTC1236 mir-138_31575mir-138AGCTGGTGTTGTGAATC256 mir-138_31575mir-138_RuvkunAGCTGGTGTTGTGAATCAGGCCG1127 mir-181b_21576mir-181bAACATTCATTGCTGTCGGTGGGTT260 mir-134 (Sanger)1289miR-134 (RFAM-TGTGACTGGTTGACCAGAGGG1240Human) mir-146 (Sanger)1577miR-146 (RFAM-TGAGAACTGAATTCCATGGGTT1241Human) mir-30e1578miR-30e (RFAM-M. mu.)TGTAAACATCCTTGACTGGA1243(RFAM/mmu) mir-30e1578miR-97 (MichaelTGTAAACATCCTTGACTGGAAG1244(RFAM/mmu)et al) mir-2991440miR-299 (RFAM-M. mu.)TGGTTTACCGTCCCACATACAT1246(RFAM/mmu) mir-34a1579mir-34c (RFAM)AGGCAGTGTAGTTAGCTGATTG1250(RFAM/mmu) mir-34a1579miR-34a (RFAM-M. mu.)AGGCAGTGTAGTTAGCTGATTGC1251(RFAM/mmu) mir-135b (Ruvkun)1580mir-135b (Ruvkun)TATGGCTTTTCATTCCTATGTG1254 mir-331 (Ruvkun)1442mir-331 (Ruvkun)GCCCCTGGGCCTATCCTAGAA1258 mir-1871443miR-187 (RFAM-TCGTGTCTTGTGTTGCAGCCG1270Human) mir-1871443mir-187TCGTGTCTTGTGTTGCAGCCGG276 collagen, type I,1581hypotheticalAGACATGTTCAGCTTTGTGGA1063alpha 1/miRNA-144hypotheticalmiRNA-144 DiGeorge syndrome1582hypotheticalTGTGATTTCCAATAATTGAGG1123critical regionmiRNA-088gene 8/hypotheticalmiRNA-088 hypothetical miR-1583miR-190TGATATGTTTGATATATTAGGT107513/miR-190 hypothetical1584hypotheticalTAAGACTTGCAGTGATGTTTA1091miRNA 039miRNA-039 hypothetical1585hypotheticalTACCAGTTGTTTTCTCTGTGA1093miRNA 041miRNA-041 hypothetical47hypotheticalTTCCACTCTGTTTATCTGACA1095miRNA 044miRNA-044 hypothetical86hypotheticalTTACATGGGGAAGCTATCATA1119miRNA 083miRNA-083 hypothetical1586hypotheticalTGACAGTTTATTGGCTTTATC1133miRNA 107miRNA-107 mir-10a1587mir-10a (Tuschl)TACCCTGTAGATCCGAATTTGT1139 mir-10a1587mir-10aTACCCTGTAGATCCGAATTTGTG267 mir-130b1588mir-130bCAGTGCAATGATGAAAGGGC273 mir-130b1588mir-266* (Kosik)CAGTGCAATGATGAAAGGGCAT1140 hypothetical1589hypotheticalAGACAAACATGCTACTCTCAC1164miRNA-177_1miRNA-177 mir_1851590miR-185TGGAGAGAAAGGCAGTTC1218 mir_194_21591miR-194TGTAACAGCAACTCCATGTGGA1221 mir-150 (Sanger)1592miR-150 (RFAM-TCTCCCAACCCTTGTACCAGTG1242Human) mir-3011593miR-301 (RFAM-M. mu.)CAGTGCAATAGTATTGTCAAAGC1247(RFAM/mmu) mir-3011593mir-301_RuvkunCAGTGCAATAGTATTGTCAAAGCAT1248(RFAM/mmu) mir_3201594miR-320AAAAGCTGGGTTGAGAGGGCGAA1252 mir_200c (RFAM)1595mir-200c (RFAM)AATACTGCCGGGTAATGATGGA1259 miR-3221596miR-322AAACATGAAGCGCTGCAACA1489 miR-3411457miR-341TCGATCGGTCGGTCGGTCAGT1494 miR-3441597miR-344TGATCTAGCCAAAGCCTGACCGT1610 miR-3501598miR-350TTCACAAAGCCCATACACTTTCAC1491 miR-3511599miR-351TCCCTGAGGAGCCCTTTGAGCCTG1493 miR-2901600miR-290CTCAAACTATGGGGGCACTTTTT1492 miR-2911601miR-291AAAGTGCTTCCACTTTGTGTGCC1481 miR-2911601miR-291CATCAAAGTGGAGGCCCTCTCT1482 miR-2921602miR-292AAGTGCCGCCAGGTTTTGAGTGT1483 miR-2921602miR-292ACTCAAACTGGGGGCTCTTTTG1484 miR-2981603miR-298GGCAGAGGAGGGCTGTTCTTCC1495 miR-3001604miR-300TATGCAAGGGCAAGCTCTCTTC1488 miR-3331605miR-333GTGGTGTGCTAGTTACTTTT1611 miR-3361606miR-336TCACCCTTCCATATCTAGTCT1612 miR-3491607miR-349CAGCCCTGCTGTCTTAACCTCT1613

A list of Drosophila pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 63. â€œPri-miRNA nameâ€ indicates the gene name for each of the pri-miRNAs, and â€œpri-miRNA sequenceâ€ indicates the sequence of the predicted primary miRNA transcript. Also given in table 63 are the name and sequence of the mature miRNA derived from the pri-miRNA. The sequences are written in the 5â€² to 3â€² direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

TABLE 63Drosophila pri-miRNA sequences and the corresponding mature miRNAsPri-SEQSEQmiRNAIDMature miRNAIDnamePri-miRNA sequenceNOnameMature miRNA sequenceNOmir-14GGAGCGAGACGGGGACTCACT1614miR-14TCAGTCTTTTTCTCTCTCCTA1616GTGCTTATTAAATAGTCAGTCTTTTTCTCTCTCCTATACAAATTGCGGGC mir-AATGATTTGACTACGAAACCG1615mir-BantamGTGAGATCATTTTGAAAGCTG1617bantamGTTTTCGATTTGGTTTGACTGTTTTTCATACAAGTGAGATCATTTTGAAAGCTGATTTTGTCAATGAATA

Oligomeric compounds targeting or mimicking pri-miRNAs, pre-miRNAs, or miRNAs were given internal numerical identifiers (ISIS Numbers) and are shown in Tables 64, 65, and 66 respectively. The sequences are written in the 5â€² to 3â€² direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.

Table 64 describes a series of oligomeric compounds designed and synthesized to target different regions of pri-miRNAs. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. In Table 64, â€œPri-miRNAâ€ indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds listed in Table 64 have phosphorothioate internucleoside linkages. In some embodiments, chimeric oligonucleotides (â€œgapmersâ€ ) are composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by five nucleotide â€œwings,â€ wherein the wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) nucleotides. These chimeric compounds are indicated in the â€œChemistryâ€ column as â€œ5-10-5 MOE gapmer.â€ In some embodiments, oligomeric compound consist of 2â€²-MOE ribonucleotides throughout, and these are indicated by â€œuniform MOE.â€

TABLE 64Phosphorothioate oligomeric compounds targeting pri-miRNAsSEQIDISIS #NOsequencechemistryPri-miRNA338615442AGAACAGCATGACGTAACCTuniform MOEmir-140, Human 338616443GCCCATCTGTGGCTTCACAGuniform MOEmir-30a, Human 338617444GAAGTCCGAGGCAGTAGGCAuniform MOEmir-30a, Human 338618445CTTCCTTACTATTGCTCACAuniform MOEmir-34, Human 338619446GCTAGATACAAAGATGGAAAuniform MOEmir-29b-1, Human 338620447CTAGACAATCACTATTTAAAuniform MOEmir-29b-2, Human 338621448GCAGCGCAGCTGGTCTCCCCuniform MOEmir-29b-2, Human 338622449TAATATATATTTCACTACGCuniform MOEmir-16-3, Human 338623450TGCTGTATCCCTGTCACACTuniform MOEmir-16-3, Human 338624451CAATTGCGCTACAGAACTGTuniform MOEmir-203, Human 338625452TCGATTTAGTTATCTAAAAAuniform MOEmir-7-1, Human 338626453CTGTAGAGGCATGGCCTGTGuniform MOEmir-7-1, Human 338627454TGACTATACGGATACCACACuniform MOEmir-10b, Human 338628455GGAACAAGGCCAATTATTGCuniform MOEmir-128a, Human 338629456AGAAATGTAAACCTCTCAGAuniform MOEmir-128a, Human 338630457AGCTGTGAGGGAGAGAGAGAuniform MOEmir-153-1, Human 338631458CTGGAGTGAGAATACTAGCTuniform MOEmir-153-1, Human 338632459ACTGGGCTCATATTACTAGCuniform MOEmir-153-2, Human 338633460TTGGATTAAATAACAACCTAuniform MOEhypothetical miR-13/miR-190, Human 338634461CCCGGAGACAGGGCAAGACAuniform MOEhypothetical miR-13/miR-190, Human 338635462AAAGCGGAAACCAATCACTGuniform MOEchromosome 9 ORF3containing mir-23b,mir-24-2 and mir-27b,Human 338636463GTCCCCATCTCACCTTCTCTuniform MOEchromosome 9 ORF3containing mir-23b,mir-24-2 and mir-27b,Human 338637464TCAGAGCGGAGAGACACAAGuniform MOEmir-96, Human 338638465TAGATGCACATATCACTACCuniform MOEmiR-17/mir-91, Human 338639466CTTGGCTTCCCGAGGCAGCTuniform MOEmiR-17/mir-91, Human 338640467AGTTTGAAGTGTCACAGCGCuniform MOEmir-123/mir-126, Human 338641468GCGTTTTCGATGCGGTGCCGuniform MOEmir-123/mir-126, Human 338642469GAGACGCGGGGGCGGGGCGCuniform MOEmir-132, Human 338643470TACCTCCAGTTCCCACAGTAuniform MOEmir-132, Human 338644471TGTGTTTTCTGACTCAGTCAuniform MOEmir-108-1, Human 338645472AGAGCACCTGAGAGCAGCGCuniform MOEchromosome 9 ORF3containing mir-23b,mir-24-2 and mir-27b,Human 338646473TCTTAAGTCACAAATCAGCAuniform MOEchromosome 9 ORF3containing mir-23b,mir-24-2 and mir-27b,Human 338647474TCTCCACAGCGGGCAATGTCuniform MOElet-7i, Human 338648475GGCGCGCTGTCCGGGCGGGGuniform MOEmir-212, Human 338649476ACTGAGGGCGGCCCGGGCAGuniform MOEmir-212, Human 338650477GTCCTCTTGCCCAAGCAACAuniform MOEhypothetical miRNA-023,Human 338651478GAAGACCAATACACTCATACuniform