PRIORITY INFORMATION

This U.S. divisional application claims priority U.S. PCT application Ser. No. 12/299,396, filed 2 May 2007, which claims priority to PCT Application Serial No. PCT/EP07/003,867, filed 2 May 2007, which claims priority to GB Application Serial No. 0608838.9, filed 4 May 2006, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND

RNA interference initially discovered in plants as Post-Transcriptional Gene Silencing (PTGS), is a highly conserved mechanism triggered by double-stranded RNA (dsRNA) and able to down regulate transcript of genes homologous to the dsRNA¹. The dsRNA is first processed by Dicer into short duplexes of 21-23 nt, called short interfering RNAs (siRNAs)². Incorporated in RNA-induced silencing complex (RISC) they are able to mediate gene silencing through cleavage of the target mRNA in the center of the region of homology by Argonaute 2, a component of RISC³. In 2001, Elbashir et al⁴demonstrated that the direct introduction of synthetic siRNAs would mediate RNA interference gene silencing in drosophila but also in mammalian cells. Since then, siRNA-mediated gene silencing has become a powerful and widely-used molecular biology tool in both target identification target validation studies. Use of siRNAs for gene silencing in animal studies has been described in a limited amount of animal models. Unmodified siRNAs were delivered locally in the eye^s, intrathecally or intracerebellarly in the central nervous system⁶, and intranasally for the inhibition of respiratory viruses'. Intravenous hydrodynamic tail vein injection of unmodified siRNAs has also been studied. This approach allows a rapid delivery, mainly to the liver⁸. A very limited number of studies have been reported on the systemic administration of unmodified siRNAs. Duxbury et al⁹administered intravenously unmodified siRNAs targeting Focal Adhesion Kinase to an orthotopic tumor xenograft mice model, and observed a tumor growth inhibition as well as a chemosensitization to gemcitabine. Soutscheck et al reported the systemic use of highly chemically modified siRNAs for the endogeneous silencing Apolipoprotein B. Intraperitoneal administration of most anti-ApoB siRNA at the high dose of 50 mg/kg reduced ApoB protein level and Lipoprotein concentration¹⁰. Despite these examples, in vivo use of siRNAs upon systemic delivery requires improvements in order to make this technology widely applicable for target validation or therapeutic applications. Indeed, unmodified siRNAs are subject to enzymatic digestion, mainly by nucleases abundant in the blood stream. In order to improve pharmacological properties of siRNAs several groups investigated chemical modification of these reagents. While the approaches described are very different among themselves and that no systematic study was yet performed, an overview of the results allows to determine the tolerance of siRNAs to chemical modifications. Several chemistries such as phosphorothioates¹¹or boranophosphates¹², 2â€²-O-Methyl¹³, 2â€²-O-allyl¹⁴, 2â€²-methoxyethyl (MOE) and 2â€²-deoxyfluoronucleotides¹⁵or Locked Nucleic Acids (LNA)¹⁶have been investigated. These studies highlighted that tolerance for modification is not only chemistry-dependent, but also position-dependent.

The present invention provides a minimally modified siRNA with improved pharmacological properties. The minimally modified siRNAs are 19 bp double-stranded RNA modified on the 3â€²-end of each strand in order to prevent 3â€²-exonuclease digestion: the 3â€²-dideoxynucleotide overhang of 21-nt siRNA has been replaced by a universal 3â€²-hydroxypropyl phosphodiester moiety and the modification of the two first base-pairing nucleotides on 3â€²-end of each strand further enhances serum stability. Applied intraperitoneally or orally to adult mice, the modified siRNAs displayed higher potency in a growth factor induce angiogenesis model which correlates with their increased serum stability.

SUMMARY

In one aspect, the present invention provides a short interfering ribonucleic acid (siRNA) for oral administration, said siRNA comprising two separate RNA strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein at least one of which strands contains at least one chemical modification.

In one embodiment, the siRNA comprises at least one modified nucleotide.

In another embodiment, the siRNA comprises at least one 3â€² end cap.

In another embodiment, said modified nucleotide is selected from among 2â€² alkoxyribonucleotide, 2â€² alkoxyalkoxy ribonucleotide, a locked nucleic acid ribonucleotide (LNA), 2â€²-fluoro ribonucleotide, morpholino nucleotide.

In another embodiment, said modified nucleotide is selected from among nucleotides having a modified internucleoside linkage selected from among phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, and amide linkages.

In another embodiment, said two RNA strands are fully complementary to each other.

In another embodiment, said siRNA comprises a 1 to 6 nucleotide overhang on at least one of the 5â€² end or 3â€² end.

In another embodiment, the siRNA contains at least one 3â€² cap, which is chemical moiety conjugated to the 3â€² end via the 3â€² carbon and is selected from among compounds of Formula I:

[Image Omitted]

wherein

X is O or S

or R₁and R₂may be of formula Y-Z where Y is O, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S.

In another embodiment, the siRNA contains at least one strand which is complementary over at least 15 nucleotides to the mRNA or pre-mRNA of VEGFR-1, VEGFR-2, VEGFR3, Tie2, bFGFR, IL8RA, IL8RB, Fas, or IGF2R.

In another embodiment, the siRNA contains at least one strand which comprises a sequence selected from SEQ ID NO 1-900.

In another embodiment, the siRNA is chosen from the group consisting of SEQ ID NO 901-930.

In another embodiment, the siRNA has a stability in a standard gastric acid assay that is greater than an unmodified siRNA with the same nucleotide sequence.

In another embodiment, the siRNA has a stability in a standard gastric acid assay that is greater than or equal to 50% after 30 minutes exposure.

In another embodiment, the siRNA has a stability in a standard serum assay greater than unmodified siRNA.

In another embodiment, the siRNA has a stability in a standard serum assay that is greater than or equal to 50% after 30 minutes exposure.

In another embodiment, the siRNA has a stability in a standard intestinal lavage assay that is greater than unmodified siRNA.

In another embodiment, the siRNA has an enhanced oral bioavailability compared to an unmodified siRNA of the same nucleotide sequence.

In one aspect, the invention provides a pharmaceutical composition comprising an siRNA with any one or more of the above properties.

In another aspect, the invention provides an siRNA with any one or more of the above properties for use as a medicament.

In another aspect, the invention provides the use of an siRNA with any one or more of the above properties in the preparation of a medicament for treating an angiogenic disorder.

In another aspect, the invention provides the use of an siRNA with any one or more of the above properties to inhibit an angiogenic process in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b, 1c, 1d and 1e: Metabolic degradation of unmodified siRNA pG13-siRNA (wild-type siRNA in mouse serum); a-c) Ion Exchange-HPLC analysis of unmodified siRNAs after incubation in mouse serum for 0â€², 30â€² and 180â€²; After 30â€² of incubation at 37Â° C., major peak in the Ion Exchange HPLC was isolated and re-injected in LC-MS, d) table of detected molecular weights and their assignments; e) ESI-MS spectrum

FIG. 2: illustration of four double-stranded RNA formats: wild-type (or unmodified) siRNA. MOE o/h siRNA, C3-siRNA and C3-MOE siRNA.

FIG. 3: Stability of siRNA in 3 different formats in mouse gastric acid. Samples were incubated at 37Â° C. in mouse gastric acid at a 2 micromolar concentration. Disappearance of parent compound was followed over a 2-6 hours period by quantifying the parent compound band.

Lane 1-7: wild-type siRNA in gastric acid at t=0, 5, 10, 15, 30, 60 and 120 min
Lane 8: ds RNA ladder (30, 21, 19, 16, 13, 10 bp)
Lane 9-15: C3 siRNA in gastric acid at t=0, 5, 10, 15, 30, 60 and 120 min
Lane 16: ds RNA ladder (30, 21, 19, 16, 13, 10 bp)
Lane 17-24: C3-MOE siRNA in gastric acid at t=0, 5, 10, 15, 30, 60 and 120 min

FIG. 4: Stability of siRNA in 4 different formats in intestinal lavage. Samples were incubated at 37Â° C. in liver microsomes at a 5 micromolar concentration.

(From Left to Right)

Lane 1: ds RNA ladder (30, 21, 19, 16, 13, 10 bp)
Lane 2-7: wild-type siRNA in intestinal lavage at t=0, 15, 30, 60, 180 and 360 min
Lane 8-13: moe o/h siRNA in intestinal lavage at t=^Ëœ0, 15, 30, 60, 180 and 360 min
Lane 14-19: C3 siRNA in intestinal lavage at t=0, 15, 30, 60, 180 and 360 min
Lane 20-25: C3-MOE siRNA in intestinal lavage at t=0, 15, 30, 60, 180 and 360 min

FIG. 5: Stability of siRNA in 4 different formats in liver microsomes. Samples were incubated at 37Â° C. in intestinal fluid from rat intestinal lavage at a 2 micromolar concentration.

(From Left to Right)Lane 1: ds

FIG. 6: Stability of siRNA in 4 different formats in mouse serum. Samples were incubated at 37Â° C. in mouse serum at a 2 micromolar concentration. Disappearance of parent compound was followed over a 6 hours period by quantifying the parent compound band.

(From Left to Right)

Lane 1: ds RNA ladder (30, 21, 19, 16, 13, 10 bp) RNA ladder (30, 21, 19, 16, 13, 10 bp)
Lane 2: wild-type siRNA untreated
Lane 3: moe o/h siRNA untreated
Lane 4: C3 siRNA untreated
Lane 5: C3-MOE siRNA untreated
Lane 6-9: same as 2-5 in liver microsomes t=0
Lane 10-13: same as 2-5 in liver microsomes t=60â€²
Lane 14-17: same as 2-5 in supernatant S12 t=0
Lane 18-21: same as 2-5 in supernatant S12 t=60â€²
Lane 2-7: wild-type siRNA in mouse serum at t=0, 15, 30, 60, 180 and 360 min
Lane 8-13: moe o/h siRNA in mouse serum at t=0, 15, 30, 60, 180 and 360 min
Lane 14-19: C3 siRNA in mouse serum at t=0, 15, 30, 60, 180 and 360 min
Lane 20-25: C3-MOE siRNA mouse serum at t=0, 15, 30, 60, 180 and 360 min

FIG. 7: Characterization in cellulo of 3 formats of anti-VEGFR2 siRNA (2 independent sequences). Wild-type siRNA, C3-siRNA and C3-MOE siRNA were transfected into MS1 cells at three concentrations (1, 5, 10 nM). Silencing potency was assessed by measuring VEGFR2 cell surface level by FACS.

FIGS. 8a and 8b: In vivo testing of wild-type siRNA, C3-siRNA and C3-Moe siRNA in a growth factor induced angiogenesis â€œAgar Chamberâ€ mouse model. FIG. 8a shows the results of controls, unmodified VEGFR2 siRNA and C3 modified VEGFR2 siRNA at 1, 5 and 25 micrograms per mouse per day. FIG. 8b shows controls, C3 modified VEGFR2 siRNA and of C3-MOE VEGFR2 siRNA at 0.2, 1 and 5 micrograms per mouse per day. In each case pools of 2 anti-VEGFR2 siRNAs were given daily intraperitoneally for three days.

FIG. 9: In vivo testing of anti-VEGFR2 C3-MOE siRNA given intraperitoneally (i.p.) in a Bl6 homograft melanoma tumor mouse model at 5 and 20 micrograms per mouse per day. FIG. 9a shows that i.p. treatment with modified VEGFR2 siRNA significantly reduces tumour development. FIG. 9b also shows that i.p. injection of VEGFR2 siRNA at 20 ug per mouse results in significant inhibition of tumour growth.

FIG. 10: In vivo testing of C3-MOE siRNA in a growth factor induced angiogenesis mouse model. anti-VEGFR2 siRNAs were given daily orally for three days at 20 micrograms per mouse per day.

FIG. 11: In vivo testing of C3-MOE siRNA in a growth factor induced angiogenesis mouse model. anti-Tie2 siRNAs were given daily intraperitoneally (1 and 0.2 micrograms per mouse per day) or orally (20 and 5 micrograms per mouse per day) for three days. FIG. 11a: weight of excised tissue; FIG. 11b: Tie2 protein knock-down

DETAILED DISCLOSURE OF THE INVENTION

The present invention relates to compositions and methods for treating angiogenic disorders in a mammal. Specifically, the invention relates to small-interfering RNA (â€œsiRNAâ€ ) which may be used to treat angiogenic disorders upon oral administration to a mammal.

Angiogenesis targets in vascular endothelial cells include the following targets/genes: VEGFR-1 (GenBank Accession #AF06365); VEGFR-2 (GenBank Accession #AF063658); VEGFR-3 (GenBank Accession #(NM_â€”002020); Tie2 (TEK) (GenBank Accession #NM_â€”000459); bFGFR (GenBank Accession #M60485); IL8RA (GenBank Accession #L19591); IL8RB (GenBank Accession #L19593); Fas (GenBank Accession #X89101); IGF2R (GenBank Accession #NM_â€”000876).

