RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/267,615, filed on Feb. 5, 2019, which is a continuation of U.S. patent application Ser. No. 15/670,132, filed on Aug. 7, 2017 now U.S. Pat. No. 10,231,988 issued on Mar. 19, 2019, which is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2016/017550, filed on Feb. 11, 2016, to U.S. Provisional Patent Application No. 62/115,724, filed on Feb. 13, 2015, and to U.S. Provisional Patent Application No. 62/266,818, filed on Dec. 14, 2015. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 12, 2021, is named 121301_03205_SL.txt and is 529,923 bytes in size.

BACKGROUND OF THE INVENTION

The accumulation of excess triglyceride in the liver is known as hepatic steatosis (or fatty liver), and is associated with adverse metabolic consequences, including insulin resistance and dyslipidemia. Fatty liver is frequently found in subjects having excessive alcohol intake and subjects having obesity, diabetes, or hyperlipidemia. However, in the absence of excessive alcohol intake (>10 g/day), nonalcoholic fatty liver disease (NAFLD) can develop. NAFLD refers to a wide spectrum of liver diseases that can progress from simple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). All of the stages of NAFLD have in common the accumulation of fat (fatty infiltration) in the liver cells (hepatocytes).

The NAFLD spectrum begins with and progress from its simplest stage, called simple fatty liver (steatosis). Simple fatty liver involves the accumulation of fat (triglyceride) in the liver cells with no inflammation (hepatitis) or scarring (fibrosis). The next stage and degree of severity in the NAFLD spectrum is NASH, which involves the accumulation of fat in the liver cells, as well as inflammation of the liver. The inflammatory cells destroy liver cells (hepatocellular necrosis), and NASH ultimately leads to scarring of the liver (fibrosis), followed by irreversible, advanced scarring (cirrhosis). Cirrhosis that is caused by NASH is the last and most severe stage in the NAFLD spectrum.

In 2008, a genomewide association study of individuals with proton magnetic resonance spectroscopy of the liver to evaluate hepatic fat content, a significant association was identified between hepatic fat content and the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) gene (see, for example, Romeo et al. (2008) Nat. Genet., 40(12):1461-1465). Studies with knock-in mice have demonstrated that expression of a sequence polymorphism (rs738409, I148M) in PNPLA3 causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of triglycerides in the liver (Smagris et al. (2015) Hepatology, 61:108-118). Specifically, the PNPLA3 I148M variant was associated with promoting the development of fibrogenesis by activating the hedgehog (Hh) signaling pathway, leading to the activation and proliferation of hepatic stellate cells and excessive generation and deposition of extracellular matrix (Chen et al. (2015) World J. Gastroenterol., 21(3):794-802).

Currently, treatments for NAFLD are directed towards weight loss and treatment of any secondary conditions, such as insulin resistance or dyslipidemia. To date, no pharmacologic treatments for NAFLD have been approved. Therefore, there is a need for therapies for subjects suffering from NAFLD.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a PNPLA3 gene. The PNPLA3 gene may be within a cell, e.g., a cell within a subject, such as a human.

In one aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2.

In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of any of the sequences in any one of Tables 3-5, 7, and 8.

In one embodiment, the double stranded RNAi agent comprises at least one modified nucleotide. In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic. In another embodiment, the modified nucleotides comprise a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In one embodiment, the region of complementarity is at least 17 nucleotides in length. In another embodiment, the region of complementarity is between 19 and 21 nucleotides in length. In another embodiment, the region of complementarity is 19 nucleotides in length. In another embodiment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In one embodiment, the double stranded RNAi agent further comprises a ligand. In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the double stranded RNAi agent. In another embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In one embodiment the ligand is

[Image Omitted]

In another embodiment, the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic:

[Image Omitted]

and, wherein X is O or S. In one embodiment, the X is O.

In one embodiment, the region of complementarity comprises one of the antisense sequences in any one of Tables 3-5, 7, and 8. In another embodiment, the region of complementarity consists of one of the antisense sequences in any one of Tables 3-5, 7, and 8.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented b formula III):

(III)

sense:

5′ n_p-N_a-(X X X)_i-Nb-Y Y Y-N_b-(Z Z Z)_l-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)k-N_b′-Y′Y′Y′-N_b′-

(Z′Z′Z′)_l-N_a′-n_q′ 5′

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, each n_p, n_p′, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; 1 is 0; k is 1; 1 is 1; both k and l are 0; or both k and l are 1. In another embodiment, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′. In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. In another embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end. In one embodiment, the Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa):

(IIIa)

sense:

5′ n_p-N_a-Y Y Y-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-Y′Y′Y′-N_a′-n_q′ 5′.