MOEmir-131-2/miR-9, Human 338652479CCGAGGGGCAACATCACTGCuniform MOElet-7b, Human 338653480TCCATAGCTTAGCAGGTCCAuniform MOEmir-1d-1, Human 338654481TTTGATAGTTTAGACACAAAuniform MOEmir-122a, Human 338655482GGGAAGGATTGCCTAGCAGTuniform MOEmir-122a, Human 338656483AGCTTTAGCTGGGTCAGGACuniform MOEmir-22, Human 338657484TACCATACAGAAACACAGCAuniform MOEmir-92-1, Human 338658485TCACAATCCCCACCAAACTCuniform MOEmir-92-1, Human 338659486TCACTCCTAAAGGTTCAAGTuniform MOEhypothetical miRNA-30,Human 338660487CACCCTCCAGTGCTGTTAGTuniform MOEmir-142, Human 338661488CTGACTGAGACTGTTCACAGuniform MOEmir-183, Human 338662489CCTTTAGGGGTTGCCACACCuniform MOEglutamate receptor,ionotrophic, AMPA 3/hypothetical miRNA-033,Human 338663490ACAGGTGAGCGGATGTTCTGuniform MOEmir-214, Human 338665492AGAGGGGAGACGAGAGCACTuniform MOEmir-192-1, Human 338666493TCACGTGGAGAGGAGTTAAAuniform MOEhypothetical miRNA-039,Human 338667494AGTGCTAATACTTCTTTCATuniform MOEhypothetical miRNA-040,Human 338668495ACCTGTGTAACAGCCGTGTAuniform MOEhypothetical miRNA-041,Human 338669496TTATCGGAACTTCACAGAGAuniform MOEhypothetical miRNA-041,Human 338670497TCCCATAGCAGGGCAGAGCCuniform MOElet-7a-3, Human 338671498GGCACTTCATTGCTGCTGCCuniform MOEhypothetical miRNA-043,Human 338672499GGAGCCTTGCGCTCAGCATTuniform MOEhypothetical miRNA-043,Human 338673500ATGGTAATTTCATTTCAGGCuniform MOEhypothetical miRNA-044,Human 338674501GATTGCACATCCACACTGTCuniform MOEhypothetical miRNA-044,Human 338675502GCTGGCCTGATAGCCCTTCTuniform MOEmir-181a, Human 338676503GTTTTTTCAAATCCCAAACTuniform MOEmir-181a, Human 338677504CCCAGTGGTGGGTGTGACCCuniform MOElet-7a-1, Human 338678505CTGGTTGGGTATGAGACAGAuniform MOEmir-205, Human 338679506TTGATCCATATGCAACAAGGuniform MOEmir-103-1, Human 338680507GCCATTGGGACCTGCACAGCuniform MOEmiR-26a-1, Human 338681508ATGGGTACCACCAGAACATGuniform MOEmir-33a, Human 338682509AGTTCAAAACTCAATCCCAAuniform MOEmir-196-2, Human 338683510GCCCTCGACGAAAACCGACTuniform MOEmir-196-2, Human 338684511TTGAACTCCATGCCACAAGGuniform MOEmir-107, Human 338685512AGGCCTATTCCTGTAGCAAAuniform MOEmir-106, Human 338686513GTAGATCTCAAAAAGCTACCuniform MOEmir-106, Human 338687514CTGAACAGGGTAAAATCACTuniform MOElet-7f-1, Human 338688515AGCAAGTCTACTCCTCAGGGuniform MOElet-7f-1, Human 338689516AATGGAGCCAAGGTGCTGCCuniform MOEhypothetical miRNA-055,Human 338690517TAGACAAAAACAGACTCTGAuniform MOEmir-29c, Human 338691518GCTAGTGACAGGTGCAGACAuniform MOEmir-130a, Human 338692519GGGCCTATCCAAAGTGACAGuniform MOEhypothetical miRNA-058,Human 338693520TACCTCTGCAGTATTCTACAuniform MOEhypothetical miRNA-058,Human 338694521TTTACTCATACCTCGCAACCuniform MOEmir-218-1, Human 338695522AATTGTATGACATTAAATCAuniform MOEmir-124a-2, Human 338696523CTTCAAGTGCAGCCGTAGGCuniform MOEmir-124a-2, Human 338697524TGCCATGAGATTCAACAGTCuniform MOEmir-21, Human 338698525ACATTGCTATCATAAGAGCTuniform MOEmir-16-1, Human 338699526TAATTTTAGAATCTTAACGCuniform MOEmir-16-1, Human 338700527AGTGTCTCATCGCAAACTTAuniform MOEmir-144, Human 338701528TGTTGCCTAACGAACACAGAuniform MOEmir-221, Human 338702529GCTGATTACGAAAGACAGGAuniform MOEmir-222, Human 338703530GCTTAGCTGTGTCTTACAGCuniform MOEmir-30d, Human 338704531GAGGATGTCTGTGAATAGCCuniform MOEmir-30d, Human 338705532CCACATATACATATATACGCuniform MOEmir-19b-2, Human 338706533AGGAAGCACACATTATCACAuniform MOEmir-19b-2, Human 338707534GACCTGCTACTCACTCTCGTuniform MOEmir-128b, Human 338708535GGTTGGCCGCAGACTCGTACuniform MOEhypothetical miRNA069/mir-219-2, Human 338709536GATGTCACTGAGGAAATCACuniform MOEhypothetical miRNA-070,Human 338710537TCAGTTGGAGGCAAAAACCCuniform MOELOC 114614/hypothetical miRNA-071,Human 338711538GGTAGTGCAGCGCAGCTGGTuniform MOEmir-29b-2, Human 338712539CCGGCTATTGAGTTATGTACuniform MOEmir-129-2, Human 338713540ACCTCTCAGGAAGACGGACTuniform MOEmir-133b, Human 338714541GAGCATGCAACACTCTGTGCuniform MOEhypothetical miRNA-075,Human 338715542CCTCCTTGTGGGCAAAATCCuniform MOElet-7d, Human 338716543CGCATCTTGACTGTAGCATGuniform MOEmir-15b, Human 338717544TCTAAGGGGTCACAGAAGGTuniform MOEmir-29a-1, Human 338718545GAAAATTATATTGACTCTGAuniform MOEmir-29a-1, Human 338719546GGTTCCTAATTAAACAACCCuniform MOEhypothetical miRNA-079,Human 338720547CCGAGGGTCTAACCCAGCCCuniform MOEmir-199b, Human 338721548GACTACTGTTGAGAGGAACAuniform MOEmir-129-1, Human 338722549TCTCCTTGGGTGTCCTCCTCuniform MOElet-7e, Human 338723550TGCTGACTGCTCGCCCTTGCuniform MOEhypothetical miRNA-083,Human 338724551ACTCCCAGGGTGTAACTCTAuniform MOElet7c-1, Human 338725552CATGAAGAAAGACTGTAGCCuniform MOEmir-204, Human 338726553GACAAGGTGGGAGCGAGTGGuniform MOEmir-145, Human 338727554TGCTCAGCCAGCCCCATTCTuniform MOEmir-124a-1, Human 338728555GCTTTTAGAACCACTGCCTCuniform MOEDiGeorge syndromecritical region gene 8/hypothetical miRNA-088,Human 338729556GGAGTAGATGATGGTTAGCCuniform MOEmir-213/mir-181a,Human 338730557ACTGATTCAAGAGCTTTGTAuniform MOEhypothetical miRNA-090,Human 338731558GTAGATAACTAAACACTACCuniform MOEmir-20, Human 338732559AATCCATTGAAGAGGCGATTuniform MOEmir-133a-1, Human 338733560GGTAAGAGGATGCGCTGCTCuniform MOEmir-138-2, Human 338734561GGCCTAATATCCCTACCCCAuniform MOEmir-98, Human 338735562GTGTTCAGAAACCCAGGCCCuniform MOEmir-196-1, Human 338736563TCCAGGATGCAAAAGCACGAuniform MOEmir-125b-1, Human 338737564TACAACGGCATTGTCCTGAAuniform MOEmir-199a-2, Human 338738565TTTCAGGCTCACCTCCCCAGuniform MOEhypothetical miRNA-099,Human 338739566AAAAATAATCTCTGCACAGGuniform MOEmir-181b, Human 338740567AGAATGAGTTGACATACCAAuniform MOEhypothetical miRNA-101,Human 338741568GCTTCACAATTAGACCATCCuniform MOEmir-141, Human 338742569AGACTCCACACCACTCATACuniform MOEmir-131-1/miR-9, Human 338743570ATCCATTGGACAGTCGATTTuniform MOEmir-133a-2, Human 338744571GGCGGGCGGCTCTGAGGCGGuniform MOEhypothetical miRNA-105,Human 338745572CTCTTTAGGCCAGATCCTCAuniform MOEhypothetical miRNA-105,Human 338746573TAATGGTATGTGTGGTGATAuniform MOEhypothetical miRNA-107,Human 338747574ATTACTAAGTTGTTAGCTGTuniform MOEmiR-1d-2, Human 338748575GATGCTAATCTACTTCACTAuniform MOEmir-18, Human 338749576TCAGCATGGTGCCCTCGCCCuniform MOEmir-220, Human 338750577TCCGCGGGGGCGGGGAGGCTuniform MOEhypothetical miRNA-111,Human 338751578AGACCACAGCCACTCTAATCuniform MOEmir-7-3, Human 338752579TCCGTTTCCATCGTTCCACCuniform MOEmir-218-2, Human 338753580GCCAGTGTACACAAACCAACuniform MOEmir-24-2, Human 338754581AAGGCTTTTTGCTCAAGGGCuniform MOEchromosome 9 ORF3containing mir-23b,mir-24-2 and mir-27b,Human 338755582TTGACCTGAATGCTACAAGGuniform MOEmir-103-2, Human 338756583TGCCCTGCTCAGAGCCCTAGuniform MOEmir-211, Human 338757584TCAATGTGATGGCACCACCAuniform MOEmir-101-3, Human 338758585ACCTCCCAGCCAATCCATGTuniform MOEmir-30b, Human 338759586TCCTGGATGATATCTACCTCuniform MOEhypothetical miRNA-120,Human 338760587TCTCCCTTGATGTAATTCTAuniform MOElet-7a-4, Human 338761588AGAGCGGAGTGTTTATGTCAuniform MOEmir-10a, Human 338762589TCATTCATTTGAAGGAAATAuniform MOEmir-19a, Human 338763590TCCAAGATGGGGTATGACCCuniform MOElet-7f-2, Human 338764591TTTTTAAACACACATTCGCGuniform MOEmir-15a-1, Human 338765592AGATGTGTTTCCATTCCACTuniform MOEmir-108-2, Human 338766593CCCCCTGCCGCTGGTACTCTuniform MOEmir-137, Human 338767594CGGCCGGAGCCATAGACTCGuniform MOEmir-219-1, Human 338768595CTTTCAGAGAGCCACAGCCTuniform MOEmir-148b, Human 338769596GCTTCCCAGCGGCCTATAGTuniform MOEmir-130b, Human 338770597CAGCAGAATATCACACAGCTuniform MOEmir-19b-1, Human 