The siRNA molecules according to the present invention mediate RNA interference (â€œRNAiâ€ ). The term â€œRNAiâ€ is well known in the art and is commonly understood to mean the inhibition of one or more target genes in a cell by siRNA with a region which is complementary to the target gene. Various assays are known in the art to test siRNA for its ability to mediate RNAi (see for instance Elbashir et al., Methods 26 (2002), 199-213). The effect of the siRNA according to the present invention on gene expression will typically result in expression of the target gene being inhibited by at least 10%, 33%, 50%, 90%, 95% or 99% when compared to a cell not treated with the RNA molecules according to the present invention.

â€œsiRNAâ€ or â€œsmall-interfering ribonucleic acidâ€ according to the invention has the meanings known in the art, including the following aspects. The siRNA consists of two strands of ribonucleotides which hybridize along a complementary region under physiological conditions. The strands are separate but they may be joined by a molecular linker in certain embodiments. The individual ribonucleotides may be unmodified naturally occurring ribonucleotides, unmodified naturally occurring deoxyribonucleotides or they may be chemically modified or synthetic as described elsewhere herein.

The siRNA molecules in accordance with the present invention comprise a double-stranded region which is substantially identical to a region of the mRNA of the target gene. A region with 100% identity to the corresponding sequence of the target gene is suitable. This state is referred to as â€œfully complementaryâ€ . However, the region may also contain one, two or three mismatches as compared to the corresponding region of the target gene, depending on the length of the region of the mRNA that is targeted, and as such may be not fully complementary. In an embodiment, the RNA molecules of the present invention specifically target one given gene. In order to only target the desired mRNA, the siRNA reagent may have 100% homology to the target mRNA and at least 2 mismatched nucleotides to all other genes present in the cell or organism. Methods to analyze and identify siRNAs with sufficient sequence identity in order to effectively inhibit expression of a specific target sequence are known in the art. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group).

Another factor affecting the efficiency of the RNAi reagent is the target region of the target gene. The region of a target gene effective for inhibition by the RNAi reagent may be determined by experimentation. A suitable mRNA target region would be the coding region. Also suitable are untranslated regions, such as the 5â€²-UTR, the 3â€²-UTR, and splice junctions. For instance, transfection assays as described in Elbashir S. M. et al, 2001 EMBO J., 20, 6877-6888 may be performed for this purpose. A number of other suitable assays and methods exist in the art which are well known to the skilled person.

The length of the region of the siRNA complementary to the target, in accordance with the present invention, may be from 10 to 100 nucleotides, 12 to 25 nucleotides, 14 to 22 nucleotides or 15, 16, 17 or 18 nucleotides. Where there are mismatches to the corresponding target region, the length of the complementary region is generally required to be somewhat longer.

Because the siRNA may carry overhanging ends (which may or may not be complementary to the target), or additional nucleotides complementary to itself but not the target gene, the total length of each separate strand of siRNA may be 10 to 100 nucleotides, 15 to 49 nucleotides, 17 to 30 nucleotides or 19 to 25 nucleotides.

The phrase â€œeach strand is 49 nucleotides or lessâ€ means the total number of consecutive nucleotides in the strand, including all modified or unmodified nucleotides, but not including any chemical moieties which may be added to the 3â€² or 5â€² end of the strand. Short chemical moieties inserted into the strand are not counted, but a chemical linker designed to join two separate strands is not considered to create consecutive nucleotides.

The phrase â€œa 1 to 6 nucleotide overhang on at least one of the 5â€² end or 3â€² endâ€ refers to the architecture of the complementary siRNA that forms from two separate strands under physiological conditions. If the terminal nucleotides are part of the double-stranded region of the siRNA, the siRNA is considered blunt ended. If one or more nucleotides are unpaired on an end, an overhang is created. The overhang length is measured by the number of overhanging nucleotides. The overhanging nucleotides can be either on the 5â€² end or 3â€² end of either strand.

The siRNA according to the present invention confer a high in vivo stability suitable for oral delivery by including at least one modified nucleotide in at least one of the strands. Thus the siRNA according to the present invention contains at least one modified or non-natural ribonucleotide. A lengthy description of many known chemical modifications are set out in published PCT patent application WO 200370918 and will not be repeated here. Suitable modifications for oral delivery are more specifically set out in the Examples and description herein. Suitable modifications include, but are not limited to modifications to the sugar moiety (i.e. the 2â€² position of the sugar moiety, such as for instance 2â€²-O-(2-methoxyethyl) or 2â€²-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group) or the base moiety (i.e. a non-natural or modified base which maintains ability to pair with another specific base in an alternate nucleotide chain). Other modifications include so-called â€˜backboneâ€™ modifications including, but not limited to, replacing the phosphoester group (connecting adjacent ribonucleotides with for instance phosphorothioates, chiral phosphorothioates or phosphorodithioates). Finally, end modifications sometimes referred to herein as 3â€² caps or 5â€² caps may be of significance. As illustrated in Table 1, caps may consist of simply adding additional nucleotides, such as â€œT-Tâ€ which has been found to confer stability on an siRNA. Caps may consist of more complex chemistries which are known to those skilled in the art.

In an embodiment used in the Examples below, the 3â€² cap is a chemical moiety conjugated to the 3â€² end via the 3â€² carbon and is selected from among compounds of Formula I.

[Image Omitted]

wherein

X is O or S

R₁and R₂are independently OH, NH₂, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms and functional groups, preferably a heteroatom selected from the group of N, O, or S or a functional group selected from the group OH, NH₂, SH, carboxylic acid or ester;
or R₁and R₂may be of formula Y-Z where Y is O, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, O, or S.

Examples of modifications on the sugar moiety include 2â€² alkoxyribonucleotide, 2â€² alkoxyalkoxy ribonucleotide, locked nucleic acid ribonucleotide (LNA), 2â€²-fluoro ribonucleotide, morpholino nucleotide.

The internucleoside linkage may also be modified. Examples of internucleoside linkage include phosphorothioate, phosphorodithioate, phosphoramidate, and amide linkages.

R₁may be OH.

R₁and R₂together may comprise from 1 to 24 C-atoms, from 1 to 12 C-atoms, from 2 to 10 C-atoms, from 1 to 8 or from 2 to 6 C-atoms. In another embodiment, R₁and R₂are independently OH, lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl can be substituted by additional heteroatoms and functional groups as defined above. In another embodiment, R₁and R₂are not both OH.

The term â€œlowerâ€ in connection with organic radicals or compounds means a compound or radical which may be branched or unbranched with up to and including 7 carbon atoms, preferably 1-4 carbon atoms. Lower alkyl represents, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and branched pentyl, n-hexyl and branched hexyl.

Examples of alkoxys include O-Met, O-Eth, O-prop, O-but, O-pent, O-hex.

Methods for the synthesis of siRNA, including siRNA containing at least one modified or non-natural ribonucleotides are well known and readily available to those of skill in the art. For example, a variety of synthetic chemistries are set out in published PCT patent applications WO2005021749 and WO200370918, both incorporated herein by reference. The reaction may be carried out in solution or, preferably, on solid phase or by using polymer supported reagents, followed by combining the synthesized RNA strands under conditions, wherein a siRNA molecule is formed, which is capable of mediating RNAi.

The present invention provides an siRNA containing at least one modified nucleotide which is suitable for oral delivery. In functional terms this means siRNA will have suitable pharmacokinetics and biodistribution upon oral administration to achieve delivery to the target tissue of concern. In particular this requires serum stability, lack of immune response, and drug like behaviour. Many of these features of siRNA can be anticipated based on the standard gastric acid assays and standard serum assays disclosed elsewhere herein.

In another aspect, the present invention provides methods for the inhibition of a target gene comprising introducing into a cell and siRNA according to the present invention, which is capable of inhibiting at least one target gene by RNAi. Also, more than one species of siRNA, which are each specific for another target region, may be introduced into a cell at the same time or sequentially.

The present invention is not limited to any type of target gene or nucleotide sequence. For example, the target gene can be a cellular gene, an endogenous gene, a pathogen-associated gene, a viral gene or an oncogene. Angiogenic genes are of particular importance to the invention because some of the Examples highlight that the orally delivered siRNA of the invention may accumulate at sites of vasculogenesis, neovascularization or angiogenesis. An updated listing of angiogenic genes at these sites of particular interest for the invention are listed in AngioDB: database of angiogenesis and angiogenesis-related molecules Tae-Kwon Sohn, Eun-Joung Moon1, Seok-Ki Lee1, Hwan-Gue Cho2 and Kyu-Won Kim3, Nucleic Acids Research, 2002, Vol. 30, No. 1 369-371 and online at http://angiodb.snu.ac.kr/. Genes of particular significance have been analyzed in detail and are set out elsewhere herein.

In another aspect, the invention also provides a kit comprising reagents for inhibiting expression of a target gene in a cell, wherein said kit comprises dsRNA according to the present invention. The kit comprises at least one of the reagents necessary to carry out the in vitro or in vivo introduction of the dsRNA according to the present invention to test samples or subjects. In a preferred embodiment, such kits also comprise instructions detailing the procedures by which the kit components are to be used.

â€œTreatment of an angiogenic disorderâ€ as used in this disclosure means use of a modified siRNA of the invention in a pharmaceutical composition for the treatment of diseases involving the physiological and pathological processes of neovascularization, vasculogenesis and/or angiogenesis. As such, these pharmaceutical compositions are useful for treating diseases, conditions and disorders that require inhibition of neovascularization, vasculogenesis or angiogenesis, including but not limited to cancer tumour growth and metastasis, neoplasm, ocular neovascularization (including macular degeneration, diabetic retinopathy, ischemic retinopathy, retinopathy of prematurity, choroidal neovascularization), rheumatoid arthritis, osteoarthritis, chronic asthma, septic shock, inflammatory diseases, synovitis, bone and cartilage destruction, pannus growth, osteophyte formation, osteomyelitis, psoriasis, obesity, haemangioma, Kaposi's sarcoma, atherosclerosis (including atherosclerotic plaque rupture), endometriosis, warts, excess hair growth, scar keloids, allergic oedema, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, endometriosis, osteomyelitis, inflammatory and infectious processes (hepatitis, pneumonia, glumerulonephtritis), asthma, nasal polyps, transplantation, liver regeneration, leukomalacia, thyroiditis, thyroid enlargement, lymphoproliferative disorders, haematologic malignancies, vascular malformations, and pre-eclampsia.

As used herein, â€œtreatmentâ€ means an action taken to inhibit or reduce a process of a disease, disorder or condition, to inhibit or reduce a symptom of a disease, disorder or condition, or to prophylactically prevent the onset or further development of a disease, disorder or condition. â€œTreatâ€ is the cognitive verb thereof.

An effective dose of the therapeutic agent of the invention is that dose required to treat a disease state. The effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of siRNA is administered dependent upon potency. The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, intraperitoneal, or intrathecal injection, or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions.

Oral administration of the compositions of the invention include all standard techniques for administering substances directly to the stomach or gut, most importantly by patient controlled swallowing of the dosage form, but also by other mechanical and assisted means of such delivery.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

Therapeutic effect of the therapeutic agents of the invention may be enhanced by combination with other agents. Typically such other agents will include agents known for use in treating similar diseases, such as angiogenic disorders. Alternatively, such agents may be used to reduce side-effects or unwanted effects caused by the therapeutic agents of the invention.

The siRNA of the invention also have important research uses. One such study includes research into an angiogenic process in vitro. By â€œangiogenic process in vitroâ€ is meant any process for studying angiogenesis or vasculogenesis which does not employ a whole animal. As such, in vitro or ex vivo methods and assays which study the steps of the angiogenic process using markers or indicators of angiogenesis are included hereby.

RNA Strand Nucleotide Sequences

The siRNA strand sequences identified in Table 1 have been identified as suitable siRNA sequences against the following targets: VEGFR-1 (GenBank Accession #AF06365); VEGFR-2 (GenBank Accession #AF063658); VEGFR-3 (GenBank Accession #(NM_â€”002020); Tie2 (TEK) (GenBank Accession #NM_â€”000459); bFGFR (GenBank Accession #M60485); IL8RA (GenBank Accession #L19591); IL8RB (GenBank Accession #L19593); Fas (GenBank Accession #X89101); IGF2R (GenBank Accession #NM_â€”000876).