In another embodiment, formula (III) is represented by formula (IIIb):

(IIIb)

sense:

5′ n_p-N_a-Y Y Y-N_b-Z Z Z-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-Y′Y′Y′-N_b′-Z′Z′Z′- N_a′-n_q′ 5′

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIIc):

(IIIc)

sense:

5′ n_p-N_a-X X X-N_b-Y Y Y-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-X′X′X′-N_b-Y′Y′Y′-N_a′-n_q′ 5′

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIId):

(IIId)

sense:

5′ n_p-N_a-X X X-N_b-Y Y Y-N_b-Z Z Z-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′-n_q′ 5′

wherein each N_band N_b′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N_aand N_a′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

In one embodiment, the double stranded region is 15-30 nucleotide pairs in length. In another embodiment, the double stranded region is 17-23 nucleotide pairs in length. In another embodiment, the double stranded region is 17-25 nucleotide pairs in length. In another embodiment, the double stranded region is 23-27 nucleotide pairs in length. In another embodiment, the double stranded region is 19-21 nucleotide pairs in length. In another embodiment, the double stranded region is 21-23 nucleotide pairs in length.

In one embodiment, each strand has 15-30 nucleotides. In another embodiment, each strand has 19-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof. In another embodiment, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In one embodiment, the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. In one embodiment, the ligand is

[Image Omitted]

In one embodiment, the ligand is attached to the 3′ end of the sense strand.

In one embodiment, the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic

[Image Omitted]

In one embodiment, the double stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand. In one embodiment, he strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand. In another embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand. In another embodiment, the strand is the antisense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In one embodiment, the Y nucleotides contain a 2′-fluoro modification. In another embodiment, the Y′ nucleotides contain a 2′-O-methyl modification. In another embodiment, p′>0. In another embodiment, p′=2. In another embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA. In another embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In one embodiment, at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage. In another embodiment, all n_p′ are linked to neighboring nucleotides via phosphorothioate linkages.

In one embodiment, the double stranded RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3-5, 7, and 8. In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In another aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III)

sense:

5′ n_p-N_a-(X X X)_i-Nb-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-

(Z′Z′Z′)_l-N_a′-n_q′5′

wherein i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, each n_p, n_p′, n_q, and n_q′, each of which may or may not be present independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.

(III)

sense:

5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′ 5′

wherein: i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand.

In another embodiment, the invention provides a double stranded-ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to part of an mRNA encoding PNPLA3, wherein each strand is about 14 to about 30 nucleotides in length, wherein the double stranded RNAi agent is represented by formula (III):

(III)

sense:

5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′ 5′

wherein i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

(III)

sense:

5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′ 5′

wherein i, j, k, and l are each independently 0 or 1; each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof, XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; modifications on N_bdiffer from the modification on Y and modifications on N_b′ differ from the modification on Y′; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

(IIIa)

sense:

5′ n_p-N_a-Y Y Y-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-Y′Y′Y′-N_a′-n_q′ 5′

wherein each n_p, n_q, and n_q′, each of which may or may not be present, independently represents an overhang nucleotide; p, q, and q′ are each independently 0-6; n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via a phosphorothioate linkage; each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; YYY and Y′Y′Y′ each independently represent one motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoro modifications; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In another aspect, the invention provides a double stranded-ribonucleic acid (RNAi) agent for inhibiting expression of PNPLA3, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached through a branched bivalent or trivalent linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. In another embodiment, each strand has 19-30 nucleotides.

In another aspect, the invention provides a cell containing the double stranded RNAi agent as described herein.

In another aspect, the invention provides a vector encoding at least one strand of a double stranded RNAi agent, wherein the double stranded RNAi agent comprises a region of complementarity to at least a part of an mRNA encoding PNPLA3, wherein the double stranded RNAi agent is 30 base pairs or less in length, and wherein the double stranded RNAi agent targets the mRNA for cleavage. In one embodiment, the region of complementarity is at least 15 nucleotides in length. In another embodiment, the region of complementarity is 19 to 21 nucleotides in length.