338771598TACAATTTGGGAGTCCTGAAuniform MOEmir-199b, Human 338772599GCCTCCTTCATATATTCTCAuniform MOEmir-204, Human 338773600CCCCATCTTAGCATCTAAGGuniform MOEmir-145, Human 338774601TTGTATGGACATTTAAATCAuniform MOEmir-124a-1, Human 338775602TTTGATTTTAATTCCAAACTuniform MOEmir-213/mir-181a,Human 338776603CAAACGGTAAGATTTGCAGAuniform MOEhypothetical miRNA-090,Human 338777604GGATTTAAACGGTAAACATCuniform MOEmir-125b-1, Human 338778605CTCTAGCTCCCTCACCAGTGuniform MOEhypothetical miRNA-099,Human 338779606GCTTGTCCACACAGTTCAACuniform MOEmir-181b, Human 338780607GCATTGTATGTTCATATGGGuniform MOEmiR-1d-2, Human 338781608TGTCGTAGTACATCAGAACAuniform MOEmir-7-3, Human 338782609AGCCAGTGTGTAAAATGAGAuniform MOEchromosome 9 ORF3containing mir-23b,mir-24-2 and mir-27b,Human 338783610TTCAGATATACAGCATCGGTuniform MOEmir-101-3, Human 338784611TGACCACAAAATTCCTTACAuniform MOEmir-10a, Human 338785612ACAACTACATTCTTCTTGTAuniform MOEmir-19a, Human 338786613TGCACCTTTTCAAAATCCACuniform MOEmir-15a-1, Human 338787614AACGTAATCCGTATTATCCAuniform MOEmir-137, Human 338788615CGTGAGGGCTAGGAAATTGCuniform MOEmir-216, Human 338789616GCAACAGGCCTCAATATCTTuniform MOEmir-100-1, Human 338790617ACGAGGGGTCAGAGCAGCGCuniform MOEmir-187, Human 338791618GGCAGACGAAAGGCTGACAGuniform MOEhypothetical miRNA-137,Human 338792619CTGCACCATGTTCGGCTCCCuniform MOEhypothetical miRNA-138,Human 338793620GGGGCCCTCAGGGCTGGGGCuniform MOEmir-124a-3, Human 338794621CCGGTCCACTCTGTATCCAGuniform MOEmir-7-2, Human 338795622GCTGGGAAAGAGAGGGCAGAuniform MOEhypothetical miRNA-142,Human 338796623TCAGATTGCCAACATTGTGAuniform MOEhypothetical miRNA-143,Human 338797624CTGGGGAGGGGGTTAGCGTCuniform MOEcollagen, type I, alpha1/hypothetical miRNA-144, Human 338798625TGGGTCTGGGGCAGCGCAGTuniform MOEmir-210, Human 338799626TTGAAGTAGCACAGTCATACuniform MOEmir-215, Human 338800627TCTACCACATGGAGTGTCCAuniform MOEmir-223, Human 338801628AGTGCCGCTGCCGCGCCGTGuniform MOEmir-131-3/miR-9, Human 338802629ACACATTGAGAGCCTCCTGAuniform MOEmir-199a-1, Human 338803630GTCGCTCAGTGCTCTCTAGGuniform MOEmir-30c-1, Human 338804631AGGCTCCTCTGATGGAAGGTuniform MOEmir-101-1, Human 338805632GCTGTGACTTCTGATATTATuniform MOEhypothetical miRNA-153,Human 338806633GACATCATGTGATTTGCTCAuniform MOEhypothetical miRNA-154,Human 338807634CACCCCAAGGCTGCAGGGCAuniform MOEmir-26b, Human 338808635TGTCAAGCCTGGTACCACCAuniform MOEhypothetical miRNA-156,Human 338809636CTGCTCCAGAGCCCGAGTCGuniform MOEmir-152, Human 338810637ACCCTCCGCTGGCTGTCCCCuniform MOEmir-135-1, Human 338811638TAGAGTGAATTTATCTTGGTuniform MOEnon-coding RNA inrhabdomyosarcoma/mir-135-2, Human 338812639TGGTGACTGATTCTTATCCAuniform MOEmir-217, Human 338813640CAATATGATTGGATAGAGGAuniform MOEhypothetical miRNA-161,Human 338814641TTTAAACACACATTCGCGCCuniform MOEmir-15a-1, Human 338815642ACCGGGTGGTATCATAGACCuniform MOElet-7g, Human 338816643TGCATACCTGTTCAGTTGGAuniform MOEhypothetical miRNA-164,Human 338817644GCCCGCCTCTCTCGGCCCCCuniform MOEsterol regulatoryelement-bindingprotein-1/mir-33b,Human 338818645TCGCCCCCTCCCAGGCCTCTuniform MOEhypothetical miRNA-166,Human 338819646ACAACTGTAGAGTATGGTCAuniform MOEmir-16-1, Human 338820647GCTGACCATCAGTACTTTCCuniform MOEhypothetical miRNA 168-1/similar to ribosomalprotein L5, Human 338821648TTATAGAACAGCCTCCAGTGuniform MOEforkhead boxP2/hypothetical miRNA-169, Human 338822649TTCAGGCACTAGCAGTGGGTuniform MOEhypothetical miRNA-170,Human 338823650AGTACTGCGAGGTTAACCGCuniform MOEglutamate receptor,ionotropic, AMPA 2/hypothetical miRNA-171,Human 338824651GGACCTTTAAGATGCAAAGTuniform MOEhypothetical miRNA-172,Human 338825652TTCATATTATCCACCCAGGTuniform MOEhypothetical miRNA-173,Human 338826653CGGATCCTGTTACCTCACCAuniform MOEmir-182, Human 338827654TGGTGCCTGCCACATCTTTGuniform MOEhypothetical miRNA-175,Human 338828655TGGGAGGCTGAATCAAGGACuniform MOEhypothetical miRNA-176,Human 338829656TGACAACCAGGAAGCTTGTGuniform MOEhypothetical miRNA-177-1, Human 338830657GCCAGGCAGCGAGCTTTTGAuniform MOEhypothetical miRNA-178,Human 338831658CAGCCTGCCACCGCCGCTTTuniform MOEhypothetical miRNA-179,Human 338832659CTGCCCCCGTGGACCGAACAuniform MOEcezanne 2/hypotheticalmiRNA-180, Human 338833660TCGTGCACCTGAGGAGTCTGuniform MOEhypothetical miRNA-181,Human 338834661CAAACGTGCTGTCTTCCTCCuniform MOEmir-148a, Human 338835662AAGGACTCAGCAGTGTTTCAuniform MOEtight junction protein1 (zona occludens 1)/hypothetical miRNA-183,Human 338836663TCCTCGGTGGCAGAGCTCAGuniform MOEmir-23a, Human 338837664AGACAATGAGTACACAGTTCuniform MOEhypothetical miRNA-185,Human 338838665CTGCAAGCACTGGTTCCCATuniform MOEhypothetical miRNA-177-2/hypothetical miRNA186, Human 338839666TTGCCTGAGCTGCCCAAACTuniform MOEmir-181c, Human 338840667TCCATCACACTGTCCTATGAuniform MOEhypothetical miRNA-188,Human 338841668GAGGGATTGTATGAACATCTuniform MOEmir-216, Human 338842669GCTTGTGCGGACTAATACCAuniform MOEmir-100-1, Human 338843670GCAGGCTAAAAGAAATAAGCuniform MOEhypothetical miRNA-138,Human 338844671ATTGTATAGACATTAAATCAuniform MOEmir-124a-3, Human 338845672GTTGAGCGCAGTAAGACAACuniform MOEmir-7-2, Human 338846673AGATGTTTCTGGCCTGCGAGuniform MOEhypothetical miRNA-142,Human 338847674GACAAACTCAGCTATATTGTuniform MOEmir-215, Human 338848675ACGGCTCTGTGGCACTCATAuniform MOEmir-131-3/miR-9, Human 338849676GCTTTCTTACTTTCCACAGCuniform MOEmir-30c-1, Human 338850677TACCTTTAGAATAGACAGCAuniform MOEmir-101-1, Human 338851678AGGCTGGACAGCACACAACCuniform MOEmir-26b, Human 338852679AGCAGGAGCCTTATCTCTCCuniform MOEhypothetical miRNA-156,Human 338853680ATGAGTGAGCAGTAGAATCAuniform MOEmir-135-1, Human 338854681TGAGACTTTATTACTATCACuniform MOEnon-coding RNA inrhabdomyosarcoma/mir-135-2, Human 338855682TACTTTACTCCAAGGTTTTAuniform MOEmir-15a-1, Human 338856683GCACCCGCCTCACACACGTGuniform MOEsterol regulatoryelement-bindingprotein-1/mir-33b,Human 338857684TTCCCGACCTGCCTTTACCTuniform MOEhypothetical miRNA-166,Human 338858685TCCTGTAATTATAGGCTAGCuniform MOEforkhead boxP2/hypothetical miRNA-169, Human 338859686GGATCATATCAATAATACCAuniform MOEhypothetical miRNA-172,Human 338860687TGCTGAGACACACAATATGTuniform MOEhypothetical miRNA-176,Human 338861688TGTTTGTCTCCAAGAAACGTuniform MOEhypothetical miRNA-177-1, Human 338862689TGTCATGGACAGGATGAATAuniform MOEhypothetical miRNA-179,Human 338863690TCTATCATACTCAGAGTCGGuniform MOEmir-148a, Human 338864691TTGTGACAGGAAGCAAATCCuniform MOEmir-23a, Human 338865692CATCAGAGTCACCAACCCCAuniform MOEhypothetical miRNA-185,Human 338866693CAAGAGATGTCTCGTTTTGCuniform MOEhypothetical miRNA-177-2/hypothetical miRNA186, Human 340342937GACTGTTGAATCTCATGGCAuniform MOEmiR-104 (Mourelatos),Human 3403441656GCATGAGCAGCCACCACAGGuniform MOEmiR-105 (Mourelatos),Human 3403461626ACGACTTGGTGTGGACCCTGuniform MOEmiR-27 (Mourelatos),Human 340347849TACTTTATATAGAACACAAGuniform MOEmir-92-2/miR-92(Mourelatos), Human 3403491632AGGTTGGGTAATCACACTACuniform MOEmiR-93 (Mourelatos),Human 3403511621AATGTAACGCATTTCAATTCuniform MOEmiR-95 (Mourelatos),Human 3403531694TGTGCGGTCCACTTCACCACuniform MOEmiR-99 (Mourelatos),Human 3403551671GTCCAGCAATTGCCCAAGTCuniform MOEmiR-25, Human 3403571662GGAAAGTCAGAAAGGTAACTuniform MOEmiR-28, Human 3403591635CAGGTTCCCAGTTCAACAGCuniform MOEmiR-31, Human 3403611636CATTGAGGCCGTGACAACATuniform MOEmiR-32, Human 3403631656GCATGAGCAGCCACCACAGG5-10-5 MOEmiR-105 (Mourelatos),gapmerHuman 3403641626ACGACTTGGTGTGGACCCTG5-10-5 