TABLEâ€ƒ1siRNAsâ€ƒagainstâ€ƒhumanâ€ƒVEGFR-1,â€ƒVEGFR-2,â€ƒVEGFR-3,â€ƒTie2,â€ƒbFGFR,IL8RA,â€ƒIL8RB,â€ƒFas,â€ƒIGF2RTargetâ€ƒNamepossiRNAâ€ƒguideâ€ƒsequenceSEQâ€ƒIDâ€ƒNOsiRNAâ€ƒcomplementSEQâ€ƒIDâ€ƒNOVEGFR-11731UAUAAGAACUUGUUAACUGTG1CAGUUAACAAGUUCUUAUATT451 VEGFR-11021UACGGUUUCAAGCACCUGCTG2GCAGGUGCUUGAAACCGUATT452 VEGFR-11209UUUAUGCUCAGCAAGAUUGTA3CAAUCUUGCUGAGCAUAAATT453 VEGFR-12904UUAUCUUCCUGAAAGCCGGAG4CCGGCUUUCAGGAAGAUAATT454 VEGFR-11363UUGAGGGAUACCAUAUGCGGT5CGCAUAUGGUAUCCCUCAATT455 VEGFR-11158UUGAUAAUUAACGAGUAGCCA6GCUACUCGUUAAUUAUCAATT456 VEGFR-11091UUAACCAUACAACUUCCGGCG7CCGGAAGUUGUAUGGUUAATT457 VEGFR-1471UUAGGUGACGUAACCCGGCAG8GCCGGGUUACGUCACCUAATT458 VEGFR-12751UUGCUCUUGAGGUAGUUGGAG9CCAACUACCUCAAGAGCAATT459 VEGFR-1636UUUGUCUUAUACAAAUGCCCA10GGCAUUUGUAUAAGACAAATT460 VEGFR-11254UUGACAAUUAGAGUGGCAGTG11CUGCCACUCUAAUUGUCAATT461 VEGFR-12375UUAUAAUUGAUAGGUAGUCAG12GACUACCUAUCAAUUAUAATT462 VEGFR-13536UUGAGUAUGUAAACCCACUAT13AGUGGGUUUACAUACUCAATT463 VEGFR-12971UUCCAUAGUGAUGGGCUCCTT14GGAGCCCAUCACUAUGGAATT464 VEGFR-11774UCUGUUAUUAACUGUCCGCAG15GCGGACAGUUAAUAACAGATT465 VEGFR-13494UUGGGAUGUAGUCUUUACCAT16GGUAAAGACUACAUCCCAATT466 VEGFR-12269UGUUAGAGUGAUCAGCUCCAG17GGAGCUGAUCACUCUAACATT467 VEGFR-1525UUUCCAUCAGGGAUCAAAGTG18CUUUGAUCCCUGAUGGAAATT468 VEGFR-1769UUGAACUCUCGUGUUCAAGGG19CUUGAACACGAGAGUUCAATT469 VEGFR-12246UAGACUUGUCCGAGGUUCCTT20GGAACCUCGGACAAGUCUATT470 VEGFR-1732UUGAGGACAAGAGUAUGGCCT21GCCAUACUCUUGUCCUCAATT471 VEGFR-13813UUACUGGUUACUCUCAAGUCA22ACUUGAGAGUAACCAGUAATT472 VEGFR-13925UUCCAGCUCAGCGUGGUCGTA23CGACCACGCUGAGCUGGAATT473 VEGFR-11414UGCUUCGGAAUGAUUAUGGTT24CCAUAAUCAUUCCGAAGCATT474 VEGFR-1615UUGACUGUUGCUUCACAGGTC25CCUGUGAAGCAACAGUCAATT475 VEGFR-13300UCAUCCAUUUGUACUCCUGGG26CAGGAGUACAAAUGGAUGATT476 VEGFR-12845UGGUUUCUUGCCUUGUUCCAG27GGAACAAGGCAAGAAACCATT477 VEGFR-12802UUAGGCUCCAUGUGUAGUGCT28CACUACACAUGGAGCCUAATT478 VEGFR-11564UCUAGAGUCAGCCACAACCAA29GGUUGUGGCUGACUCUAGATT479 VEGFR-11154UAAUUAACGAGUAGCCACGAG30CGUGGCUACUCGUUAAUUATT480 VEGFR-11090UAACCAUACAACUUCCGGCGA31GCCGGAAGUUGUAUGGUUATT481 VEGFR-11260UUCACAUUGACAAUUAGAGTG32CUCUAAUUGUCAAUGUGAATT482 VEGFR-13530AUGUAAACCCACUAUUUCCTG33GGAAAUAGUGGGUUUACAUTT483 VEGFR-11177AUCCUCUUCAGUUACGUCCTT34GGACGUAACUGAAGAGGAUTT484 VEGFR-11193UUGUAUAAUUCCCUGCAUCCT35GAUGCAGGGAAUUAUACAATT485 VEGFR-11092UUUAACCAUACAACUUCCGGC36CGGAAGUUGUAUGGUUAAATT486 VEGFR-1627UACAAAUGCCCAUUGACUGTT37CAGUCAAUGGGCAUUUGUATT487 VEGFR-1474AUGUUAGGUGACGUAACCCGG38GGGUUACGUCACCUAACAUTT488 VEGFR-12761UAAGUCACGUUUGCUCUUGAG39CAAGAGCAAACGUGACUUATT489 VEGFR-12752UUUGCUCUUGAGGUAGUUGGA40CAACUACCUCAAGAGCAAATT490 VEGFR-13516UUUCCUGUCAGUAUGGCAUTG41AUGCCAUACUGACAGGAAATT491 VEGFR-11790UACUGUAGUGCAUUGUUCUGT42AGAACAAUGCACUACAGUATT492 VEGFR-11155AUAAUUAACGAGUAGCCACGA43GUGGCUACUCGUUAAUUAUTT493 VEGFR-11370UUGUAGGUUGAGGGAUACCAT44GGUAUCCCUCAACCUACAATT494 VEGFR-12227UUGAACAGUGAGGUAUGCUGA45AGCAUACCUCACUGUUCAATT495 VEGFR-13481UUUACCAUCCUGUUGUACATT46UGUACAACAGGAUGGUAAATT496 VEGFR-11261UUUCACAUUGACAAUUAGAGT47UCUAAUUGUCAAUGUGAAATT497 VEGFR-11791AUACUGUAGUGCAUUGUUCTG48GAACAAUGCACUACAGUAUTT498 VEGFR-13805UACUCUCAAGUCAAUCUUGAG49CAAGAUUGACUUGAGAGUATT499 VEGFR-12764AAAUAAGUCACGUUUGCUCTT50GAGCAAACGUGACUUAUUUTT500 VEGFR-2617UAAUAGACUGGUAACUUUCAT51GAAAGUUACCAGUCUAUUATT501 VEGFR-22686UAGAAGGUUGACCACAUUGAG52CAAUGUGGUCAACCUUCUATT502 VEGFR-2561UAGCUGAUCAUGUAGCUGGGA53CCAGCUACAUGAUCAGCUATT503 VEGFR-2525UUGCUGUCCCAGGAAAUUCTG54GAAUUUCCUGGGACAGCAATT504 VEGFR-22277AUGAUUUCCAAGUUCGUCUTT55AGACGAACUUGGAAAUCAUTT505 VEGFR-2395UAAUGUACACGACUCCAUGTT56CAUGGAGUCGUGUACAUUATT506 VEGFR-22410UUCAUCUGGAUCCAUGACGAT57CGUCAUGGAUCCAGAUGAATT507 VEGFR-22007UGAUUCUCCAGGUUUCCUGTG58CAGGAAACCUGGAGAAUCATT508 VEGFR-21323UAGACCGUACAUGUCAGCGTT59CGCUGACAUGUACGGUCUATT509 VEGFR-23382UUCUGGUGUAGUAUAAUCAGG60UGAUUAUACUACACCAGAATT510 VEGFR-23078UUUCGUGCCGCCAGGUCCCTG61GGGACCUGGCGGCACGAAATT511 VEGFR-21432UUCUUCACAAGGGUAUGGGTT62CCCAUACCCUUGUGAAGAATT512 VEGFR-21817UCAAUUUCCAAAGAGUAUCCA63GAUACUCUUUGGAAAUUGATT513 VEGFR-2688UAGUUCAAUUCCAUGAGACGG64GUCUCAUGGAAUUGAACUATT514 VEGFR-22310AACAUGGCAAUCACCGCCGTG65CGGCGGUGAUUGCCAUGUUTT515 VEGFR-22130UCCUUCAAUACAAUGCCUGAG66CAGGCAUUGUAUUGAAGGATT516 VEGFR-2799UACAAGUUUCUUAUGCUGATG67UCAGCAUAAGAAACUUGUATT517 VEGFR-23523UGAUAUCGGAAGAACAAUGTA68CAUUGUUCUUCCGAUAUCATT518 VEGFR-21843UGUGCUAUUAGAGAACAUGGT69CAUGUUCUCUAAUAGCACATT519 VEGFR-22941UUCUACAUCACUGAGGGACTT70GUCCCUCAGUGAUGUAGAATT520 VEGFR-22088UCUUUAAACCACAUGAUCUGT71AGAUCAUGUGGUUUAAAGATT521 VEGFR-2472UCUUGCACAAAGUGACACGTT72CGUGUCACUUUGUGCAAGATT522 VEGFR-2180UGAUUAUUGGGCCAAAGCCAG73GGCUUUGGCCCAAUAAUCATT523 VEGFR-21568AUUUGUACAAAGCUGACACAT74GUGUCAGCUUUGUACAAAUTT524 VEGFR-23141UAAAUAUCCCGGGCCAAGCCA75GCUUGGCCCGGGAUAUUUATT525 VEGFR-23769AACCAUACCACUGUCCGUCTG76GACGGACAGUGGUAUGGUUTT526 VEGFR-23920UGUCAUCGGAGUGAUAUCCGG77GGAUAUCACUCCGAUGACATT527 VEGFR-21718UCUCAAACGUAGAUCUGUCTG78GACAGAUCUACGUUUGAGATT528 VEGFR-22919UCCUCCACAAAUCCAGAGCTG79GCUCUGGAUUUGUGGAGGATT529 VEGFR-2324UAAAUGACCGAGGCCAAGUCA80ACUUGGCCUCGGUCAUUUATT530 VEGFR-21050UAACCAAGGUACUUCGCAGGG81CUGCGAAGUACCUUGGUUATT531 VEGFR-256UAGGCAAACCCACAGAGGCGG82GCCUCUGUGGGUUUGCCUATT532 VEGFR-22453UGGCAUCAUAAGGCAGUCGTT83CGACUGCCUUAUGAUGCCATT533 VEGFR-21303UUGAGUGGUGCCGUACUGGTA84CCAGUACGGCACCACUCAATT534 VEGFR-21813UUUCCAAAGAGUAUCCAAGTT85CUUGGAUACUCUUUGGAAATT535 VEGFR-22015UUGUCGUCUGAUUCUCCAGGT86CUGGAGAAUCAGACGACAATT536 VEGFR-23088UAAGAGGAUAUUUCGUGCCGC87GGCACGAAAUAUCCUCUUATT537 VEGFR-2625UAUGUACAUAAUAGACUGGTA88CCAGUCUAUUAUGUACAUATT538 VEGFR-2800UUACAAGUUUCUUAUGCUGAT89CAGCAUAAGAAACUUGUAATT539 VEGFR-2811UAGGUCUCGGUUUACAAGUTT90ACUUGUAAACCGAGACCUATT540 VEGFR-2812UUAGGUCUCGGUUUACAAGTT91CUUGUAAACCGAGACCUAATT541 VEGFR-23093UCCGAUAAGAGGAUAUUUCGT92GAAAUAUCCUCUUAUCGGATT542 VEGFR-2801UUUACAAGUUUCUUAUGCUGA93AGCAUAAGAAACUUGUAAATT543 VEGFR-22009UCUGAUUCUCCAGGUUUCCTG94GGAAACCUGGAGAAUCAGATT544 VEGFR-22127UUCAAUACAAUGCCUGAGUCT95ACUCAGGCAUUGUAUUGAATT545 VEGFR-21585UUUGUUGACCGCUUCACAUTT96AUGUGAAGCGGUCAACAAATT546 VEGFR-2562AUAGCUGAUCAUGUAGCUGGG97CAGCUACAUGAUCAGCUAUTT547 VEGFR-23906UAUCCGGACUGGUAGCCGCTT98GCGGCUACCAGUCCGGAUATT548 VEGFR-21316UACAUGUCAGCGUUUGAGUGG99ACUCAAACGCUGACAUGUATT549 VEGFR-23520UAUCGGAAGAACAAUGUAGTC100CUACAUUGUUCUUCCGAUATT550 VEGFR-3453UUCCUGUUGACCAAGAGCGTG101CGCUCUUGGUCAACAGGAATT551 VEGFR-32694UUGAGCUCCGACAUCAGCGCG102CGCUGAUGUCGGAGCUCAATT552 VEGFR-31689UUGGAUUCGAUGGUGAAGCCG103GCUUCACCAUCGAAUCCAATT553 VEGFR-3988UUCAUGCACAAUGACCUCGGT104CGAGGUCAUUGUGCAUGAATT554 VEGFR-34374UUACCAAGGAAUAAUCGGCGG105GCCGAUUAUUCCUUGGUAATT555 VEGFR-32142UCUUUGUACCACACGAUGCTG106GCAUCGUGUGGUACAAAGATT556 VEGFR-31833UUGCAGUCGAGCAGAAGCGGG107CGCUUCUGCUCGACUGCAATT557 VEGFR-33903UUCAGCUACCUGAAGCCGCTT108GCGGCUUCAGGUAGCUGAATT558 VEGFR-33273UACACCUUGUCGAAGAUGCTT109GCAUCUUCGACAAGGUGUATT559 