MOEmiR-27 (Mourelatos),gapmerHuman 3403661632AGGTTGGGTAATCACACTAC5-10-5 MOEmiR-93 (Mourelatos),gapmerHuman 3403671621AATGTAACGCATTTCAATTC5-10-5 MOEmiR-95 (Mourelatos),gapmerHuman 3403681694TGTGCGGTCCACTTCACCAC5-10-5 MOEmiR-99 (Mourelatos),gapmerHuman 3403691671GTCCAGCAATTGCCCAAGTC5-10-5 MOEmiR-25, Humangapmer 3403701662GGAAAGTCAGAAAGGTAACT5-10-5 MOEmiR-28, Humangapmer 3403711635CAGGTTCCCAGTTCAACAGC5-10-5 MOEmiR-31, Humangapmer 3403721636CATTGAGGCCGTGACAACAT5-10-5 MOEmiR-32, Humangapmer 3418171630AGCCACCTTGAGCTCACAGCuniform MOEmiR-30c-2, Human 3418181695TGTGTGCGGCGAAGGCCCCGuniform MOEmiR-99b, Human 3418191657GCCAGGCTCCCAAGAACCTCuniform MOEMiR-125a, Human 3418201653GATGTTACTAAAATACCTCAuniform MOEMiR-125b-2, Human 3418221679TCCGATGATCTTTCTGAATCuniform MOEmiR-127, Human 3418251646CTTAAAATAAAACCAGAAAGuniform MOEmiR-186, Human 3418261618AAAATCACAGGAACCTATCTuniform MOEmiR-198, Human 3418271688TGGAATGCTCTGGAGACAACuniform MOEmiR-191, Human 3418281677TCCATAGCAAAGTAATCCATuniform MOEmiR-206, Human 3418291668GGTAGCACGGAGAGGACCACuniform MOEmiR-94, Human 3418301624ACACTTACAGTCACAAAGCTuniform MOEmiR-184, Human 3418311654GCAGACTCGCTTCCCTGTGCuniform MOEmiR-195, Human 3418321684TGATCCGACACCCTCATCTCuniform MOEmiR-193, Human 3418331641CCTGGGGAGGGGACCATCAGuniform MOEmiR-185, Human 3418341676TCAGAAAGCTCACCCTCCACuniform MOEmiR-188, Human 3418351648GAGCTCTTACCTCCCACTGCuniform MOEmiR-197, Human 3418361686TGGAAATTGGTACACAGTCCuniform MOEmiR-194-1, Human 3418371642CGTGAGCATCAGGTATAACCuniform MOEmiR-208, Human 3418381687TGGAACCAGTGGGCACTTCCuniform MOEmiR-194-2, Human 3418391638CCAGCCTCCGAGCCACACTGuniform MOEmiR-139, Human 3418401628AGACCTGACTCCATCCAATGuniform MOEmiR-200b, Human 3418411629AGAGTCAAGCTGGGAAATCCuniform MOEmiR-200a, Human 3418431630AGCCACCTTGAGCTCACAGC5-10-5 MOEmiR-30c-2, Humangapmer 3418441695TGTGTGCGGCGAAGGCCCCG5-10-5 MOEmiR-99b, Humangapmer 3418451657GCCAGGCTCCCAAGAACCTC5-10-5 MOEMiR-125a, Humangapmer 3418461653GATGTTACTAAAATACCTCA5-10-5 MOEMiR-125b-2, Humangapmer 3418481679TCCGATGATCTTTCTGAATC5-10-5 MOEmiR-127, Humangapmer 3418511646CTTAAAATAAAACCAGAAAG5-10-5 MOEmiR-186, Humangapmer 3418521618AAAATCACAGGAACCTATCT5-10-5 MOEmiR-198, Humangapmer 3418531688TGGAATGCTCTGGAGACAAC5-10-5 MOEmiR-191, Humangapmer 3418541677TCCATAGCAAAGTAATCCAT5-10-5 MOEmiR-206, Humangapmer 3418551668GGTAGCACGGAGAGGACCAC5-10-5 MOEmiR-94, Humangapmer 3418561624ACACTTACAGTCACAAAGCT5-10-5 MOEmiR-184, Humangapmer 3418571654GCAGACTCGCTTCCCTGTGC5-10-5 MOEmiR-195, Humangapmer 3418581684TGATCCGACACCCTCATCTC5-10-5 MOEmiR-193, Humangapmer 3418591641CCTGGGGAGGGGACCATCAG5-10-5 MOEmiR-185, Humangapmer 3418601676TCAGAAAGCTCACCCTCCAC5-10-5 MOEmiR-188, Humangapmer 3418611648GAGCTCTTACCTCCCACTGC5-10-5 MOEmiR-197, Humangapmer 3418621686TGGAAATTGGTACACAGTCC5-10-5 MOEmiR-194-1, Humangapmer 3418631642CGTGAGCATCAGGTATAACC5-10-5 MOEmiR-208, Humangapmer 3418641687TGGAACCAGTGGGCACTTCC5-10-5 MOEmiR-194-2, Humangapmer 3418651638CCAGCCTCCGAGCCACACTG5-10-5 MOEmiR-139, Humangapmer 3418661628AGACCTGACTCCATCCAATG5-10-5 MOEmiR-200b, Humangapmer 3418671629AGAGTCAAGCTGGGAAATCC5-10-5 MOEmiR-200a, Humangapmer 3447311619AACGGTTTATGACAAACATTuniform MOEmir-240* (Kosik), Human 3447321665GGGCTGTATGCACTTTCTCCuniform MOEmir-232* (Kosik), Human 3447331667GGGTCTCCAGCTTTACACCAuniform MOEmir-227* (Kosik)/mir-226* (Kosik), Human 3447341649GAGTCGCCTGAGTCATCACTuniform MOEmir-244* (Kosik), Human 3447351658GCCATAAATAAAGCGAACGCuniform MOEmir-224* (Kosik), Human 3447361678TCCATTAACCATGTCCCTCAuniform MOEmir-248* (Kosik), Human 3447371619AACGGTTTATGACAAACATT5-10-5 MOEmir-240* (Kosik), Humangapmer 3447381665GGGCTGTATGCACTTTCTCC5-10-5 MOEmir-232* (Kosik), Humangapmer 3447391667GGGTCTCCAGCTTTACACCA5-10-5 MOEmir-227* (Kosik)/mir-gapmer226* (Kosik), Human 3447401649GAGTCGCCTGAGTCATCACT5-10-5 MOEmir-244* (Kosik), Humangapmer 3447411658GCCATAAATAAAGCGAACGC5-10-5 MOEmir-224* (Kosik), Humangapmer 3447421678TCCATTAACCATGTCCCTCA5-10-5 MOEmir-248* (Kosik), Humangapmer 3467871689TGGCTTCCATAGTCTGGTGTuniform MOEmiR-147 (Sanger), Human 3467881623ACAATGCACAATCATCTACTuniform MOEmiR-224 (Sanger), Human 3467891669GGTGAACACAGTGCATGCCCuniform MOEmiR-134 (Sanger), Human 3467901682TCTGACACTGACACAACCCAuniform MOEmiR-146 (Sanger), Human 3467911631AGGGTCTGAGCCCAGCACTGuniform MOEmiR-150 (Sanger), Human 3467921637CCAAGAGACGTTTCATTTTGuniform MOEhypothetical miRNA-177-3, Human 3467931683TCTGATTGGCAACGGCCTGAuniform MOEmir-138-3, Human 3467941627ACTGTCCATCTTAGTTCAGAuniform MOEmir-138-4, Human 3467951634AGTTGATTCAGACTCAAACCuniform MOEmir-181b-2, Human 3467961655GCATAAGCAGCCACCACAGGuniform MOEmiR-105-2, Human 3467971691TGTATGATATCTACCTCAGGuniform MOEhypothetical miRNA-120-2, Human 3467981689TGGCTTCCATAGTCTGGTGT5-10-5 MOEmiR-147 (Sanger), Humangapmer 3467991623ACAATGCACAATCATCTACT5-10-5 MOEmiR-224 (Sanger), Humangapmer 3468001669GGTGAACACAGTGCATGCCC5-10-5 MOEmiR-134 (Sanger), Humangapmer 3468011682TCTGACACTGACACAACCCA5-10-5 MOEmiR-146 (Sanger), Humangapmer 3468021631AGGGTCTGAGCCCAGCACTG5-10-5 MOEmiR-150 (Sanger), Humangapmer 3468031637CCAAGAGACGTTTCATTTTG5-10-5 MOEhypothetical miRNA-177-gapmer3, Human 3468041683TCTGATTGGCAACGGCCTGA5-10-5 MOEmir-138-3, Humangapmer 3468051627ACTGTCCATCTTAGTTCAGA5-10-5 MOEmir-138-4, Humangapmer 3468061634AGTTGATTCAGACTCAAACC5-10-5 MOEmir-181b-2, Humangapmer 3468071655GCATAAGCAGCCACCACAGG5-10-5 MOEmiR-105-2, Humangapmer 3468081691TGTATGATATCTACCTCAGG5-10-5 MOEhypothetical miRNA-120-gapmer2, Human 3482251620AAGAGAAGGCGGAGGGGAGC5-10-5 MOEmiR-320, Humangapmer 3482261643CTCGAACCCACAATCCCTGG5-10-5 MOEmiR-321-1, Humangapmer 3540061650GAGTTTGGGACAGCAATCAC5-10-5 MOEmir-135b (Ruvkun),gapmerHuman 3540071633AGTAGGGGATGAGACATACT5-10-5 MOEmir-151* (Ruvkun),gapmerHuman 3540081639CCCACAAACGACATATGACA5-10-5 MOEmir-340 (Ruvkun), Humangapmer 3540091664GGCCTGGTTTGATCTGGGAT5-10-5 MOEmir-331 (Ruvkun), Humangapmer 3540101647GAGACTCCCAACCGCACCCA5-10-5 MOEmiR-200c (RFAM-Human)gapmer 3540111700TTGTAACCACCACAGTACAA5-10-5 MOEmiR-34b (RFAM-Human)gapmer 3540121663GGAGGACAGGGAGAGCGGCC5-10-5 MOEmir-339-1 (RFAM-Human)gapmer 3540131675TCACAGGCAGGCACACGTGA5-10-5 MOEmir-339-1 (RFAM-Human)gapmer 3540141698TTCAGAGCTACAGCATCGGT5-10-5 MOEmir-101-3, Mousegapmer 3540151670GTAGAACTCAAAAAGCTACC5-10-5 MOEmir-106, Mousegapmer 3540161673TAGATGCACACATCACTACC5-10-5 MOEmiR-17/mir-91, Mousegapmer 3540171690TGTACAATTTGGGAGTCCTG5-10-5 MOEmir-199b, Humangapmer 3540181644CTCTTTAGACCAGATCCACA5-10-5 MOEhypothetical miRNA-105,gapmerMouse 3540191640CCTCACTCAGAGGCCTAGGC5-10-5 MOEmir-211, Mousegapmer 3540201666GGGGATTAAGTCTTATCCAG5-10-5 MOEmir-217, Mousegapmer 3540211622ACAATGCACAAACCATCTAC5-10-5 MOEmiR-224 (Sanger), Mousegapmer 3540221693TGTCATATCATATCAGAACA5-10-5 MOEmir-7-3, Mousegapmer 3540231672TAGATGACGACACACTACCT5-10-5 MOEmir-20, Ratgapmer 3540241692TGTCACAAACACTTACTGGA5-10-5 MOEmir-325 (Ruvkun), Humangapmer 3540251625ACGAATTATGTCACAAACAC5-10-5 MOEmir-325 (Ruvkun), Mousegapmer 3540261651GATCTGAGCACCACCCGCCT5-10-5 MOEmir-326 (Ruvkun), Humangapmer 3540271652GATCTGAGCATAACCCGCCT5-10-5 MOEmir-326 (Ruvkun), Mousegapmer 3540281697TGTTTCGTCCTCATTAAAGA5-10-5 MOEmir-329-1 (Ruvkun),gapmerHuman 3540291699TTCTCATCAAAGAAACAGAG5-10-5 MOEmir-329-1 (Ruvkun),gapmerMouse 3540301696TGTTTCGTCCTCAATAAAGA5-10-5 MOEmir-329-2 (Ruvkun),gapmerHuman 3540311681TCGGTTGATCTTGCAGAGCC5-10-5 MOEmir-330 (Ruvkun), Humangapmer 3540321685TGCTCGTTGGATCTTGAAGA5-10-5 MOEmir-330 (Ruvkun), Mousegapmer 3540331661GCTGGATAACTGTGCATCAA5-10-5 MOEmir-337 (Ruvkun), Humangapmer 3540341645CTGAATGGCTGTGCAATCAA5-10-5 MOEmir-337 (Ruvkun), Mousegapmer 3540351659GCCCACCAGCCATCACGAGC5-10-5 MOEmir-345 (Ruvkun), Humangapmer 3540361660GCCCAGTAGCCACCACAAGC5-10-5 MOEmir-345 (Ruvkun), Mousegapmer 3540371680TCCTTCAGAGCAACAGAGAG5-10-5 MOEmir-346 (Ruvkun), Humangapmer 3540381674TAGTAGGGAGGAGACATACT5-10-5 MOEmir-151* (Ruvkun),gapmerMouse 3540391701TTGTCAGCACCGCACTACAA5-10-5 MOEmiR-34b (RFAM-Mouse)gapmer

In accordance with the present invention, a further series of oligomeric compounds were designed and synthesized to target different regions of miRNAs. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. The compounds are shown in Table 65, where â€œpri-miRNAâ€ indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. Oligomeric compounds having phosphorothioate internucleoside linkages are indicated by â€œPSâ€ in the â€œChemistryâ€ column of Table 65, whereas compounds having phosphodiester internucleoside linkages are indicated by â€œPO.â€ In some embodiments, chimeric oligonucleotides (â€œgapmersâ€ ) are composed of a central â€œgapâ€ region consisting of ten 2â€²-deoxynucleotides, which is flanked on both sides (5â€² and 3â€² directions) by nucleotide â€œwingsâ€ two to ten nucleotides in length. The wings are composed of 2â€²-methoxyethoxy (2â€²-MOE) ribonucleotides. In some embodiments, chimeric oligonucleotides are of the â€œopen endâ€ type wherein the â€œgapâ€ segment is located at either the 3â€² or the 5â€² terminus of the oligomeric compound. Chimeric oligonucleotides of this type are also known in the art and are indicated in Table 65 as â€œhemimers.â€ For example, â€œPO/6MOE-10deoxy hemimer,â€ describes a chimeric oligomeric compound consisting of six 2â€²-MOE ribonucleotides at the 5â€²-terminus, followed by ten deoxyribonucleotides on the 3â€²-terminal end, with a phosphodiester backbone throughout the hemimer

TABLE 65Oligomeric compounds targeting miRNAsSEQIDISIS #NOsequenceChemistryPri-miRNA3403431780ACAGGAGTCTGAGCATTTGAPS/MOEmiR-105(Mourelatos) 3403451882GGAACTTAGCCACTGTGAAPS/MOEmiR-27(Mourelatos) 340350855TGCTCAATAAATACCCGTTGAAPS/MOEmiR-95(Mourelatos) 3403521821CACAAGATCGGATCTACGGGTTPS/MOEmiR-99(Mourelatos) 3403541903TCAGACCGAGACAAGTGCAATGPS/MOEmiR-25 (Tuschl) 3403561853CTCAATAGACTGTGAGCTCCTTPS/MOEmiR-28 (Tuschl) 3403581825CAGCTATGCCAGCATCTTGCCPS/MOEmiR-31 (Tuschl) 3403601865GCAACTTAGTAATGTGCAATAPS/MOEmiR-32 (Tuschl) 340924298ACAAATTCGGTTCTACAGGGTAPS/MOE 5-10-7mir-10bgapmer 340925307GTGGTAATCCCTGGCAATGTGATPS/MOE 5-10-8mir-23bgapmer 340928322ACTCACCGACAGCGTTGAATGTTPS/MOE 5-10-8mir-181agapmer 340929331AACCGATTTCAAATGGTGCTAGPS/MOE 5-10-7mir-29cgapmer 340930342GCAAGCCCAGACCGCAAAAAGPS/MOE 5-10-6mir-129gapmer 340931346AACCGATTTCAGATGGTGCTAGPS/MOE 5-10-7mir-29agapmer 340932349AACCATACAACCTACTACCTCAPS/MOE 5-10-7let-7cgapmer 340933352GGTACAATCAACGGTCGATGGTPS/MOE 5-10-7mir-213gapmer 340934356AACAATACAACTTACTACCTCAPS/MOE 5-10-7mir-98gapmer 340935373GCCCTTTCATCATTGCACTGPS/MOE 5-10-5mir-130bgapmer 340936385ACTGTACAAACTACTACCTCAPS/MOE 5-10-6let-7ggapmer 341785854GGAGTGAAGACACGGAGCCAGAPS/MOEmiR-149 3417861845CGCAAGGTCGGTTCTACGGGTGPS/MOEmiR-99b 341787852CACAGGTTAAAGGGTCTCAGGGAPS/MOEmiR-125a 341788853AGCCAAGCTCAGACGGATCCGAPS/MOEmiR-127 3417891909TCCATCATCAAAACAAATGGAGTPS/MOEmiR-136 3417901843CGAAGGCAACACGGATAACCTAPS/MOEmiR-154 3417911880GCTTCCAGTCGAGGATGTTTACAPS/MOEmiR-30a-s 3417921911TCCGTGGTTCTACCCTGTGGTAPS/MOEmiR-140-as 3417931836CCATAAAGTAGGAAACACTACAPS/MOEmiR-142-as 3417941761AACAGGTAGTCTGAACACTGGGPS/MOEmiR-199-s 3417951762AACCAATGTGCAGACTACTGTAPS/MOEmiR-199-as 3417961904TCATACAGCTAGATAACCAAAGAPS/MOEmiR-9 3417971773ACAAGTGCCTTCACTGCAGTPS/MOEmiR-17 3417981871GCATTATTACTCACGGTACGAPS/MOEmiR-126a 3417991787ACCTAATATATCAAACATATCAPS/MOEmiR-190 3418001766AAGCCCAAAAGGAGAATTCTTTGPS/MOEmiR-186 3418011839CCTATCTCCCCTCTGGACCPS/MOEmiR-198a 3418021806AGCTGCTTTTGGGATTCCGTTGPS/MOEmiR-191c 341803760CCACACACTTCCTTACATTCCAPS/MOEmiR-206d 341804761ATCTGCACTGTCAGCACTTTPS/MOEmiR-94 341805762ACCCTTATCAGTTCTCCGTCCAPS/MOEmiR-184 341806763GCCAATATTTCTGTGCTGCTAPS/MOEmiR-195 341807764CTGGGACTTTGTAGGCCAGTTPS/MOEmiR-193 3418081861GAACTGCCTTTCTCTCCAPS/MOEmiR-185 3418091786ACCCTCCACCATGCAAGGGATGPS/MOEmiR-188 3418101879GCTGGGTGGAGAAGGTGGTGAAPS/MOEmiR-197a 3418111906TCCACATGGAGTTGCTGTTACAPS/MOEmiR-194 3418121771ACAAGCTTTTTGCTCGTCTTATPS/MOEmiR-208 3418141887GTCATCATTACCAGGCAGTATTAPS/MOEmiR-200b 3418151831CATCGTTACCAGACAGTGTTAPS/MOEmiR-200a 3429461897TAGGAGAGAGAAAAAGACTGAPS/MOEmiR-14 3429471827CAGCTTTCAAAATGATCTCACPS/MOEmiR-Bantam 343875321AACTATACAACCTACTACCTCAPO/MOElet-7a 3442671769ACAAATTCGGATCTACAGGGTAPS/MOEmiR-10 (Tuschl) 3442681774ACACAAATTCGGTTCTACAGGGPS/MOEmiR-10b(Tuschl) 3442691890TAACCGATTTCAAATGGTGCTAPS/MOEmiR-29c(Tuschl) 3442701867GCACGAACAGCACTTTGPS/MOEmiR-93 (Tuschl) 3442711770ACAAGATCGGATCTACGGGTPS/MOEmiR-99a(Tuschl) 3442721816CAAACACCATTGTCACACTCCAPS/MOEmiR-122a,b(Tuschl) 3442731920TGTCAATTCATAGGTCAGPS/MOEmiR-192(Tuschl) 3442741832CCAACAACATGAAACTACCTAPS/MOEmiR-196(Tuschl) 3442751912TCTAGTGGTCCTAAACATTTCAPS/MOEmiR-203(Tuschl) 3442761828CAGGCATAGGATGACAAAGGGAAPS/MOEmiR-204(Tuschl) 3442771767AATACATACTTCTTTACATTCCAPS/MOEmiR-1d (Tuschl) 3442781769ACAAATTCGGATCTACAGGGTAPS/MOE 5-10-7miR-10 (Tuschl)gapmer 3442791774ACACAAATTCGGTTCTACAGGGPS/MOE 5-10-7miR-10bgapmer(Tuschl) 3442801890TAACCGATTTCAAATGGTGCTAPS/MOE 5-10-7miR-29cgapmer(Tuschl) 3442811867GCACGAACAGCACTTTGPS/MOE 5-10-2miR-93 (Tuschl)gapmer 3442821770ACAAGATCGGATCTACGGGTPS/MOE 5-10-5miR-99agapmer(Tuschl) 3442831816CAAACACCATTGTCACACTCCAPS/MOE 5-10-7miR-122a,bgapmer(Tuschl) 3442841920TGTCAATTCATAGGTCAGPS/MOE 5-10-3miR-192gapmer(Tuschl) 3442851832CCAACAACATGAAACTACCTAPS/MOE 5-10-6miR-196gapmer(Tuschl) 3442861912TCTAGTGGTCCTAAACATTTCAPS/MOE 5-10-7miR-203gapmer(Tuschl) 3442871828CAGGCATAGGATGACAAAGGGAAPS/MOE 5-10-8miR-204gapmer(Tuschl) 3442881767AATACATACTTCTTTACATTCCAPS/MOE 5-10-8miR-1d (Tuschl)gapmer 3443361918TGGCATTCACCGCGTGCCTTAPS/MOEmir-124a(Kosik) 3443371754AAAGAGACCGGTTCACTGTGAPS/MOEmir-128 (Kosik) 3443381812ATGCCCTTTTAACATTGCACTGPS/MOEmir-130 (Kosik) 3443391854CTCACCGACAGCGTTGAATGTTPS/MOEmir-178 (Kosik) 3443401921TGTCCGTGGTTCTACCCTGTGGTAPS/MOEmir-239*(Kosik) 3443411823CACATGGTTAGATCAAGCACAAPS/MOEmir-253*(Kosik) 3443421814ATGCTTTTTGGGGTAAGGGCTTPS/MOEmir-129as/mir-258* (Kosik) 3443431811ATGCCCTTTCATCATTGCACTGPS/MOEmir-266*(Kosik) 3443441918TGGCATTCACCGCGTGCCTTAPS/MOE 5-10-6mir-124agapmer(Kosik) 3443451754AAAGAGACCGGTTCACTGTGAPS/MOE 5-10-6mir-128 (Kosik)gapmer 3443461812ATGCCCTTTTAACATTGCACTGPS/MOE 5-10-7mir-130 (Kosik)gapmer 3443471854CTCACCGACAGCGTTGAATGTTPS/MOE 5-10-7mir-178 (Kosik)gapmer 3443481921TGTCCGTGGTTCTACCCTGTGGTAPS/MOE 5-10-9mir-239*gapmer(Kosik) 3443491823CACATGGTTAGATCAAGCACAAPS/MOE 5-10-7mir-253*gapmer(Kosik) 3443501814ATGCTTTTTGGGGTAAGGGCTTPS/MOE 5-10-7mir-129as/mir-gapmer258* (Kosik) 3443511811ATGCCCTTTCATCATTGCACTGPS/MOE 5-10-7mir-266*gapmer(Kosik) 3446111785ACATTTTTCGTTATTGCTCTTGAPS/MOEmir-240*(Kosik) 