VEGFR-31107UACCACUGGAACUCGGGCGGG110CGCCCGAGUUCCAGUGGUATT560 VEGFR-3336UAGCAGACGUAGCUGCCUGTG111CAGGCAGCUACGUCUGCUATT561 VEGFR-32607UUGUGGAUGCCGAAAGCGGAG112CCGCUUUCGGCAUCCACAATT562 VEGFR-31556UCACAGUCUUAUUCUUUCCCT113GGAAAGAAUAAGACUGUGATT563 VEGFR-3108UCCGUGAUGUUCAAGGUCGGG114CGACCUUGAACAUCACGGATT564 VEGFR-31954AUAGUGGCCCUCGUGCUCGGG115CGAGCACGAGGGCCACUAUTT565 VEGFR-32100AAGCACUGCAUCUCCAGCGAG116CGCUGGAGAUGCAGUGCUUTT566 VEGFR-3693UCAUAGAGCUCGUUGCCUGTG117CAGGCAACGAGCUCUAUGATT567 VEGFR-32337AGGAUCACGAUCUCCAUGCTG118GCAUGGAGAUCGUGAUCCUTT568 VEGFR-32054UCAAGUUCUGCGUGAGCCGAG119CGGCUCACGCAGAACUUGATT569 VEGFR-3860UCUGUUGGGAGCGUCGCUCGG120GAGCGACGCUCCCAACAGATT570 VEGFR-32436UAGCCCGUCUUGAUGUCUGCG121CAGACAUCAAGACGGGCUATT571 VEGFR-33759UUCAUCCUGGAGGAACCACGG122GUGGUUCCUCCAGGAUGAATT572 VEGFR-3288AACACCUUGCAGUAGGGCCTG123GGCCCUACUGCAAGGUGUUTT573 VEGFR-31485UGCGUGGUCACCGCCCUCCAG124GGAGGGCGGUGACCACGCATT574 VEGFR-32502UCGUAGGACAGGUAUUCGCAT125GCGAAUACCUGUCCUACGATT575 VEGFR-3925AUACGAGCCCAGGUCGUGCTG126GCACGACCUGGGCUCGUAUTT576 VEGFR-3426UUGUUGAUGAAUGGCUGCUCA127AGCAGCCAUUCAUCAACAATT577 VEGFR-33189UAGAUGUCCCGGGCAAGGCCA128GCCUUGCCCGGGACAUCUATT578 VEGFR-32274UUGACGCAGCCCUUGGGUCTG129GACCCAAGGGCUGCGUCAATT579 VEGFR-32196UUCUGGUUGGAGUCCGCCAAG130UGGCGGACUCCAACCAGAATT580 VEGFR-32019UGCACCGACAGGUACUUCUTG131AGAAGUACCUGUCGGUGCATT581 VEGFR-3360AUGCGUGCCUUGAUGUACUTG132AGUACAUCAAGGCACGCAUTT582 VEGFR-31755UACUUGUAGCUGUCGGCUUGG133AAGCCGACAGCUACAAGUATT583 VEGFR-33037UUCCAUGGUCAGCGGGCUCAG134GAGCCCGCUGACCAUGGAATT584 VEGFR-31018UUUGAGCCACUCGACGCUGAT135CAGCGUCGAGUGGCUCAAATT585 VEGFR-31684UUCGAUGGUGAAGCCGUCGGG136CGACGGCUUCACCAUCGAATT586 VEGFR-34373UACCAAGGAAUAAUCGGCGGG137CGCCGAUUAUUCCUUGGUATT587 VEGFR-3987UCAUGCACAAUGACCUCGGTG138CCGAGGUCAUUGUGCAUGATT588 VEGFR-33267UUGUCGAAGAUGCUUUCAGGG139CUGAAAGCAUCUUCGACAATT589 VEGFR-34387UGUAUUACUCAUAUUACCAAG140UGGUAAUAUGAGUAAUACATT590 VEGFR-33883UUCUUGUCUAUGCCUGCUCTC141GAGCAGGCAUAGACAAGAATT591 VEGFR-34376UAUUACCAAGGAAUAAUCGGC142CGAUUAUUCCUUGGUAAUATT592 VEGFR-32140UUUGUACCACACGAUGCUGGG143CAGCAUCGUGUGGUACAAATT593 VEGFR-3978AUGACCUCGGUGCUCUCCCGA144GGGAGAGCACCGAGGUCAUTT594 VEGFR-32427UUGAUGUCUGCGUGGGCCGGC145CGGCCCACGCAGACAUCAATT595 VEGFR-31109UGUACCACUGGAACUCGGGCG146CCCGAGUUCCAGUGGUACATT596 VEGFR-3319UGUGUCGUUGGCAUGUACCTC147GGUACAUGCCAACGACACATT597 VEGFR-31843AUGCACGUUCUUGCAGUCGAG148CGACUGCAAGAACGUGCAUTT598 VEGFR-3317UGUCGUUGGCAUGUACCUCGT149GAGGUACAUGCCAACGACATT599 VEGFR-3700CUGGAUGUCAUAGAGCUCGTT150CGAGCUCUAUGACAUCCAGTT600 Tie-21223UAAGCUUACAAUCUGGCCCGT151GGGCCAGAUUGUAAGCUUATT601(TEK) Tie-22350UAUCUUCACAUCAACGUGCTG152GCACGUUGAUGUGAAGAUATT602(TEK) Tie-2706UAUGUUCACGUUAUCUCCCTT153GGGAGAUAACGUGAACAUATT603(TEK) Tie-23561UUUAAGGACACCAAUAUCUGG154AGAUAUUGGUCUCCUUAAATT604(TEK) Tie-22763UGAAAUUUGAUGUCAUUCCAG155GGAAUGACAUCAAAUUUCATT605(TEK) Tie-2174UUGUUUACAAGUUAGAGGCAA156GCCUCUAACUUGUAAACAATT606(TEK) Tie-21183UUCAUUGCACUGCAGACCCTT157GGGUCUGCAGUGCAAUGAATT607(TEK) Tie-2805UAGAAUAUCAGGUACUUCATG158UGAAGUACCUGAUAUUCUATT608(TEK) Tie-22601UUCAAUUGCAAUAUGAUCAGA159UGAUCAUAUUGCAAUUGAATT609(TEK) Tie-22277UAGCCAUCCAAUAUUGUCCAA160GGACAAUAUUGGAUGGCUATT610(TEK) Tie-21366UACUUCUAUAUGAUCUGGCAA161GCCAGAUCAUAUAGAAGUATT611(TEK) Tie-232UUUGGUAUCAGCAGGGCUGGG162CAGCCCUGCUGAUACCAAATT612(TEK) Tie-24085UGUACUAUCAGGGUCAUUGTT163CAAUGACCCUGAUAGUACATT613(TEK) Tie-23881UUCUGAUUUCAGCCCAUUCTT164GAAUGGGCUGAAAUCAGAATT614(TEK) Tie-2646UUGUUGACGCAUCUUCAUGGT165CAUGAAGAUGCGUCAACAATT615(TEK) Tie-24021AUAGCAUUCAACAUAAAGGTA166CCUUUAUGUUGAAUGCUAUTT616(TEK) Tie-2209UUUGUGACUUUCCAUUAGCAT167GCUAAUGGAAAGUCACAAATT617(TEK) Tie-24223UAAAUGAAACGGGACUGGCTG168GCCAGUCCCGUUUCAUUUATT618(TEK) Tie-23961UACUAAUUGUACUCACGCCTT169GGCGUGAGUACAAUUAGUATT619(TEK) Tie-21771UUGAAUAUGUUGCCAAGCCTC170GGCUUGGCAACAUAUUCAATT620(TEK) Tie-23909UUAUUGCAUAUGAAACCACAA171GUGGUUUCAUAUGCAAUAATT621(TEK) Tie-23606UAAAGCGUGGUAUUCACGUAG172ACGUGAAUACCACGCUUUATT622(TEK) Tie-2477AUUAAGGCUUCAAAGUCCCTT173GGGACUUUGAAGCCUUAAUTT623(TEK) Tie-23421UUCUGCACAAGUCAUCCCGCA174CGGGAUGACUUGUGCAGAATT624(TEK) Tie-22730UAAAUUGUAGGAUCUGGGUTG175ACCCAGAUCCUACAAUUUATT625(TEK) Tie-21800UAGUUGAGUGUAACAAUCUCA176AGAUUGUUACACUCAACUATT626(TEK) Tie-23385UAAGCUAACAAUCUCCCAUAG177AUGGGAGAUUGUUAGCUUATT627(TEK) Tie-21692UAAGGCUCAGAGCUGAUGUTG178ACAUCAGCUCUGAGCCUUATT628(TEK) Tie-21657AUGUCCAGUGUCAAUCACGTT179CGUGAUUGACACUGGACAUTT629(TEK) Tie-23665UUCUGUCCUAGGCCGCUUCTT180GAAGCGGCCUAGGACAGAATT630(TEK) Tie-22091UUAAGUAGCACCGAAGUCAAG181UGACUUCGGUGCUACUUAATT631(TEK) Tie-22827UAACCCAUCCUUCUUGAUGCG182CAUCAAGAAGGAUGGGUUATT632(TEK) Tie-21979UUGGUUGCCAGGUCAAAUUTA183AAUUUGACCUGGCAACCAATT633(TEK) Tie-267UAGAUUAGGAUGGGAAAGGCT184CCUUUCCCAUCCUAAUCUATT634(TEK) Tie-23459UUCUCCAGUCUGUAGCCCUGG185AGGGCUACAGACUGGAGAATT635(TEK) Tie-22764UUGAAAUUUGAUGUCAUUCCA186GAAUGACAUCAAAUUUCAATT636(TEK) Tie-23560UUAAGGACACCAAUAUCUGGG187CAGAUAUUGGUGUCCUUAATT637(TEK) Tie-2715UUUGAAAGAUAUGUUCACGTT188CGUGAACAUAUCUUUCAAATT638(TEK) Tie-21368UUUACUUCUAUAUGAUCUGGC189CAGAUCAUAUAGAAGUAAATT639(TEK) Tie-22351UUAUCUUCACAUCAACGUGCT190CACGUUGAUGUGAAGAUAATT640(TEK) Tie-2205UGACUUUCCAUUAGCAUCGTC191CGAUGCUAAUGGAAAGUCATT641(TEK) Tie-23957AAUUGUACUCACGCCUUCCTA192GGAAGGCGUGAGUACAAUUTT642(TEK) Tie-23962AUACUAAUUGUACUCACGCCT193GCGUGAGUACAAUUAGUAUTT643(TEK) Tie-22352UUUAUCUUCACAUCAACGUGC194ACGUUGAUGUGAAGAUAAATT644(TEK) Tie-23963UAUACUAAUUGUACUCACGCC195CGUGAGUACAAUUAGUAUATT645(TEK) Tie-21777UGUCACUUGAAUAUGUUGCCA196GCAACAUAUUCAAGUGACATT646(TEK) Tie-23388UCCUAAGCUAACAAUCUCCCA197GGAGAUUGUUAGCUUAGGATT647(TEK) Tie-2636AUCUUCAUGGUUCGUAUCCTG198GGAUACGAACCAUGAAGAUTT648(TEK) Tie-274UCCUUUGUAGAUUAGGAUGGG199CAUCCUAAUCUACAAAGGATT649(TEK) Tie-2707AUAUGUUCACGUUAUCUCCCT200GGAGAUAACGUGAACAUAUTT650(TEK) bFGFR3814UAAAUCUCUGGUAACGACCCT201GGUCGUUACCAGAGAUUUATT651 bFGFR1478UUACACAUGAACUCCACGUTG202ACGUGGAGUUCAUGUGUAATT652 bFGFR3773UAUACUCAGAUUUAUCAACTT203GUUGAUAAAUCUGAGUAUATT653 bFGFR715UAGCGGUGCAGAGUGUGGCTG204GCCACACUCUGCACCGCUATT654 bFGFR575UUCAAACUGACCCUCGCUCGG205GAGCGAGGGUCAGUUUGAATT655 bFGFR646UUCUGCAGUUAGAGGUUGGTG206CCAACCUCUAACUGCAGAATT656 bFGFR3625AUCGGAAUUAAUAAGCCACTG207GUGGCUUAUUAAUUCCGAUTT657 bFGFR2318UACAAGGGACCAUCCUGCGTG208CGCAGGAUGGUCCCUUGUATT658 bFGFR1439UUGUUGGCGGGCAACCCUGCT209CAGGGUUGCCCGCCAACAATT659 bFGFR3860AUAGCAACUGAUGCCUCCCAG210GGGAGGCAUCAGUUGCUAUTT660 bFGFR3163UGAGGGUUACAGCUGACGGTG211CCGUCAGCUGUAACCCUCATT661 bFGFR2600UCGAUGUGGUGAAUGUCCCGT212GGGACAUUCACCACAUCGATT662 bFGFR2513UCUCGGUGUAUGCACUUCUTG213AGAAGUGCAUACACCGAGATT663 bFGFR2214UUUCUCUGUUGCGUCCGACTT214GUCGGACGCAACAGAGAAATT664 bFGFR1346UUCUCCACAAUGCAGGUGUAG215ACACCUGCAUUGUGGAGAATT665 bFGFR1556UUGUCUGGGCCAAUCUUGCTC216GCAAGAUUGGCCCAGACAATT666 bFGFR2671UCCGGUCAAAUAAUGCCUCGG217GAGGCAUUAUUUGACCGGATT667 bFGFR3105UUUGAGUCCGCCAUUGGCAAG218UGCCAAUGGCGGACUCAAATT668 bFGFR2091UUUGCCUAAGACCAGUCUGTC219CAGACUGGUCUUAGGCAAATT669 bFGFR1590UCCAGCAGUCUUCAAGAUCTG220GAUCUUGAAGACUGCUGGATT670 bFGFR1689UCCGAUAGAGUUACCCGCCAA221GGCGGGUAACUCUAUCGGATT671 bFGFR1319UUGUCAGAGGGCACCACAGAG222CUGUGGUGCCCUCUGACAATT672 bFGFR2342UUGGAGGCAUACUCCACGATG223UCGUGGAGUAUGCCUCCAATT673 bFGFR107UCUCGGUCCCGACCGGACGTG224CGUCCGGUCGGGACCGAGATT674 bFGFR3662UCUGGUACCAGGCAUUUGGTC225CCAAAUGCCUGGUACCAGATT675 bFGFR2150UUGUCCAGCCCGAUAGCCUCT226AGGCUAUCGGGCUGGACAATT676 bFGFR1517UUUAGCCACUGGAUGUGCGGC227CGCACAUCCAGUGGCUAAATT677 bFGFR1264UGUAGCCUCCAAUUCUGUGGT228CACAGAAUUGGAGGCUACATT678 bFGFR3576UUCAAUCGUGGCUCGAAGCAC229GCUUCGAGCCACGAUUGAATT679 bFGFR613AUCUCCAUGGAUACUCCACAG230GUGGAGUAUCCUAGGAGAUTT680 bFGFR1221UUUCAACCAGCGCAGUGUGGG231CACACUGCGCUGGUUGAAATT681 bFGFR3004UAGAGCUCCGGGUGUCGGGAA232CCCGACACCCGGAGCUCUATT682 bFGFR3825UUACCGAUGGGUAAAUCUCTG233GAGAUUUACCCAUCGGUAATT683 bFGFR3813AAAUCUCUGGUAACGACCCTT234GGGUCGUUACCAGAGAUUUTT684 bFGFR3861UAUAGCAACUGAUGCCUCCCA235GGAGGCAUCAGUUGCUAUATT685 bFGFR576UUUCAAACUGACCCUCGCUCG236AGCGAGGGUCAGUUUGAAATT686 bFGFR3772AUACUCAGAUUUAUCAACUTT237AGUUGAUAAAUCUGAGUAUTT687 bFGFR3824UACCGAUGGGUAAAUCUCUGG238AGAGAUUUACCCAUCGGUATT688 bFGFR2319AUACAAGGGACCAUCCUGCGT239GCAGGAUGGUCCCUUGUAUTT689 bFGFR3771UACUCAGAUUUAUCAACUUTG240AAGUUGAUAAAUCUGAGUATT690 bFGFR2511UCGGUGUAUGCACUUCUUGGA241CAAGAAGUGCAUACACCGATT691 bFGFR2333UACUCCACGAUGACAUACAAG242UGUAUGUCAUCGUGGAGUATT692 bFGFR3624UCGGAAUUAAUAAGCCACUGG243AGUGGCUUAUUAAUUCCGATT693 bFGFR1304ACAGAGUCCAUUAUGAUGCTC244GCAUCAUAAUGGACUCUGUTT694 bFGFR1608UUUGUCGGUGGUAUUAACUCC245AGUUAAUACCACCGACAAATT695 bFGFR1301GAGUCCAUUAUGAUGCUCCAG246GGAGCAUCAUAAUGGACUCTT696 bFGFR3626UAUCGGAAUUAAUAAGCCACT247UGGCUUAUUAAUUCCGAUATT697 bFGFR2672AUCCGGUCAAAUAAUGCCUCG248AGGCAUUAUUUGACCGGAUTT698 bFGFR2213UUCUCUGUUGCGUCCGACUTC249AGUCGGACGCAACAGAGAATT699 bFGFR2597AUGUGGUGAAUGUCCCGUGCG250CACGGGACAUUCACCACAUTT700 IL8RA1971UUUAUUAGGAACAUCUGCCTG251GGCAGAUGUUCCUAAUAAATT701 IL8RA75UUGAUCUAACUGAAGCACCGG252GGUGCUUCAGUUAGAUCAATT702 IL8RA645AUUGUUUGGAUGGUAAGCCTG253GGCUUACCAUCCAAACAAUTT703 IL8RA1431UAAUUAGCCAGUUAGUGGGTT254CCCACUAACUGGCUAAUUATT704 IL8RA1378UUCGUUUCCAUGGAGGUGCAA255GCACCUCCAUGGAAACGAATT705 IL8RA1470UCAUCUAAUGUCAGAUUCGGG256CGAAUCUGACAUUAGAUGATT706 IL8RA218UACUUGUUGAGUGUCUCAGTT257CUGAGACACUCAACAAGUATT707 IL8RA1101AUGACGUGCCAAGAACUCCTT258GGAGUUCUUGGCACGUCAUTT708 IL8RA677UUUCCCAGGACCUCAUAGCAA259GCUAUGAGGUCCUGGGAAATT709 IL8RA1178AAGAGAUAUUCCUUCAUCGAT260CGAUGAAGGAAUAUCUCUUTT710 IL8RA1543UUGAGGAGAUGCUCCUGUGAG261CACAGGAGCAUCUCCUCAATT711 IL8RA1783UCUUGUGGCAUAGAUCUGGCT262CCAGAUCUAUGCCACAAGATT712 IL8RA1249AUAGUGCCUGUCCAGAGCCAG263GGCUCUGGACAGGCACUAUTT713 IL8RA1520UCAACGAGAGCAUCCAGCCCT264GGCUGGAUGCUCUCGUUGATT714 IL8RA1068AUGCAUAGCCAGGAUCUUGAG265CAAGAUCCUGGCUAUGCAUTT715 IL8RA1347UUGGAGGUACCUCAACAGCTC266GCUGUUGAGGUACCUCCAATT716 IL8RA1208UCAGGGUGUUGGUUAUUCUTT267AGAAUAACCAACACCCUGATT717 IL8RA117AUCUGUAAUAUUUGACAUGTC268CAUGUCAAAUAUUACAGAUTT718 IL8RA1862UGCUUGUCUCGUUCCACUUGG269AAGUGGAACGAGACAAGCATT719 IL8RA1153UUCAGAGGUUGGAAGAGACAT270GUCUCUUCCAACCUCUGAATT720 IL8RA640UUGGAUGGUAAGCCUGGCGGA271CGCCAGGCUUACCAUCCAATT721 IL8RA1411UAAAGAUGUGACGUUCAACGG272GUUGAACGUCACAUCUUUATT722 IL8RA71UCUAACUGAAGCACCGGCCAG273GGCCGGUGCUUCAGUUAGATT723 IL8RA1397UCAACGGGAAUGAUGGUGCTT274GCACCAUCAUUCCCGUUGATT724 IL8RA644UUGUUUGGAUGGUAAGCCUGG275AGGCUUACCAUCCAAACAATT725 IL8RA641UUUGGAUGGUAAGCCUGGCGG276GCCAGGCUUACCAUCCAAATT726 IL8RA76UUUGAUCUAACUGAAGCACCG277GUGCUUCAGUUAGAUCAAATT727 IL8RA1398UUCAACGGGAAUGAUGGUGCT278CACCAUCAUUCCCGUUGAATT728 IL8RA1381UGCUUCGUUUCCAUGGAGGTG279CCUCCAUGGAAACGAAGCATT729 IL8RA1769UCUGGCUUCCAAACCCUCUTT280AGAGGGUUUGGAAGCCAGATT730 IL8RA1435AUGCUAAUUAGCCAGUUAGTG281CUAACUGGCUAAUUAGCAUTT731 IL8RA1175AGAUAUUCCUUCAUCGAUGGT282CAUCGAUGAAGGAAUAUCUTT732 IL8RA1970UUAUUAGGAACAUCUGCCUGC283AGGCAGAUGUUCCUAAUAATT733 IL8RA1432CUAAUUAGCCAGUUAGUGGGT284CCACUAACUGGCUAAUUAGTT734 IL8RA74UGAUCUAACUGAAGCACCGGC285CGGUGCUUCAGUUAGAUCATT735 IL8RA646AAUUGUUUGGAUGGUAAGCCT286GCUUACCAUCCAAACAAUUTT736 IL8RA639UGGAUGGUAAGCCUGGCGGAA287CCGCCAGGCUUACCAUCCATT737 IL8RA1082UUGCUGACCAGGCCAUGCATA288UGCAUGGCCUGGUCAGCAATT738 IL8RA1770AUCUGGCUUCCAAACCCUCTT289GAGGGUUUGGAAGCCAGAUTT739 IL8RA81AAUGGUUUGAUCUAACUGAAG290UCAGUUAGAUCAAACCAUUTT740 IL8RA1372UCCAUGGAGGUGCAAAGGCCG291GCCUUUGCACCUCCAUGGATT741 IL8RA1388AUGAUGGUGCUUCGUUUCCAT292GGAAACGAAGCACCAUCAUTT742 IL8RA643UGUUUGGAUGGUAAGCCUGGC293CAGGCUUACCAUCCAAACATT743 IL8RA1784UUCUUGUGGCAUAGAUCUGGC294CAGAUCUAUGCCACAAGAATT744 IL8RA1524AGGGUCAACGAGAGCAUCCAG295GGAUGCUCUCGUUGACCCUTT745 IL8RA237AUAGGCGAUGAUCACAACATA296UGUUGUGAUCAUCGCCUAUTT746 IL8RA219AUACUUGUUGAGUGUCUCAGT297UGAGACACUCAACAAGUAUTT747 IL8RA1389AAUGAUGGUGCUUCGUUUCCA298GAAACGAAGCACCAUCAUUTT748 IL8RA1972CUUUAUUAGGAACAUCUGCCT299GCAGAUGUUCCUAAUAAAGTT749 IL8RA1115UAGGAGGUAACACGAUGACGT300GUCAUCGUGUUACCUCCUATT750 IL8RB2648UUAAGUGUCAAUUUAGUGGCA301CCACUAAAUUGACACUUAATT751 IL8RB2184UUUCUUGUGGGUCAAUUCCTA302GGAAUUGACCCACAAGAAATT752 IL8RB2250UUGGGUCUUGUGAAUAAGCTG303GCUUAUUCACAAGACCCAATT753 IL8RB1746UUCACUUCUUAGAACAUAGAG304CUAUGUUCUAAGAAGUGAATT754 IL8RB960UUGGAUGAGUAGACGGUCCTT305GGACCGUCUACUCAUCCAATT755 IL8RB454AUUACUAAGAUCUUCACCUTT306AGGUGAAGAUCUUAGUAAUTT756 IL8RB2750UUGGUUUAAUCAGCCUUGGTG307CCAAGGCUGAUUAAACCAATT757 IL8RB2604AUCACUACUGUUUAUCUGCAG308GCAGAUAAACAGUAGUGAUTT758 IL8RB1026AUCCGUAACAGCAUCCGCCAG309GGCGGAUGCUGUUACGGAUTT759 IL8RB1384AUGUAUAGCUAGAAUCUUGAG310CAAGAUUCUAGCUAUACAUTT760 IL8RB1149AAGAUGACCCGCAUGGCCCGG311GGGCCAUGCGGGUCAUCUUTT761 IL8RB2464UCUCAGUACCUCAUGUAGGTG312CCUACAUGAGGUACUGAGATT762 IL8RB877UUUGACCAAGUAGCGCUUCTG313GAAGCGCUACUUGGUCAAATT763 IL8RB2324UUCGUUAGGUACAUAUCACAT314GUGAUAUGUACCUAACGAATT764 IL8RB2360AUGAGUACUUCAUUCCUCUTT315AGAGGAAUGAAGUACUCAUTT765 IL8RB265UUGGGUGGUAGUCAGAGCUGT316AGCUCUGACUACCACCCAATT766 IL8RB1642UUUCUAAACCAUGCAAGGGAA317CCCUUGCAUGGUUUAGAAATT767 IL8RB2146UCAUGUGUUAAUUCUAUGUCT318ACAUAGAAUUAACACAUGATT768 IL8RB2627UUAAGUCACAUUGCGGUACAA319GUACCGCAAUGUGACUUAATT769 IL8RB1000UGUAUUGUUGCCCAUGUCCTC320GGACAUGGGCAACAAUACATT770 IL8RB315UGACCUGCUGUUAUUGGAGTG321CUCCAAUAACAGCAGGUCATT771 IL8RB2774AAAUAUAGGCAGGUGGUUCTA322GAACCACCUGCCUAUAUUUTT772 IL8RB219ACCUUGACGAUGAAACUUCTG323GAAGUUUCAUCGUCAAGGUTT773 IL8RB2389UUUCAAGGUUCGUCCGUGUTG324ACACGGACGAACCUUGAAATT774 IL8RB385UGAGGUAAACUUAAAUCCUGA325AGGAUUUAAGUUUACCUCATT775 IL8RB1347UUCUGGCCAAUGAAGGCGUAG326ACGCCUUCAUUGGCCAGAATT776 IL8RB2649UUUAAGUGUCAAUUUAGUGGC327CACUAAAUUGACACUUAAATT777 IL8RB1737UAGAACAUAGAGUGCCAUGGG328CAUGGCACUCUAUGUUCUATT778 IL8RB455AAUUACUAAGAUCUUCACCTT329GGUGAAGAUCUUAGUAAUUTT779 IL8RB965UAACAUUGGAUGAGUAGACGG330GUCUACUCAUCCAAUGUUATT780 IL8RB1740UCUUAGAACAUAGAGUGCCAT331GGCACUCUAUGUUCUAAGATT781 IL8RB2632UGGCAUUAAGUCACAUUGCGG332GCAAUGUGACUUAAUGCCATT782 IL8RB2755UAGCCUUGGUUUAAUCAGCCT333GCUGAUUAAACCAAGGCUATT783 