3446121790ACGGAAGGGCAGAGAGGGCCAGPS/MOEmir-232*(Kosik) 3446131775ACACCAATGCCCTAGGGGATGCGPS/MOEmir-227*(Kosik) 3446141834CCAGCAGCACCTGGGGCAGTPS/MOEmir-226*(Kosik) 3446151900TCAACAAAATCACTGATGCTGGAPS/MOEmir-244*(Kosik) 3446161800AGAGGTCGACCGTGTAATGTGCPS/MOEmir-224*(Kosik) 3446171862GACGGGTGCGATTTCTGTGTGAGAPS/MOEmir-248*(Kosik) 3446181785ACATTTTTCGTTATTGCTCTTGAPS/MOE 5-10-8mir-240*gapmer(Kosik) 3446191790ACGGAAGGGCAGAGAGGGCCAGPS/MOE 5-10-7mir-232*gapmer(Kosik) 3446201775ACACCAATGCCCTAGGGGATGCGPS/MOE 5-10-8mir-227*gapmer(Kosik) 3446211834CCAGCAGCACCTGGGGCAGTPS/MOE 5-10-5mir-226*gapmer(Kosik) 3446221900TCAACAAAATCACTGATGCTGGAPS/MOE 5-10-8mir-244*gapmer(Kosik) 3446231800AGAGGTCGACCGTGTAATGTGCPS/MOE 5-10-7mir-224*gapmer(Kosik) 3446241862GACGGGTGCGATTTCTGTGTGAGAPS/MOE 5-10-9mir-248*gapmer(Kosik) 345344291CTACCATAGGGTAAAACCACTPS/MOE 5-10-6mir-140gapmer 345345292GCTGCAAACATCCGACTGAAAGPS/MOE 5-10-7mir-30agapmer 345346293ACAACCAGCTAAGACACTGCCAPS/MOE 5-10-7mir-34gapmer 345347294AACACTGATTTCAAATGGTGCTAPS/MOE 5-10-8mir-29bgapmer 345348295CGCCAATATTTACGTGCTGCTAPS/MOE 5-10-7mir-16gapmer 345350297AACAAAATCACTAGTCTTCCAPS/MOE 5-10-6mir-7gapmer 345351299AAAAGAGACCGGTTCACTGTGAPS/MOE 5-10-7mir-128agapmer 345352300TCACTTTTGTGACTATGCAAPS/MOE 5-10-5mir-153gapmer 345353301CAGAACTTAGCCACTGTGAAPS/MOE 5-10-5mir-27bgapmer 345354302GCAAAAATGTGCTAGTGCCAAAPS/MOE 5-10-7mir-96gapmer 345355303ACTACCTGCACTGTAAGCACTTTGPS/MOE 5-10-9mir-17as/mir-91gapmer 345356304CGCGTACCAAAAGTAATAATGPS/MOE 5-10-6mir-123/mir-gapmer126as 345357305GCGACCATGGCTGTAGACTGTTAPS/MOE 5-10-8mir-132gapmer 345358306AATGCCCCTAAAAATCCTTATPS/MOE 5-10-6mir-108gapmer 345359308AGCACAAACTACTACCTCAPS/MOE 5-10-4let-7igapmer 345360309GGCCGTGACTGGAGACTGTTAPS/MOE 5-10-6mir-212gapmer 345361311AACCACACAACCTACTACCTCAPS/MOE 5-10-7let-7bgapmer 345362312ATACATACTTCTTTACATTCCAPS/MOE 5-10-7mir-1dgapmer 345363313ACAAACACCATTGTCACACTCCAPS/MOE 5-10-8mir-122agapmer 345364314ACAGTTCTTCAACTGGCAGCTTPS/MOE 5-10-7mir-22gapmer 345365315ACAGGCCGGGACAAGTGCAATAPS/MOE 5-10-7mir-92gapmer 345366316GTAGTGCTTTCTACTTTATGPS/MOE 5-10-5mir-142gapmer 345367317CAGTGAATTCTACCAGTGCCATAPS/MOE 5-10-8mir-183gapmer 345368318CTGCCTGTCTGTGCCTGCTGTPS/MOE 5-10-6mir-214gapmer 345369320GGCTGTCAATTCATAGGTCAGPS/MOE 5-10-6mir-192gapmer 345370321AACTATACAACCTACTACCTCAPS/MOE 5-10-7let-7agapmer 345371323CAGACTCCGGTGGAATGAAGGAPS/MOE 5-10-7mir-205gapmer 345372324TCATAGCCCTGTACAATGCTGCTPS/MOE 5-10-8mir-103gapmer 345373325AGCCTATCCTGGATTACTTGAAPS/MOE 5-10-7mir-26agapmer 345374326CAATGCAACTACAATGCACPS/MOE 5-10-4mir-33agapmer 345375327CCCAACAACATGAAACTACCTAPS/MOE 5-10-7mir-196gapmer 345376328TGATAGCCCTGTACAATGCTGCTPS/MOE 5-10-8mir-107gapmer 345377329GCTACCTGCACTGTAAGCACTTTTPS/MOE 5-10-9mir-106gapmer 345378330AACTATACAATCTACTACCTCAPS/MOE 5-10-7let-7fgapmer 345379332GCCCTTTTAACATTGCACTGPS/MOE 5-10-5mir-130agapmer 345380333ACATGGTTAGATCAAGCACAAPS/MOE 5-10-6mir-218gapmer 345381334TGGCATTCACCGCGTGCCTTAAPS/MOE 5-10-7mir-124agapmer 345382335TCAACATCAGTCTGATAAGCTAPS/MOE 5-10-7mir-21gapmer 345383336CTAGTACATCATCTATACTGTAPS/MOE 5-10-7mir-144gapmer 345384337GAAACCCAGCAGACAATGTAGCTPS/MOE 5-10-8mir-221gapmer 345385338GAGACCCAGTAGCCAGATGTAGCTPS/MOE 5-10-9mir-222gapmer 345386339CTTCCAGTCGGGGATGTTTACAPS/MOE 5-10-7mir-30dgapmer 345387340TCAGTTTTGCATGGATTTGCACAPS/MOE 5-10-8mir-19bgapmer 345388341GAAAGAGACCGGTTCACTGTGAPS/MOE 5-10-7mir-128bgapmer 345389343TAGCTGGTTGAAGGGGACCAAPS/MOE 5-10-6mir-133bgapmer 345390344ACTATGCAACCTACTACCTCTPS/MOE 5-10-6let-7dgapmer 345391345TGTAAACCATGATGTGCTGCTAPS/MOE 5-10-7mir-15bgapmer 345392347GAACAGATAGTCTAAACACTGGGPS/MOE 5-10-8mir-199bgapmer 345393348ACTATACAACCTCCTACCTCAPS/MOE 5-10-6let-7egapmer 345394350AGGCATAGGATGACAAAGGGAAPS/MOE 5-10-7mir-204gapmer 345395351AAGGGATTCCTGGGAAAACTGGACPS/MOE 5-10-9mir-145gapmer 345396353CTACCTGCACTATAAGCACTTTAPS/MOE 5-10-8mir-20gapmer 345397354ACAGCTGGTTGAAGGGGACCAAPS/MOE 5-10-7mir-133agapmer 345398355GATTCACAACACCAGCTPS/MOE 5-10-2mir-138gapmer 345399357TCACAAGTTAGGGTCTCAGGGAPS/MOE 5-10-7mir-125bgapmer 345400358GAACAGGTAGTCTGAACACTGGGPS/MOE 5-10-8mir-199agapmer 345401359AACCCACCGACAGCAATGAATGTTPS/MOE 5-10-9mir-181bgapmer 345402360CCATCTTTACCAGACAGTGTTPS/MOE 5-10-6mir-141gapmer 345403361TATCTGCACTAGATGCACCTTAPS/MOE 5-10-7mir-18gapmer 345404362AAAGTGTCAGATACGGTGTGGPS/MOE 5-10-6mir-220gapmer 345405363CTGTTCCTGCTGAACTGAGCCAPS/MOE 5-10-7mir-24gapmer 345406364AGGCGAAGGATGACAAAGGGAAPS/MOE 5-10-7mir-211gapmer 345407365TCAGTTATCACAGTACTGTAPS/MOE 5-10-5mir-101gapmer 345408366GCTGAGTGTAGGATGTTTACAPS/MOE 5-10-6mir-30bgapmer 345409367CACAAATTCGGATCTACAGGGTAPS/MOE 5-10-8mir-10agapmer 345410368TCAGTTTTGCATAGATTTGCACAPS/MOE 5-10-8mir-19agapmer 345411369CACAAACCATTATGTGCTGCTAPS/MOE 5-10-7mir-15agapmer 345412370CTACGCGTATTCTTAAGCAATAPS/MOE 5-10-7mir-137gapmer 345413371AGAATTGCGTTTGGACAATCAPS/MOE 5-10-6mir-219gapmer 345414372ACAAAGTTCTGTGATGCACTGAPS/MOE 5-10-7mir-148bgapmer 345415374CACAGTTGCCAGCTGAGATTAPS/MOE 5-10-6mir-216gapmer 345416375CACAAGTTCGGATCTACGGGTTPS/MOE 5-10-7mir-100gapmer 345417376CCGGCTGCAACACAAGACACGAPS/MOE 5-10-7mir-187gapmer 345418377CAGCCGCTGTCACACGCACAGPS/MOE 5-10-6mir-210gapmer 345419378GTCTGTCAATTCATAGGTCATPS/MOE 5-10-6mir-215gapmer 345420379GGGGTATTTGACAAACTGACAPS/MOE 5-10-6mir-223gapmer 345421380GCTGAGAGTGTAGGATGTTTACAPS/MOE 5-10-8mir-30cgapmer 345422381AACCTATCCTGAATTACTTGAAPS/MOE 5-10-7mir-26bgapmer 345423382CCAAGTTCTGTCATGCACTGAPS/MOE 5-10-6mir-152gapmer 345424383ATCACATAGGAATAAAAAGCCATAPS/MOE 5-10-9mir-135gapmer 345425384ATCCAATCAGTTCCTGATGCAGTAPS/MOE 5-10-9mir-217gapmer 345426386CAATGCAACAGCAATGCACPS/MOE 5-10-4mir-33bgapmer 345427387TGTGAGTTCTACCATTGCCAAAPS/MOE 5-10-7mir-182gapmer 345428388ACAAAGTTCTGTAGTGCACTGAPS/MOE 5-10-7mir-148agapmer 345429389GGAAATCCCTGGCAATGTGATPS/MOE 5-10-6mir-23agapmer 345430390ACTCACCGACAGGTTGAATGTTPS/MOE 5-10-7mir-181cgapmer 345431391ACTGTAGGAATATGTTTGATAPS/MOE 5-10-6hypotheticalgapmermiRNA-013 345432392ATTAAAAAGTCCTCTTGCCCAPS/MOE 5-10-6hypotheticalgapmermiRNA-023 345433393GCTGCCGTATATGTGATGTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-030 345434394GGTAGGTGGAATACTATAACAPS/MOE 5-10-6hypotheticalgapmermiRNA-033 345435395TAAACATCACTGCAAGTCTTAPS/MOE 5-10-6hypotheticalgapmermiRNA-039 345436396TTGTAAGCAGTTTTGTTGACAPS/MOE 5-10-6hypotheticalgapmermiRNA-040 345437397TCACAGAGAAAACAACTGGTAPS/MOE 5-10-6hypotheticalgapmermiRNA-041 345438398CCTCTCAAAGATTTCCTGTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-043 345439399TGTCAGATAAACAGAGTGGAAPS/MOE 5-10-6hypotheticalgapmermiRNA-044 345440400GAGAATCAATAGGGCATGCAAPS/MOE 5-10-6hypotheticalgapmermiRNA-055 345441401AAGAACATTAAGCATCTGACAPS/MOE 5-10-6hypotheticalgapmermiRNA-058 345442402AATCTCTGCAGGCAAATGTGAPS/MOE 5-10-6hypotheticalgapmermiRNA-070 345443403AAACCCCTATCACGATTAGCAPS/MOE 5-10-6hypotheticalgapmermiRNA-071 345444404GCCCCATTAATATTTTAACCAPS/MOE 5-10-6hypotheticalgapmermiRNA-075 345445405CCCAATATCAAACATATCAPS/MOE 5-10-4hypotheticalgapmermiRNA-079 345446406TATGATAGCTTCCCCATGTAAPS/MOE 5-10-6hypotheticalgapmermiRNA-083 345447407CCTCAATTATTGGAAATCACAPS/MOE 5-10-6hypotheticalgapmermiRNA-088 345448408ATTGATGCGCCATTTGGCCTAPS/MOE 5-10-6hypotheticalgapmermiRNA-090 345449409CTGTGACTTCTCTATCTGCCTPS/MOE 5-10-6hypotheticalgapmermiRNA-099 345450410AAACTTGTTAATTGACTGTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-101 345451411AAAGAAGTATATGCATAGGAAPS/MOE 5-10-6hypotheticalgapmermiRNA-105 345452412GATAAAGCCAATAAACTGTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-107 345453413TCCGAGTCGGAGGAGGAGGAAPS/MOE 5-10-6hypotheticalgapmermiRNA-111 345454414ATCATTACTGGATTGCTGTAAPS/MOE 5-10-6hypotheticalgapmermiRNA-120 345455415CAAAAATTATCAGCCAGTTTAPS/MOE 5-10-6hypotheticalgapmermiRNA-137 345456416AATCTCATTTTCATACTTGCAPS/MOE 5-10-6hypotheticalgapmermiRNA-138 345457417AGAAGGTGGGGAGCAGCGTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-142 345458418CAAAATTGCAAGCAAATTGCAPS/MOE 5-10-6hypotheticalgapmermiRNA-143 345459419TCCACAAAGCTGAACATGTCTPS/MOE 5-10-6hypotheticalgapmermiRNA-144 345460420TATTATCAGCATCTGCTTGCAPS/MOE 5-10-6hypotheticalgapmermiRNA-153 345461421AATAACACACATCCACTTTAAPS/MOE 5-10-6hypotheticalgapmermiRNA-154 345462422AAGAAGGAAGGAGGGAAAGCAPS/MOE 5-10-6hypotheticalgapmermiRNA-156 345463423ATGACTACAAGTTTATGGCCAPS/MOE 5-10-6hypotheticalgapmermiRNA-161 345464424CAAAACATAAAAATCCTTGCAPS/MOE 5-10-6hypotheticalgapmermiRNA-164 345465425TTACAGGTGCTGCAACTGGAAPS/MOE 5-10-6hypotheticalgapmermiRNA-166 345466426AGCAGGTGAAGGCACCTGGCTPS/MOE 5-10-6hypotheticalgapmermiRNA-168 345467427TATGAAATGCCAGAGCTGCCAPS/MOE 5-10-6hypotheticalgapmermiRNA-169 345468428CCAAGTGTTAGAGCAAGATCAPS/MOE 5-10-6hypotheticalgapmermiRNA-170 345469429AACGATAAAACATACTTGTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-171 345470430AGTAACTTCTTGCAGTTGGAPS/MOE 5-10-5hypotheticalgapmermiRNA-172 345471431AGCCTCCTTCTTCTCGTACTAPS/MOE 5-10-6hypotheticalgapmermiRNA-173 345472432ACCTCAGGTGGTTGAAGGAGAPS/MOE 5-10-6hypotheticalgapmermiRNA-175 345473433ATATGTCATATCAAACTCCTAPS/MOE 5-10-6hypotheticalgapmermiRNA-176 345474434GTGAGAGTAGCATGTTTGTCTPS/MOE 5-10-6hypotheticalgapmermiRNA-177 345475435TGAAGGTTCGGAGATAGGCTAPS/MOE 5-10-6hypotheticalgapmermiRNA-178 345476436AATTGGACAAAGTGCCTTTCAPS/MOE 5-10-6hypotheticalgapmermiRNA-179 345477437ACCGAACAAAGTCTGACAGGAPS/MOE 5-10-6hypotheticalgapmermiRNA-180 345478438AACTACTTCCAGAGCAGGTGAPS/MOE 5-10-6hypotheticalgapmermiRNA-181 345479439GTAAGCGCAGCTCCACAGGCTPS/MOE 5-10-6hypotheticalgapmermiRNA-183 345480440GAGCTGCTCAGCTGGCCATCAPS/MOE 5-10-6hypotheticalgapmermiRNA-185 345481441TACTTTTCATTCCCCTCACCAPS/MOE 5-10-6hypotheticalgapmermiRNA-188 345482236TAGCTTATCAGACTGATGTTGAPS/MOE 5-10-7miR-104gapmer(Mourelatos) 3454831780ACAGGAGTCTGAGCATTTGAPS/MOE 5-10-5miR-105gapmer(Mourelatos) 3454841882GGAACTTAGCCACTGTGAAPS/MOE 5-10-4miR-27gapmer(Mourelatos) 345485848CTACCTGCACGAACAGCACTTTPS/MOE 5-10-7miR-93gapmer(Mourelatos) 345486855TGCTCAATAAATACCCGTTGAAPS/MOE 5-10-7miR-95gapmer(Mourelatos) 3454871821CACAAGATCGGATCTACGGGTTPS/MOE 5-10-7miR-99gapmer(Mourelatos) 3454881903TCAGACCGAGACAAGTGCAATGPS/MOE 5-10-7miR-25 (Tuschl)gapmer 3454891853CTCAATAGACTGTGAGCTCCTTPS/MOE 5-10-7miR-28 (Tuschl)gapmer 3454901825CAGCTATGCCAGCATCTTGCCPS/MOE 5-10-6miR-31 (Tuschl)gapmer 3454911865GCAACTTAGTAATGTGCAATAPS/MOE 5-10-6miR-32 (Tuschl)gapmer 3454921897TAGGAGAGAGAAAAAGACTGAPS/MOE 5-10-6miR-14gapmer 345493854GGAGTGAAGACACGGAGCCAGAPS/MOE 5-10-7miR-149gapmer 3454941845CGCAAGGTCGGTTCTACGGGTGPS/MOE 5-10-7miR-99bgapmer 345495852CACAGGTTAAAGGGTCTCAGGGAPS/MOE 5-10-8miR-125agapmer 345496853AGCCAAGCTCAGACGGATCCGAPS/MOE 5-10-7miR-127gapmer 3454971909TCCATCATCAAAACAAATGGAGTPS/MOE 5-10-8miR-136gapmer 3454981843CGAAGGCAACACGGATAACCTAPS/MOE 5-10-7miR-154gapmer 3454991880GCTTCCAGTCGAGGATGTTTACAPS/MOE 5-10-8miR-30a-sgapmer 3455001911TCCGTGGTTCTACCCTGTGGTAPS/MOE 5-10-7miR-140-asgapmer 3455011836CCATAAAGTAGGAAACACTACAPS/MOE 5-10-7miR-142-asgapmer 3455021761AACAGGTAGTCTGAACACTGGGPS/MOE 5-10-7miR-199-sgapmer 3455031762AACCAATGTGCAGACTACTGTAPS/MOE 5-10-7miR-199-asgapmer 3455041904TCATACAGCTAGATAACCAAAGAPS/MOE 5-10-8miR-9gapmer 3455051773ACAAGTGCCTTCACTGCAGTPS/MOE 5-10-5miR-17gapmer 3455061871GCATTATTACTCACGGTACGAPS/MOE 5-10-6miR-126agapmer 3455071787ACCTAATATATCAAACATATCAPS/MOE 5-10-7miR-190gapmer 3455081766AAGCCCAAAAGGAGAATTCTTTGPS/MOE 5-10-8miR-186gapmer 3455091839CCTATCTCCCCTCTGGACCPS/MOE 5-10-4miR-198agapmer 3455101806AGCTGCTTTTGGGATTCCGTTGPS/MOE 5-10-7miR-191cgapmer 345511760CCACACACTTCCTTACATTCCAPS/MOE 5-10-7miR-206dgapmer 345512761ATCTGCACTGTCAGCACTTTPS/MOE 5-10-5miR-94gapmer 345513762ACCCTTATCAGTTCTCCGTCCAPS/MOE 5-10-7miR-184gapmer 345514763GCCAATATTTCTGTGCTGCTAPS/MOE 5-10-6miR-195gapmer 345515764CTGGGACTTTGTAGGCCAGTTPS/MOE 5-10-6miR-193gapmer 3455161861GAACTGCCTTTCTCTCCAPS/MOE 5-10-3miR-185gapmer 3455171786ACCCTCCACCATGCAAGGGATGPS/MOE 5-10-7miR-188gapmer 3455181879GCTGGGTGGAGAAGGTGGTGAAPS/MOE 5-10-7miR-197agapmer 3455191906TCCACATGGAGTTGCTGTTACAPS/MOE 5-10-7miR-194gapmer 3455201771ACAAGCTTTTTGCTCGTCTTATPS/MOE 5-10-7miR-208gapmer 345521938AGACACGTGCACTGTAGAPS/MOE 5-10-3miR-139gapmer 3455221887GTCATCATTACCAGGCAGTATTAPS/MOE 5-10-8miR-200bgapmer 3455231831CATCGTTACCAGACAGTGTTAPS/MOE 5-10-6miR-200agapmer 3455241827CAGCTTTCAAAATGATCTCACPS/MOE 5-10-6miR-Bantamgapmer 3459221783ACAGTGCTTCATCTCAPO/6MOE-10deoxymir-143hemimer 3459231848CTACAGTGCTTCATCTCPO/6MOE-11deoxymir-143hemimer 3459241876GCTACAGTGCTTCATCTPO/6MOE-11deoxymir-143hemimer 3459251875GCTACAGTGCTTCATCPO/6MOE-10deoxymir-143hemimer 3459261803AGCTACAGTGCTTCATPO/6MOE-10deoxymir-143hemimer 3459271863GAGCTACAGTGCTTCAPO/6MOE-10deoxymir-143hemimer 3459281916TGAGCTACAGTGCTTCPO/6MOE-10deoxymir-143hemimer 3466851884GGCGGAACTTAGCCACTGTGAAPS/MOEmiR-27a (RFAM-Human) 3466861857CTTCAGTTATCACAGTACTGTAPS/MOEmiR-101 (RFAM-Human) 3466871802AGCAAGCCCAGACCGCAAAAAGPS/MOEmiR-129b (RFAM-Human) 3466881898TAGTTGGCAAGTCTAGAACCAPS/MOEmiR-182* (RFAM-Human) 3466891830CATCATTACCAGGCAGTATTAGAGPS/MOEmiR-200a (RFAM-Human) 3466901792ACTGATATCAGCTCAGTAGGCACPS/MOEmiR-189 (RFAM-Human) 3466911870GCAGAAGCATTTCCACACACPS/MOEmiR-147 (RFAM-Human) 3466921889TAAACGGAACCACTAGTGACTTGPS/MOEmiR-224 (RFAM-Human) 3466931838CCCTCTGGTCAACCAGTCACAPS/MOEmiR-134 (RFAM-Human) 3466941763AACCCATGGAATTCAGTTCTCAPS/MOEmiR-146 (RFAM-Human) 3466951824CACTGGTACAAGGGTTGGGAGAPS/MOEmiR-150 (RFAM-Human) 3466961893TACCTGCACTATAAGCACTTTAPS/MOEmir-20 3466971788ACCTATCCTGAATTACTTGAAPS/MOEmir-26b 3466981793ACTGATTTCAAATGGTGCTAPS/MOEmir-29b 3466991847CGGCTGCAACACAAGACACGAPS/MOEmiR-187 (RFAM-Human) 3467001844CGACCATGGCTGTAGACTGTTAPS/MOEmiR-132 (RFAM-Human) 3467011901TCACATAGGAATAAAAAGCCATAPS/MOEmiR-135 (RFAM-Human) 3467021893TACCTGCACTATAAGCACTTTAPS/MOE 5-10-7mir-20gapmer 3467031788ACCTATCCTGAATTACTTGAAPS/MOE 5-10-6mir-26bgapmer 3467041884GGCGGAACTTAGCCACTGTGAAPS/MOE 5-10-7miR-27a (RFAM-gapmerHuman) 3467051857CTTCAGTTATCACAGTACTGTAPS/MOE 5-10-7miR-101 (RFAM-gapmerHuman) 3467061793ACTGATTTCAAATGGTGCTAPS/MOE 5-10-5mir-29bgapmer 3467071847CGGCTGCAACACAAGACACGAPS/MOE 5-10-6miR-187 (RFAM-gapmerHuman) 3467081844CGACCATGGCTGTAGACTGTTAPS/MOE 5-10-7miR-132 (RFAM-gapmerHuman) 3467091901TCACATAGGAATAAAAAGCCATAPS/MOE 5-10-8miR-135 (RFAM-gapmerHuman) 3467101802AGCAAGCCCAGACCGCAAAAAGPS/MOE 5-10-7miR-129b (RFAM-gapmerHuman) 3467111898TAGTTGGCAAGTCTAGAACCAPS/MOE 5-10-6miR-182* (RFAM-gapmerHuman) 3467121830CATCATTACCAGGCAGTATTAGAGPS/MOE 5-10-9miR-200a (RFAM-gapmerHuman) 3467131792ACTGATATCAGCTCAGTAGGCACPS/MOE 5-10-8miR-189 (RFAM-gapmerHuman) 3467141870GCAGAAGCATTTCCACACACPS/MOE 5-10-5miR-147 (RFAM-gapmerHuman) 