IL8RB2183UUCUUGUGGGUCAAUUCCUAT334AGGAAUUGACCCACAAGAATT784 IL8RB2605UAUCACUACUGUUUAUCUGCA335CAGAUAAACAGUAGUGAUATT785 IL8RB2340UCAGGCUGAAGGAUACUUCGT336GAAGUAUCCUUCAGCCUGATT786 IL8RB2143UGUGUUAAUUCUAUGUCUGAA337CAGACAUAGAAUUAACACATT787 IL8RB998UAUUGUUGCCCAUGUCCUCAT338GAGGACAUGGGCAACAAUATT788 IL8RB2180UUGUGGGUCAAUUCCUAUAAG339UAUAGGAAUUGACCCACAATT789 IL8RB2185AUUUCUUGUGGGUCAAUUCCT340GAAUUGACCCACAAGAAAUTT790 IL8RB307UGUUAUUGGAGUGGCCACCGA341GGUGGCCACUCCAAUAACATT791 IL8RB2481UCUGUAAAUUUGUUCACUCTC342GAGUGAACAAAUUUACAGATT792 IL8RB2617UUGCGGUACAACUAUCACUAC343AGUGAUAGUUGUACCGCAATT793 IL8RB956AUGAGUAGACGGUCCUUCGGA344CGAAGGACCGUCUACUCAUTT794 IL8RB456UAAUUACUAAGAUCUUCACCT345GUGAAGAUCUUAGUAAUUATT795 IL8RB226UGAAACAACCUUGACGAUGAA346CAUCGUCAAGGUUGUUUCATT796 IL8RB1394UGAUCAAGCCAUGUAUAGCTA347GCUAUACAUGGCUUGAUCATT797 IL8RB458UGUAAUUACUAAGAUCUUCAC348GAAGAUCUUAGUAAUUACATT798 IL8RB881UGAAUUUGACCAAGUAGCGCT349CGCUACUUGGUCAAAUUCATT799 IL8RB2327UACUUCGUUAGGUACAUAUCA350AUAUGUACCUAACGAAGUATT800 Fas109UGUAGUAACAGUCUUCCUCAA351GAGGAAGACUGUUACUACATT801 Fas41UGGACGAUAAUCUAGCAACAG352GUUGCUAGAUUAUCGUCCATT802 Fas161UAUGGCAGAAUUGGCCAUCAT353GAUGGCCAAUUCUGCCAUATT803 Fas182UUUCACCUGGAGGACAGGGCT354CCCUGUCCUCCAGGUGAAATT804 Fas62UCACUUGGGCAUUAACACUTT355AGUGUUAAUGCCCAAGUGATT805 Fas377ACUUCCUCUUUGCACUUGGTG356CCAAGUGCAAAGAGGAAGUTT806 Fas349UGAGUGUGCAUUCCUUGAUGA357AUCAAGGAAUGCACACUCATT807 Fas245UCCCUUCUUGGCAGGGCACGC358GUGCCCUGCCAAGAAGGGATT808 Fas205GACUGUGCAGUCCCUAGCUTT359AGCUAGGGACUGCACAGUCTT809 Fas145AUCAUGAUGCAGGCCUUCCAA360GGAAGGCCUGCAUCAUGAUTT810 Fas123UUCUGAGUCUCAACUGUAGTA361CUACAGUUGAGACUCAGAATT811 Fas34UAAUCUAGCAACAGACGUAAG362UACGUCUGUUGCUAGAUUATT812 Fas114UCAACUGUAGUAACAGUCUTC363AGACUGUUACUACAGUUGATT813 Fas115CUCAACUGUAGUAACAGUCTT364GACUGUUACUACAGUUGAGTT814 Fas28AGCAACAGACGUAAGAACCAG365GGUUCUUACGUCUGUUGCUTT815 Fas122UCUGAGUCUCAACUGUAGUAA366ACUACAGUUGAGACUCAGATT816 Fas186UUCCUUUCACCUGGAGGACAG367GUCCUCCAGGUGAAAGGAATT817 Fas42UUGGACGAUAAUCUAGCAACA368UUGCUAGAUUAUCGUCCAATT818 Fas111ACUGUAGUAACAGUCUUCCTC369GGAAGACUGUUACUACAGUTT819 Fas144UCAUGAUGCAGGCCUUCCAAG370UGGAAGGCCUGCAUCAUGATT820 Fas92UCAAUUCCAAUCCCUUGGAGT371UCCAAGGGAUUGGAAUUGATT821 Fas201GUGCAGUCCCUAGCUUUCCTT372GGAAAGCUAGGGACUGCACTT822 Fas128CCAAGUUCUGAGUCUCAACTG373GUUGAGACUCAGAACUUGGTT823 Fas36GAUAAUCUAGCAACAGACGTA374CGUCUGUUGCUAGAUUAUCTT824 Fas162UUAUGGCAGAAUUGGCCAUCA375AUGGCCAAUUCUGCCAUAATT825 Fas127CAAGUUCUGAGUCUCAACUGT376AGUUGAGACUCAGAACUUGTT826 Fas202UGUGCAGUCCCUAGCUUUCCT377GAAAGCUAGGGACUGCACATT827 Fas82UCCCUUGGAGUUGAUGUCAGT378UGACAUCAACUCCAAGGGATT828 Fas160AUGGCAGAAUUGGCCAUCATG379UGAUGGCCAAUUCUGCCAUTT829 Fas150UGGCCAUCAUGAUGCAGGCCT380GCCUGCAUCAUGAUGGCCATT830 Fas63GUCACUUGGGCAUUAACACTT381GUGUUAAUGCCCAAGUGACTT831 Fas164GCUUAUGGCAGAAUUGGCCAT382GGCCAAUUCUGCCAUAAGCTT832 Fas37CGAUAAUCUAGCAACAGACGT383GUCUGUUGCUAGAUUAUCGTT833 Fas116UCUCAACUGUAGUAACAGUCT384ACUGUUACUACAGUUGAGATT834 Fas32AUCUAGCAACAGACGUAAGAA385CUUACGUCUGUUGCUAGAUTT835 Fas64AGUCACUUGGGCAUUAACACT386UGUUAAUGCCCAAGUGACUTT836 Fas167AGGGCUUAUGGCAGAAUUGGC387CAAUUCUGCCAUAAGCCCUTT837 Fas120UGAGUCUCAACUGUAGUAACA388UUACUACAGUUGAGACUCATT838 Fas125AGUUCUGAGUCUCAACUGUAG389ACAGUUGAGACUCAGAACUTT839 Fas43UUUGGACGAUAAUCUAGCAAC390UGCUAGAUUAUCGUCCAAATT840 Fas94CCUCAAUUCCAAUCCCUUGGA391CAAGGGAUUGGAAUUGAGGTT841 Fas159UGGCAGAAUUGGCCAUCAUGA392AUGAUGGCCAAUUCUGCCATT842 Fas110CUGUAGUAACAGUCUUCCUCA393AGGAAGACUGUUACUACAGTT843 Fas31UCUAGCAACAGACGUAAGAAC394UCUUACGUCUGUUGCUAGATT844 Fas38ACGAUAAUCUAGCAACAGACG395UCUGUUGCUAGAUUAUCGUTT845 Fas118AGUCUCAACUGUAGUAACAGT396UGUUACUACAGUUGAGACUTT846 Fas169ACAGGGCUUAUGGCAGAAUTG397AUUCUGCCAUAAGCCCUGUTT847 Fas33AAUCUAGCAACAGACGUAAGA398UUACGUCUGUUGCUAGAUUTT848 Fas163CUUAUGGCAGAAUUGGCCATC399UGGCCAAUUCUGCCAUAAGTT849 Fas233AGGGCACGCAGUCUGGUUCAT400GAACCAGACUGCGUGCCCUTT850 IGF2R6340UUUGUCACCUAUGACACCCAG401GGGUGUCAUAGGUGACAAATT851 IGF2R2936UUAUAGAGCAAGCCUGGUCTG402GACCAGGCUUGCUCUAUAATT852 IGF2R1331UCUGAUUGUGGUAUCUUCCTG403GGAAGAUACCACAAUCAGATT853 IGF2R4491UAUUUCAGGACAAUUAUGCCA404GCAUAAUUGUCCUGAAAUATT854 IGF2R2562UUAAUGUAGUAUUUCCUCCAC405GGAGGAAAUACUACAUUAATT855 IGF2R1456UUUCCCAUCGUUACCUGCGGT406CGCAGGUAACGAUGGGAAATT856 IGF2R2253UAGUUCAGUUGGAUCAUCCCA407GGAUGAUCCAACUGAACUATT857 IGF2R3570UUGCCUUCUGACACUAAGCAA408GCUUAGUGUCAGAAGGCAATT858 IGF2R2274UUAUAGGGUGUGCCGCCUCTG409GAGGCGGCACACCCUAUAATT859 IGF2R1197UUUCCAUCUGAAAUAUAGGAT410CCUAUAUUUCAGAUGGAAATT860 IGF2R897UUGCGCACCAGCUUCAGUCCG411GACUGAAGCUGGUGCGCAATT861 IGF2R5205UUGAUGUAGAAAUCAGGGUTG412ACCCUGAUUUCUACAUCAATT862 IGF2R8904UUCUCAGCAAUAGAACACCAG413GGUGUUCUAUUGCUGAGAATT863 IGF2R8604UAAGGCUUCUUAUAGGUCGAA414CGACCUAUAAGAAGCCUUATT864 IGF2R3629UCAAAGAUCCAUUCGCCGCGG415GCGGCGAAUGGAUCUUUGATT865 IGF2R4344UUGAUGAGGUAGUGCUCCGGG416CGGAGCACUACCUCAUCAATT866 IGF2R1419UUUAUGACGCUCAUCCGCUGA417AGCGGAUGAGCGUCAUAAATT867 IGF2R7185UAUUUGUAGGACACGUUGGAA418CCAACGUGUCCUACAAAUATT868 IGF2R4447UACCCUGCCGAGGUUCACGGG419CGUGAACCUCGGCAGGGUATT869 IGF2R3706UAUCUGAGCACACUCAAACGT420GUUUGAGUGUGCUCAGAUATT870 IGF2R6422UCUUUGUACAGGUCAAUUCTA421GAAUUGACCUGUACAAAGATT871 IGF2R1306UUUGACUUGAGAGGUAUCGCT422CGAUACCUCUCAAGUCAAATT872 IGF2R6129UUGUGUUUCUGGACGAAUUTG423AAUUCGUCCAGAAACACAATT873 IGF2R5105UAGAGCUUCCAUUCCUCACGG424GUGAGGAAUGGAAGCUCUATT874 IGF2R4572UUCACUUGGCUCUCGCUGCAG425GCAGCGAGAGCCAAGUGAATT875 IGF2R5308UACCCGGCCGAUAUCUAUGGG426CAUAGAUAUCGGCCGGGUATT876 IGF2R3153UUCUCAAUUCCGACUGGCCTT427GGCCAGUCGGAAUUGAGAATT877 IGF2R9029UAUUACAGUAAAGUUGAUUGA428AAUCAACUUUACUGUAAUATT878 IGF2R1530UUAACACAGGCGUAUUCCGTG429CGGAAUACGCCUGUGUUAATT879 IGF2R8364AAAUGUGCUCUGUACGCCCAG430GGGCGUACAGAGCACAUUUTT880 IGF2R5400UAGUUGAAAUGCUUGUCCGCT431CGGACAAGCAUUUCAACUATT881 IGF2R6702UUGGCUCCAGAGCACGCCGGG432CGGCGUGCUCUGGAGCCAATT882 IGF2R8479UUCUCUGACACCUCAACUCCA433GAGUUGAGGUGUCAGAGAATT883 IGF2R4723UAAGGAGCUCAGAUCAAACAG434GUUUGAUCUGAGCUCCUUATT884 IGF2R4237UGAACAUUCAGUCAGAUCGAA435CGAUCUGACUGAAUGUUCATT885 IGF2R6203UAUAGUACGAGACUCCGUUGT436AACGGAGUCUCGUACUAUATT886 IGF2R753AUGAAUAGAGAAGUGUCCGGA437CGGACACUUCUCUAUUCAUTT887 IGF2R8554AUAAGCACAGUAAAGGUGGTA438CCACCUUUACUGUGCUUAUTT888 IGF2R5462UUAACAGCUUAGGCGUUCCCA439GGAACGCCUAAGCUGUUAATT889 IGF2R1460UUCCUUUCCCAUCGUUACCTG440GGUAACGAUGGGAAAGGAATT890 IGF2R5206AUUGAUGUAGAAAUCAGGGTT441CCCUGAUUUCUACAUCAAUTT891 IGF2R2559AUGUAGUAUUUCCUCCACGTG442CGUGGAGGAAAUACUACAUTT892 IGF2R8605UUAAGGCUUCUUAUAGGUCGA443GACCUAUAAGAAGCCUUAATT893 IGF2R4345AUUGAUGAGGUAGUGCUCCGG444GGAGCACUACCUCAUCAAUTT894 IGF2R1187AAAUAUAGGAUGAACCUCCGC445GGAGGUUCAUCCUAUAUUUTT895 IGF2R1184UAUAGGAUGAACCUCCGCUCT446AGCGGAGGUUCAUCCUAUATT896 IGF2R7190UUGAGUAUUUGUAGGACACGT447GUGUCCUACAAAUACUCAATT897 IGF2R7182UUGUAGGACACGUUGGAACTT448GUUCCAACGUGUCCUACAATT898 IGF2R2941AUCCCUUAUAGAGCAAGCCTG449GGCUUGCUCUAUAAGGGAUTT899 IGF2R3693UCAAACGUGAUCCUGGUGGAG450CCACCAGGAUCACGUUUGATT900

Chemical Modification of RNA Strand Nucleotides

The siRNA according to the invention may comprise at least one modified nucleotide in at least one of the RNA strands. A range of potential modified nucleotides are disclosed elsewhere herein. Useful modifications and combinations of modifications for use according to the invention are shown in Table 2:

TABLEâ€ƒ2Chemicalâ€ƒModificationsâ€ƒandâ€ƒSequenceâ€ƒArchitecture#Modificationâ€ƒNameFormat1PSâ€ƒDNAâ€ƒo/hâ€ƒâ€ƒâ€ƒâ€ƒNNNNNNNNNNNNNNNNNNNsnsnnsnsNNNNNNNNNNNNNNNNNNN 2Fullâ€ƒPSâ€ƒâ€ƒâ€ƒâ€ƒNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsN 3RNAâ€ƒo/hâ€ƒâ€ƒNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 4Blunt-endedNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 52â€²-OMeâ€ƒo/hâ€ƒâ€ƒâ€ƒâ€ƒNNNNNNNNNNNNNNNNNNNN^pN^pN^pN^pNNNNNNNNNNNNNNNNNNN 62â€²-OMe/2â€²Fâ€ƒâ€ƒâ€ƒâ€ƒNNNNNNNNNNNNNNNNNNNN^pN^pN^pN^pNNNNNNNNNNNNNNNNNNN 7LNAâ€ƒ(3-7â€ƒâ€ƒâ€ƒâ€ƒNNNNNNNNNNNNNNNNNNNsnsnincorporationsnsnsNNNNNNNNNNNNNNNNNNNinâ€ƒdsâ€ƒregion)N = any unmodified RNA nucleotiden = unmodified DNA nucleotideN^p = modified RNA nucleotides = identifies phosphorothioate internucleoside linkageo/h = overhang

The following modifications added to the 3â€² position of the 3â€²-terminus of the siRNA strands, sometimes referred to as a â€˜3â€² end capâ€™ are also recognized as useful embodiments of the invention and may be used with any of the siRNA according to the invention:

[Image Omitted]

Specific compounds with activity according to the invention include the following, shown in Table 3:

TABLEâ€ƒ3Sequencesâ€ƒandâ€ƒChemistriesâ€ƒofâ€ƒsiRNAâ€ƒusedâ€ƒinâ€ƒExamplesSequenceâ€ƒ(N:â€ƒRNA;â€ƒdN:â€ƒDNA;Namestrand2'-moeâ€ƒRNA;â€ƒs:â€ƒphosphorothioate)SEQâ€ƒIDâ€ƒNOpGI3-siRNAguideâ€ƒstrandUCGâ€ƒAAGâ€ƒUACâ€ƒUCAâ€ƒGCGâ€ƒUAAâ€ƒGdTdT901complementâ€ƒstrandCUUâ€ƒACGâ€ƒCUGâ€ƒAGUâ€ƒACUâ€ƒUCGâ€ƒAdTdT902 pGL3â€ƒMOEâ€ƒo/hguideâ€ƒstrandCUUâ€ƒACGâ€ƒCUGâ€ƒAGUâ€ƒACUâ€ƒUCGâ€ƒAtst903siRNAcomplementâ€ƒstrandUCGâ€ƒAAGâ€ƒUACâ€ƒUCAâ€ƒGCGâ€ƒUAAâ€ƒGtst904 pGI3-C3-siRNAguideâ€ƒstrandUCGâ€ƒAAGâ€ƒUACâ€ƒUCAâ€ƒGCGâ€ƒUAAâ€ƒG-C3905complementâ€ƒstrandCUUâ€ƒACGâ€ƒCUGâ€ƒAGUâ€ƒACUâ€ƒUCGâ€ƒA-C3906 pGI3-C3-MOE-guideâ€ƒstrandUCGâ€ƒAAGâ€ƒUACâ€ƒUCAâ€ƒGCGâ€ƒUAaâ€ƒg-C3907siRNAcomplementâ€ƒstrandCUUâ€ƒACGâ€ƒCUGâ€ƒAGUâ€ƒACUâ€ƒUCgâ€ƒa-C3908 VEGFR2-siRNA1guideâ€ƒstrandUUGâ€ƒAGGâ€ƒUUUâ€ƒGAAâ€ƒAUCâ€ƒGACâ€ƒCdCdT909complementâ€ƒstrandGGUâ€ƒCGAâ€ƒUUUâ€ƒCAAâ€ƒACCâ€ƒUCAâ€ƒAdTdT910 VEGFR2-siRNA2guideâ€ƒstrandUAAâ€ƒUUUâ€ƒGUUâ€ƒCCUâ€ƒGUCâ€ƒUUCâ€ƒCdAdG911complementâ€ƒstrandGGAâ€ƒAGAâ€ƒCAGâ€ƒGAAâ€ƒCAAâ€ƒAUUâ€ƒAdTdT912 siRNAâ€ƒcontrolguideâ€ƒstrandACGâ€ƒUGAâ€ƒCACâ€ƒGUUâ€ƒCGGâ€ƒAGAâ€ƒAdTdT913complementâ€ƒstrandUUCâ€ƒUCCâ€ƒGAAâ€ƒCGUâ€ƒGUCâ€ƒACGâ€ƒUdTdT914 VEGFR2-C3-guideâ€ƒstrandUUGâ€ƒAGGâ€ƒUUUâ€ƒGAAâ€ƒAUCâ€ƒGACâ€ƒC-C3915siRNA1complementâ€ƒstrandGGUâ€ƒCGAâ€ƒUUUâ€ƒCAAâ€ƒACCâ€ƒUCAâ€ƒA-C3916 VEGFR2-C3-guideâ€ƒstrandUAAâ€ƒUUUâ€ƒGUUâ€ƒCCUâ€ƒGUCâ€ƒUUCâ€ƒC-C3917siRNA2complementâ€ƒstrandGGAâ€ƒAGAâ€ƒCAGâ€ƒGAAâ€ƒCAAâ€ƒAUUâ€ƒA-C3918 C3-siRNAâ€ƒcontrolguideâ€ƒstrandACGâ€ƒUGAâ€ƒCACâ€ƒGUUâ€ƒCGGâ€ƒAGAâ€ƒA-C3919complementâ€ƒstrandUUCâ€ƒUCCâ€ƒGAAâ€ƒCGUâ€ƒGUCâ€ƒACGâ€ƒU-C3920 VEGFR2-C3-MOE-guideâ€ƒstrandUUGâ€ƒAGGâ€ƒUUUâ€ƒGAAâ€ƒAUCâ€ƒGAcâ€ƒc-C3921siRNA1complementâ€ƒstrandGGUâ€ƒCGAâ€ƒUUUâ€ƒCAAâ€ƒACCâ€ƒUCaâ€ƒa-C3922 VEGFR2-C3-MOE-guideâ€ƒstrandUAAâ€ƒUUUâ€ƒGUUâ€ƒCCUâ€ƒGUCâ€ƒUUcâ€ƒc-C3923siRNA2complementâ€ƒstrandGGAâ€ƒAGAâ€ƒCAGâ€ƒGAAâ€ƒCAAâ€ƒAUuâ€ƒA-C3924 Tie2-C3-MOE-guideâ€ƒstrandUUCâ€ƒUUCâ€ƒUUUâ€ƒAAUâ€ƒUAAâ€ƒCAcâ€ƒc-C3925siRNA1complementâ€ƒstrandGGUâ€ƒGUUâ€ƒAAUâ€ƒUAAâ€ƒAGAâ€ƒAGaâ€ƒa-C3926 Tie2-C3-MOE-guideâ€ƒstrandUCUâ€ƒGAGâ€ƒUUUâ€ƒGUAâ€ƒAAUâ€ƒAUcâ€ƒg-C3927siRNA2complementâ€ƒstrandCGAâ€ƒUAUâ€ƒUUAâ€ƒCAAâ€ƒACUâ€ƒCAgâ€ƒa-C3928 C3-MOE-siRNAguideâ€ƒstrandACGâ€ƒUGAâ€ƒCACâ€ƒGUUâ€ƒCGGâ€ƒAGaâ€ƒa-C3929controlcomplementâ€ƒstrandUUCâ€ƒUCCâ€ƒGAAâ€ƒCGUâ€ƒGUCâ€ƒAGgâ€ƒt-C3930

EXAMPLES

The following Examples illustrate aspects of the invention, and are not intended to limit the embodiments included in the claims recited below. The results and discussion section further below refers to experiments conducted according to the following protocols and employing the following materials. Materials and protocols that are not specifically described are considered to be routinely available to those skilled in the art.

Example 1Preparation of siRNAs

Single strand siRNA derivatives were synthesized by standard 2â€²-O-TOM phosphoamidite technology and purified by OasisÂ® HLB Extraction Plates (Waters). Sense- and antisense stranded siRNA were mixed in hybridization buffer (100 mM potassium acetate, 2 mM magnesium acetate, 30 mM Hepes, pH 7.6) heat-denatured at 90Â° C. for 3 min and annealed at 37Â° C. for 60 min. 100 Î¼M stock solutions of siRNA duplexes were stored at âˆ’20Â° C.

Example 2Incubation in Serum and Analysis by IE-HPLC (Lc-Ms)

In a standard serum assay, 6 Î¼L 20 Î¼M of each siRNA were mixed with 54 Î¼L serum or CSF and heated at 37Â° C. in an incubator. 50 Î¼L of the cooled mixture was loaded on an analytical DNA-pac PA-100 Column (Dionex) and analyzed with a NaCl gradient (0-0.6 M in 30 min) in a 1:10 Acetonitrile:Buffer (20 mM sodium acetate, 1 mM magnesium acetate, pH 6.5) solution.

For LC-MS analysis 100 Î¼L (20 Î¼M or 50 Î¼M) each siRNA was mixed with 900 Î¼L sterile fetal bovine serum (GIBCO) incubated at 37Â° C. and separated by HPLC as indicated previously (except of the NaCl gradient: 0M-0.36M in 9â€²/0.36M-0.6M in 12â€²). Degradation products were desalted on NAP columns and analyzed by LC-ESIâ€³-MS.

Example 3Incubation in Gastric Acid

To prepare a standard gastric acid assay, FVB and C57BL6 mice, weighing 18 to 20 g (6 to 8 weeks old), were obtained from Charles River Laboratories (Les Oncins, France). Animals were sacrificed using CO₂, and then stomachs were quickly recovered. Gastric fluid as well as stomach contents were collected and pooled, then loaded on centrifugal filter devices (Ultrafree MC, Millipores). Filter units were spun for 10 minutes according to manufacturer's recommendations. The filtrate, corresponding to mouse gastric fluid, was recovered, aliquoted and frozen prior further experiments.

For each assay, 20 Î¼M of siRNA solutions were diluted in 9Ã— volume of gastric acid as above described and incubated at 37Â° C. for 0, 5, 10, 15, 30, 60 and 120 min.

Example 4Incubation in Intestinal Lavage

To prepare a standard intestinal lavage assay, Male Wistar rat were fasted, anesthetized with isoflurane. Intestinal lavage was obtained by in situ perfusion of the small intestine (duodenum, jejunum, ileum) with 10 mL saline (0.5 mL/min) followed by 20 mL water (1 mL/min). Outlet collected was centrifuged (3000Ã—g, 15 min, 22Â° C.), and supernatant passed through a 1.2-Î¼m filter and stored at âˆ’20Â° C. For each assay, 20 Î¼M siRNA solutions were diluted in 9Ã— volume of intestinal lavage and incubated at 37Â° C. for 0, 15, 30, 60, 180 and 360 min.

Example 5Incubation in Mouse Liver Microsomes

In a standard liver microsome assay, to 10 Î¼l of a 250 Î¼M solution of siRNA were added 25 Î¼l of mouse liver microsomes (GEntest 452701 Charge 11) at 20 mg protein/ml, 365 Î¼l of 100 mM phosphate buffer (pH 7.4), 50 Î¼l of UDPGA cofactor (24 mM in water), 50 Î¼l of NADPH. Incubation was quenched by freezing at t=0 min and t=60 min.