3467151889TAAACGGAACCACTAGTGACTTGPS/MOE 5-10-8miR-224 (RFAM-gapmerHuman) 3467161838CCCTCTGGTCAACCAGTCACAPS/MOE 5-10-6miR-134 (RFAM-gapmerHuman) 3467171763AACCCATGGAATTCAGTTCTCAPS/MOE 5-10-7miR-146 (RFAM-gapmerHuman) 3467181824CACTGGTACAAGGGTTGGGAGAPS/MOE 5-10-7miR-150 (RFAM-gapmerHuman) 3469051907TCCAGTCAAGGATGTTTACAPS/MOEmiR-30e (RFAM-M. musculus) 3469061781ACAGGATTGAGGGGGGGCCCTPS/MOEmiR-296 (RFAM-M. musculus) 3469071815ATGTATGTGGGACGGTAAACCAPS/MOEmiR-299 (RFAM-M. musculus) 3469081881GCTTTGACAATACTATTGCACTGPS/MOEmiR-301 (RFAM-M. musculus) 3469091902TCACCAAAACATGGAAGCACTTAPS/MOEmiR-302 (RFAM-M. musculus) 3469101866GCAATCAGCTAACTACACTGCCTPS/MOEmiR-34a (RFAM-M. musculus) 3469111776ACACTGATTTCAAATGGTGCTAPS/MOEmiR-29b (RFAM-M. musculus) 3469121851CTAGTGGTCCTAAACATTTCAPS/MOEmiR-203 (RFAM-M. musculus) 3469131795AGAAAGGCAGCAGGTCGTATAGPS/MOElet-7d* (RFAM-M. musculus) 3469141810ATCTGCACTGTCAGCACTTTAPS/MOEmiR-106b (RFAM-M. musculus) 3469151784ACATCGTTACCAGACAGTGTTAPS/MOEmiR-200a (RFAM-M. musculus) 3469161874GCGGAACTTAGCCACTGTGAAPS/MOEmiR-27a (RFAM-M. musculus) 3469171826CAGCTATGCCAGCATCTTGCCTPS/MOEmiR-31 (RFAM-M. musculus) 3469181829CAGGCCGGGACAAGTGCAATAPS/MOEmiR-92 (RFAM-M. musculus) 3469191849CTACCTGCACGAACAGCACTTTGPS/MOEmiR-93 (RFAM-M. musculus) 3469201801AGCAAAAATGTGCTAGTGCCAAAPS/MOEmiR-96 (RFAM-M. musculus) 3469211759AACAACCAGCTAAGACACTGCCAPS/MOEmiR-172 (RFAM-M. musculus) 3469221907TCCAGTCAAGGATGTTTACAPS/MOE 5-10-5miR-30e (RFAM-gapmerM. musculus) 3469231781ACAGGATTGAGGGGGGGCCCTPS/MOE 5-10-6miR-296 (RFAM-gapmerM. musculus) 3469241815ATGTATGTGGGACGGTAAACCAPS/MOE 5-10-7miR-299 (RFAM-gapmerM. musculus) 3469251881GCTTTGACAATACTATTGCACTGPS/MOE 5-10-8miR-301 (RFAM-gapmerM. musculus) 3469261902TCACCAAAACATGGAAGCACTTAPS/MOE 5-10-8miR-302 (RFAM-gapmerM. musculus) 3469271866GCAATCAGCTAACTACACTGCCTPS/MOE 5-10-8miR-34a (RFAM-gapmerM. musculus) 3469281776ACACTGATTTCAAATGGTGCTAPS/MOE 5-10-7miR-29b (RFAM-gapmerM. musculus) 3469291851CTAGTGGTCCTAAACATTTCAPS/MOE 5-10-6miR-203 (RFAM-gapmerM. musculus) 3469301795AGAAAGGCAGCAGGTCGTATAGPS/MOE 5-10-7let-7d* (RFAM-gapmerM. musculus) 3469311810ATCTGCACTGTCAGCACTTTAPS/MOE 5-10-6miR-106b (RFAM-gapmerM. musculus) 3469321784ACATCGTTACCAGACAGTGTTAPS/MOE 5-10-7miR-200a (RFAM-gapmerM. musculus) 3469331874GCGGAACTTAGCCACTGTGAAPS/MOE 5-10-6miR-27a (RFAM-gapmerM. musculus) 3469341826CAGCTATGCCAGCATCTTGCCTPS/MOE 5-10-7miR-31 (RFAM-M. musculus)gapmer 3469351829CAGGCCGGGACAAGTGCAATAPS/MOE 5-10-6miR-92 (RFAM-M. musculus)gapmer 3469361849CTACCTGCACGAACAGCACTTTGPS/MOE 5-10-8miR-93 (RFAM-M. musculus)gapmer 3469371801AGCAAAAATGTGCTAGTGCCAAAPS/MOE 5-10-8miR-96 (RFAM-M. musculus)gapmer 3469381759AACAACCAGCTAAGACACTGCCAPS/MOE 5-10-8miR-172 (RFAM-gapmerM. musculus) 3473851782ACAGTGCTTCATCTCPO/6MOE-9deoxymir-143hemimer 3473861848CTACAGTGCTTCATCTCPO/6MOE-11deoxymir-143hemimer 3473871876GCTACAGTGCTTCATCTPO/6MOE-11deoxymir-143hemimer 3473881875GCTACAGTGCTTCATCPO/6MOE-10deoxymir-143hemimer 3473891803AGCTACAGTGCTTCATPO/6MOE-10deoxymir-143hemimer 3473901863GAGCTACAGTGCTTCAPO/6MOE-10deoxymir-143hemimer 3473911916TGAGCTACAGTGCTTCPO/6MOE-10deoxymir-143hemimer 3474521783ACAGTGCTTCATCTCAPO/6MOE-10deoxymir-143hemimer 3474531783ACAGTGCTTCATCTCAPO/6MOE-10deoxymir-143hemimer 3481161922TTCGCCCTCTCAACCCAGCTTTTPS/MOEmiR-320 3481171860GAACCCACAATCCCTGGCTTAPS/MOEmiR-321-1 3481181886GTAAACCATGATGTGCTGCTAPS/MOEmiR-15b(Michael et al) 3481191908TCCATAAAGTAGGAAACACTACAPS/MOEmiR-142as(Michael et al) 3481201864GAGCTACAGTGCTTCATCTCAPS/MOEmiR-143(Michael et al) 3481211883GGATTCCTGGGAAAACTGGACPS/MOEmiR-145(Michael et al) 3481221905TCATCATTACCAGGCAGTATTAPS/MOEmiR-200b(Michael et al) 3481231791ACTATACAATCTACTACCTCAPS/MOElet-7f (Michaelet al) 3481241820CACAAATTCGGTTCTACAGGGTAPS/MOEmiR-10b(Michael et al) 3481251878GCTGGATGCAAACCTGCAAAACTPS/MOEmiR-19b(Michael et al) 3481261873GCCTATCCTGGATTACTTGAAPS/MOEmiR-26a(Michael et al) 3481271869GCAGAACTTAGCCACTGTGAAPS/MOEmiR-27*(Michael et al) 3481281858CTTCCAGTCAAGGATGTTTACAPS/MOEmiR-97 (Michaelet al) 3481291855CTGGCTGTCAATTCATAGGTCAPS/MOEmiR-192(Michael et al) 3481301922TTCGCCCTCTCAACCCAGCTTTTPS/MOE 5-10-8miR-320gapmer 3481311860GAACCCACAATCCCTGGCTTAPS/MOE 5-10-6miR-321-1gapmer 3481321886GTAAACCATGATGTGCTGCTAPS/MOE 5-10-6miR-15bgapmer(Michael et al) 3481331908TCCATAAAGTAGGAAACACTACAPS/MOE 5-10-8miR-142asgapmer(Michael et al) 3481341864GAGCTACAGTGCTTCATCTCAPS/MOE 5-10-6miR-143gapmer(Michael et al) 3481351883GGATTCCTGGGAAAACTGGACPS/MOE 5-10-6miR-145gapmer(Michael et al) 3481361905TCATCATTACCAGGCAGTATTAPS/MOE 5-10-7miR-200bgapmer(Michael et al) 3481371791ACTATACAATCTACTACCTCAPS/MOE 5-10-6let-7f (Michaelgapmeret al) 3481381820CACAAATTCGGTTCTACAGGGTAPS/MOE 5-10-8miR-10bgapmer(Michael et al) 3481391878GCTGGATGCAAACCTGCAAAACTPS/MOE 5-10-8miR-19bgapmer(Michael et al) 3481401873GCCTATCCTGGATTACTTGAAPS/MOE 5-10-6miR-26agapmer(Michael et al) 3481411869GCAGAACTTAGCCACTGTGAAPS/MOE 5-10-6miR-27*gapmer(Michael et al) 3481421858CTTCCAGTCAAGGATGTTTACAPS/MOE 5-10-7miR-97 (Michaelgapmeret al) 3481431855CTGGCTGTCAATTCATAGGTCAPS/MOE 5-10-7miR-192gapmer(Michael et al) 3540401751AAACCACACAACCTACTACCTCAPS/MOElet-7b-Ruvkun 3540411752AAACCATACAACCTACTACCTCAPS/MOElet-7c-Ruvkun 3540421764AACTATGCAACCTACTACCTCTPS/MOElet-7d-Ruvkun 3540431765AACTGTACAAACTACTACCTCAPS/MOElet-7gL-Ruvkun 3540441760AACAGCACAAACTACTACCTCAPS/MOElet-7i-Ruvkun 3540451924TTGGCATTCACCGCGTGCCTTAAPS/MOEmir-124a-Ruvkun 3540461833CCAAGCTCAGACGGATCCGAPS/MOEmir-127-Ruvkun 3540471896TACTTTCGGTTATCTAGCTTTAPS/MOEmir-131-Ruvkun 3540481846CGGCCTGATTCACAACACCAGCTPS/MOEmir-138-Ruvkun 3540491768ACAAACCATTATGTGCTGCTAPS/MOEmir-15-Ruvkun 3540501789ACGCCAATATTTACGTGCTGCTAPS/MOEmir-16-Ruvkun 3540511852CTATCTGCACTAGATGCACCTTAPS/MOEmir-18-Ruvkun 3540521779ACAGCTGCTTTTGGGATTCCGTTGPS/MOEmir-191-Ruvkun 3540531891TAACCGATTTCAGATGGTGCTAPS/MOEmir-29a-Ruvkun 3540541813ATGCTTTGACAATACTATTGCACTGPS/MOEmir-301-Ruvkun 3540551805AGCTGAGTGTAGGATGTTTACAPS/MOEmir-30b-Ruvkun 3540561804AGCTGAGAGTGTAGGATGTTTACAPS/MOEmir-30c-Ruvkun 3540571807AGCTTCCAGTCGGGGATGTTTACAPS/MOEmir-30d-Ruvkun 3540581835CCAGCAGCACCTGGGGCAGTGGPS/MOEmir-324-3p-Ruvkun 3540591899TATGGCAGACTGTGATTTGTTGPS/MOEmir-7-1*-Ruvkun 3540601850CTACCTGCACTGTAAGCACTTTGPS/MOEmir-91-Ruvkun 3540611822CACATAGGAATGAAAAGCCATAPS/MOEmir-135b(Ruvkun) 3540621895TACTAGACTGTGAGCTCCTCGAPS/MOEmir-151*(Ruvkun) 3540631885GGCTATAAAGTAACTGAGACGGAPS/MOEmir-340(Ruvkun) 3540641923TTCTAGGATAGGCCCAGGGGCPS/MOEmir-331(Ruvkun) 3540651892TACATACTTCTTTACATTCCAPS/MOEmiR-1 (RFAM) 3540661817CAATCAGCTAACTACACTGCCTPS/MOEmiR-34c (RFAM) 3540671837CCCCTATCACGATTAGCATTAAPS/MOEmiR-155 (RFAM) 3540681910TCCATCATTACCCGGCAGTATTPS/MOEmiR-200c (RFAM) 3540691818CAATCAGCTAATGACACTGCCTPS/MOEmiR-34b (RFAM) 3540701753AAACCCAGCAGACAATGTAGCTPS/MOEmir-221 (RFAM-M. musculus) 3540711796AGACCCAGTAGCCAGATGTAGCTPS/MOEmir-222 (RFAM-M. musculus) 3540721917TGAGCTCCTGGAGGACAGGGAPS/MOEmir-339-1(RFAM) 3540731925TTTAAGTGCTCATAATGCAGTPS/MOEmiR-20* (human) 3540741926TTTTCCCATGCCCTATACCTCTPS/MOEmiR-202 (human) 3540751856CTTCAGCTATCACAGTACTGTAPS/MOEmiR-101b 3540761894TACCTGCACTGTTAGCACTTTGPS/MOEmiR-106a 3540771772ACAAGTGCCCTCACTGCAGTPS/MOEmiR-17-3p 3540781859GAACAGGTAGTCTAAACACTGGGPS/MOEmiR-199b(mouse) 3540791915TCTTCCCATGCGCTATACCTCT