Example 6Incubation in Rat S12 Supernatant

For a standard rat S12 supernatant assay, 10 Î¼l of a 250 Î¼M solution of siRNA were added to 17 Î¼l of rat liver S12 at 29.9 mg protein/ml, 373 Î¼l of 100 mM phosphate buffer (pH 7.4), 50 Î¼l of UDPGA cofactor (24 mM in water), 50 Î¼l of NADPH. Incubation was quenched by freezing at t=0 min and t=60 min.

Example 7Incubation in Mouse Serum

For a standard incubation in mouse serum, 20 M siRNA solutions were diluted in 9Ã— volume of murine serum (Harlan nude mouse) and incubated at 37Â° C. for 0, 15, 30, 60, 180 and 360 min.

Example 8Gel Electrophoresis Stability Assay

A 10 Î¼L aliquot of incubation solution was taken immediately after shaking and shock-frozen on dry ice, the mixtures were incubated at 37Â° C. and aliquots were shock frozen at various time points. Aliquots were thawed in 30 Î¼L (15 Î¼L respectively) Loading Buffer (Elchrom Sc., Cham, Switzerland) and separated on a SF50 gels (Elchrom Sc., Cham, Switzerland) at 120 V, 8Â° C. for 240 min. Bands were stained with SYBR Gold (Molecular Probes) and picture were taken with a BIORAD ChemiDocâ„¢ XRS system.

Example 9Cell Culture

The mouse immortalized endothelial cell line MS1 (ATCC CRL-2279) was grown in DMEM high glucose (4.5 g/l) supplemented with L-Glutamine and 10% heat-inactivated FCS (AMIMED, Switzerland) on 1.5% Gelatine-coated culture dishes. MS1 cells were transfected in 24 well-format with siRNA using HiPerfect (QIAGEN) according to manufacturer procedure (tetraplicate, final siRNA concentration was 10 nM or as indicated).

Example 10FACS Analysis

Non-transfected and siRNA transfected MS1 cells were analyzed by FACS for VEGFR2 levels. Briefly, cells were trypsinized from duplicate or triplicate wells, pooled for each conditions, then washed twice with PBS+10% FCS and incubated 10 minutes on ice prior addition of RPE-conjugated anti-VEGFR2 Ab (1 Î¼g/10⁶cells; Avas 12Î±1, BD Pharmingen). RPE-labeled isotype IgG2Î± were used as FACS control (BD Pharmingen). FACS acquisition and analysis were performed on a FACScalibur using Cell Quest Software (Becton-Dickinson).

Example 11Animal Studies

Female FVB mice (6 to 8 weeks old), were obtained from Charles River Laboratories (Les Oncins, France). Mice were identified via ear markings and kept in groups (6 animals per cage) under normal conditions and observed daily. Six mice were used per treatment group and all animal experiments were performed in strict adherence to the Swiss law for animal protection.

The reference chamber model has been described in publications (e.g. Wood J, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000; 60:2178-89) In brief, porous tissue chambers made of perfluoro-alkoxy-Teflon (TeflonÂ®-PFA, 21 mmÃ—8 mm diameter, 550 Î¼l volume) were filled with 0.8% agar (BBLÂ® Nr. 11849, Becton Dickinson, Meylan, France) and 20 U/ml heparin, (Novo Nordisk A/S, Bagsvaerd, Denmark) supplemented with or without 3 Î¼g/ml recombinant human VEGF and siRNAs as indicated. Solutions were maintained at 42Â° C. prior the filling procedure. Mice were anesthetized using 3% Isoflurane (ForeneÂ®, Abbott AG, Cham, Switzerland) inhalation. For subcutaneous implantation, a small skin incision was made at the base of the tail to allow the insertion of an implant trocar. The chamber was implanted under aseptic conditions through the small incision onto the back of the animal. The skin incision was closed by wound clips (Autoclip 9 mm Clay Adams). Depending on the required dose, siRNAs were diluted in â€œinjectable quality gradeâ€ 0.9% saline solution then delivered to animals either i.p. (200 Î¼L/dose) or p.o. by gavage (100 Î¼L/dose). The mice were receiving the first dose 2 to 4 hours before implanting chambers; then treated daily for 2 days. If not otherwise indicated, mice were sacrificed three days after implantation, chambers excised and the vascularized fibrous tissue formed around each implant carefully removed. Body weight was used to monitor the general condition of the mice. Statistical analysis was done using one-way ANOVA followed by Dunnett test.

Example 12Bl6 Melanoma Xenograft Model

The syngeneic Bl6/BL6 murine melanoma model, previously identified to be responsive to antiangiogenic therapy (e.g. LaMontagne K, Littlewood-Evans A, Schnell C, O'Reilly T, Wyder L, Sanchez T, Probst B, Butler J, Wood A, Liau G, Billy E, Theuer A, Hla T, Wood J. Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization. Cancer Res. 2006 Jan. 1; 66(1):221-31), was used to evaluate the antitumor activity of standard or modified siRNAs. Tumor cells (1 Î¼L, 5Ã—10⁴/Î¼L) were injected intradermally into the dorsal pinna of both ears of syngeneic female C57BL/6 mice. Measurements of primary tumor area (mm²) were carried out on days 7, 14, and 21 after tumor cell inoculation using computer-assisted image analysis software (KS-400 3.0 imaging system, Zeiss) and a specifically designed macro. From days 7 to 21, mice were receiving siRNAs diluted in â€œinjectable quality gradeâ€ 0.9% saline solution either i.p. (200 Î¼L/dose) or p.o. by gavage (100 Î¼L/dose) once a day. Mice were sacrificed on day 21, and cranial lymph node metastases were weighed and then frozen.

In these results, actual siRNA sequences and chemistries employed may be determined by reference to Table 3.

Wild-Type siRNAs are Degraded in Mouse Serum from Both 3â€²-Ends

Oligonucleotide degradation by nucleases is predominantly 3â€²-exonucleolytic. Modification of antisense oligonucleotides at their termini by the introduction of aromatic or lipophilic residues delays their nucleolytic degradation¹⁷. To verify whether this metabolic pathway would also be dominant for siRNA, we incubated at 37Â° C. a unmodified siRNA (wild-type siRNA) in mouse serum for up to 3 hours.

The unmodified siRNA sequence employed was pGl3-siRNA (see Table 3)

The mixtures were analyzed with Strong Anion Exchange HPLC at t=0 min., t=30 min, t=180 min.

As shown in FIGS. 1a, 1b and 1c, at t=30 min, a well defined peak corresponding to blunt ended siRNA was observed. By t=3h substantial degradation is observed. FIGS. 1d and 1e illustrate the metabolites identified by HPLC-ESI-MS analysis. This analysis revealed the presence of several metabolites corresponding to the loss of the 3â€² overhangs and of the 3â€²-terminal first base pairing ribonucleotide on both strands. Digestion of the 5â€²-terminal ribonucleotide of the guide strand was also observed.

FIG. 1 suggests the degradation pathway of unmodified siRNAs in serum. DNA overhangs are first digested, possibly by 3â€²-exonucleases. In the LC-MS, additional metabolites were also detected which correspond to the loss of the first base-pairing 3â€²-ribonucleotide of both strands and also the first 5â€²-base-pairing ribonucleotide of the guide strand.

3â€²-Modified siRNAs are Stable Through the GI Tract

siRNAs with 2â€²-methoxyethyl ribonucleotides overhangs (MOE o/h siRNA), blunt-ended siRNAs 3â€²-capped with a hydroxypropoxy phosphodiester moiety (C3-siRNA), and hydroxypropoxy phosphodiester 3â€²-capped siRNAs where the two first base paring nucleotide at 3â€²-end of each strand were modified by 2â€²-methoxyethyl ribonucleotides residues (C3-MOE siRNA) were synthesized. These compounds are illustrated schematically in FIG. 2.

First siRNAs were incubated in mouse gastric acid for 2 h (FIG. 3). No degradation was observed in the cases of C3 siRNA and C3-MOE sRNA, while degradation of wild-type siRNA was observed after 30 minutes.

Stability in intestinal fluid obtained from intestinal lavage of rats revealed almost complete degradation of wild-type siRNA after 15 minutes whereas parent compound in the MOE o/h siRNA, C3-siRNA and C3-Moe siRNA were observed for 60 minutes. (FIG. 4)

Stability in liver was evaluated using a liver microsome assay and a S12 assay (representative of liver cytosolic enzymatic activity). Results are shown in FIG. 5. In both cases, no degradation was observed after 60 minutes of incubation.

Finally, siRNAs were tested in mouse serum by incubation at 2 micromolar for up to 6 hours at 37Â° C. (results in FIG. 6). Parent compound stability was followed by gel electrophoresis. In the cases of modified siRNAs (C3 siRNA, C3-MOE siRNA of MOE o/h siRNA), no significant degradation was observed while the wild-type siRNA

This study indicate that wild type (unmodified) siRNAs are metabolized in mouse gastric acid and in mouse serum. In case of 3â€²-ends modified siRNAs, no degradation was observed in the GI tract. Therefore it is likely that 3â€²-modified siRNAs will have a higher oral bioavailability than wild-type siRNAs

Systemically Delivered 3â€²-Modified siRNAs are More Active in an In Vivo Growth Factor Induced Angiogenesis Mode¹⁸.

Firstly, the ability of modified siRNAs (C3-siRNA and CE-MOE siRNA) to down regulate a target gene was checked in cellulo by measuring VEGFR2 surface level of MS1 cells transfected with anti-VEGFR2 siRNAs.

Pools of 2 anti VEGFR2 siRNAs as wild-type siRNAs, C3-siRNAs and C3-MOE siRNAs were administered intraperitoneally. Results are shown in FIG. 7. Pooled Wild type siRNAs reduced significantly the VEGF induced vascularization at the higher dose of 25 micrograms per mice per day. The same level of inhibition was observed at a 5-fold lower dose with C3-siRNA. In the case of the C3-MOE siRNAs pool, significant reduction of vascularized tissue weight was observed at all tested doses including the lowest 0.2 microgram per mouse per day.

FIGS. 8a and 8b show that, when given intraperitoneally, both VEGFR2â€”C3 and C3-MOE siRNAs were active at below 1 microgram per mouse per day dose.

In vivo testing of anti-VEGFR2C3-MOE siRNA given intraperitoneally (i.p.) in a Bl6 homograft melanoma tumor mouse model. FIG. 9a shows that i.p. treatment with modified VEGFR2â€”C3-MOE-siRNA significantly reduces tumour development. FIG. 9b also shows that i.p. injection of VEGFR2-C3-MOE-siRNA at 20 ug per mouse results in significant inhibition of tumour growth.

Oral Delivery of siRNA for Treatment of Angiogenic Disorders

FIG. 10 shows that given orally, at a dose of 20 micrograms per mouse per day, the VEGFR2-C3-MOE-siRNA1 reduced vascularization weight down to basal level (e.g. weight without growth factor induction). Actual siRNA sequences used are referred to in Table 3.

Anti Tie2 C3-MOE siRNAs were also tested in the growth factor induced angiogenesis model under both intraperitoneal and oral deliveries. FIG. 11a and 11b show that given orally, both C3-MOE siRNAs directed at Tie2 were active at 20 microgram per mouse per day. Actual siRNA sequences used may be determined by reference to Table 3.

The data shows that 3â€²-end modified siRNAs with or without additional internal modifications are able to demonstrate therapeutic effect at reasonable doses upon oral administration.

REFERENCES

1. a) Y. Tomari et al. Genes and Development 19 (2005), 517; b) P. Shankar et al. JAMA 11 (2005), 1367; c) Y. Dorsett et al. Nature Reviews 3 (2004), 318
2. a) P. D. Zamore et al. Cell 101, (2000), 25; b) S. M. Hammond et al. Nature 404 (2000), 293
3. a) G. Meister et al. Molecular Cell 15 (2004), 185.
4. S. M. Elbashir et al. Genes Dev. 15 (2001), 188.
5. S. J. Reich et al. Molecular Vision 9 (2003), 210.
6. a) Dorn et al. Nucleic Acids Research 32 (2004), e49; b) D. R. Thakker et al. PNAS101 (2004), 17270; c) D. R. Thakker et al. Molecular Psychiatry 10 (2005), 714
7. V. Bitko et al. Nature Medicine 11 (2005), 50.
8. E. Song et al. Nature Medicine 9 (2003), 347.
9. D. A. Braasch et al. Biochemistry 42 (2003), 7967.
10. Harborth, Antisense Nucleic Acid Drug Devt, 2003
11. A. H. S. Hall et al. Nucleic Acids Research 32 (2004), 5991.
12. M. Amarzguioui et al. Nucleic Acids Research 31 (2003), 589.
13. F. Czauderna et al. Nucleic Acids Research 31 (2003), 2705.
14. T. Prakash et al. Journal of Medicinal Chemistry 48 (2005), 4247.
15. J. Elmen et al. Nucleic Acids Research 33 (2005), 439.
16. A. S. Boutorin, L. V. Guskova, E. M. Ivanova, N. D. Kobetz, V. F. Zafytova, A. S. Ryte, L. V. Yurchenko and V. V. Vlassov FEBS Lett. 254 (1989), p. 129
17. J. Wood et al. Cancer Research 60 (2000), 2178.
18. K. LaMontagne et al. Cancer Res. 66 (2006), 221.