In another aspect, the invention provides a cell comprising a vector as described herein.

In another aspect, the invention provides a pharmaceutical composition for inhibiting expression of a PNPLA3 gene comprising the double stranded RNAi agent of the invention. In one embodiment, the double stranded RNAi agent is administered in an unbuffered solution. In another embodiment, the unbuffered solution is saline or water. In another embodiment, the double stranded RNAi agent is administered with a buffer solution. In another embodiment, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In another embodiment, the buffer solution is phosphate buffered saline (PBS).

In another aspect, the invention provides a pharmaceutical composition comprising the double stranded RNAi agent of the invention and a lipid formulation. In one embodiment, the lipid formulation comprises a LNP. In another embodiment, the lipid formulation comprises a MC3.

In another aspect, the invention provides a method of inhibiting PNPLA3 expression in a cell, the method comprising (a) contacting the cell with the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the cell. In one embodiment, the cell is within a subject. In another embodiment, the subject is a human. In one embodiment, the subject is a female human. In another embodiment, the subject is a male human. In one embodiment, the PNPLA3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

In another aspect, the invention provides a method of treating a subject having a disease or disorder that would benefit from reduction in PNPLA3 expression, the method comprising administering to the subject a therapeutically effective amount of the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

In another aspect, the invention provides a method of preventing at least one symptom in a subject having a disease or disorder that would benefit from reduction in PNPLA3 expression, the method comprising administering to the subject a prophylactically effective amount of the double stranded RNAi agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in PNPLA3 expression.

In one embodiment, the administration of the double stranded RNAi to the subject causes a decrease in the hedgehog signaling pathway.

In one embodiment, the PNPLA3-associated disease is a PNPLA3-associated disease. In another embodiment, the PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is fatty liver (steatosis). In another embodiment, the PNPLA3-associated disease is nonalcoholic steatohepatitis (NASH). In another embodiment, the PNPLA3-associated disease is obesity. In one embodiment, the subject is human. In another embodiment, the subject is a female human. In another embodiment, the subject is a male human. In one embodiment, the subject has a PNPLA3 I148M mutation. In one embodiment, the mutation is heterozygous. In another embodiment, the mutation is homozygous.

In another embodiment, the invention further comprises administering an anti-PNPLA3 antibody, or antigen-binding fragment thereof, to the subject.

In one embodiment, the double stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In one embodiment, the dsRNA agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In another embodiment, the dsRNA agent is administered at a dose selected from the group consisting of 0.5 mg/kg 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg.

In one embodiment, the double stranded RNAi agent is administered to the subject once a week. In another embodiment, the double stranded RNAi agent is administered to the subject once a month.

In one embodiment, the double stranded RNAi agent is administered to the subject subcutaneously.

In another embodiment, the methods of the invention further comprise measuring hedgehog signaling pathway levels in the subject. In one embodiment, a decrease in the levels of expression or activity of the hedgehog (Hh) signaling pathway indicate that the PNPLA3-associated disease is being treated or prevented.

The present invention is further illustrated by the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the percentage of PNPLA3 mRNA remaining in the liver of ob/ob mice following administration of a single dose of 0.3 mg/kg, 1.5 mg/kg, or 3.0 mg/kg of the indicated iRNA agents.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (PNPLA3 gene) in mammals.

The RNAi agents of the invention have been designed to target the human PNPLA3 gene, including portions of the gene that are conserved in the PNPLA3 orthologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites and/or the specific modifications in these RNAi agents confer to the RNAi agents of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention also provides methods for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a PNPLA3 gene, e.g., an PNPLA3-associated disease, such as Nonalcoholic Fatty Liver Disease (NAFLD), using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a PNPLA3 gene.

Very low dosages of the iRNAs of the invention, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of the corresponding gene (PNPLA3 gene).

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a PNPLA3 gene.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an angiotensinogen gene as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of a PNPLA3 gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, “Patatin-Like Phospholipase Domain Containing 3,” used interchangeably with the term “PNPLA3,” refers to the naturally occurring gene that encodes a triacylglycerol lipase that mediates triacyl glycerol hydrolysis in adipocytes. The amino acid and complete coding sequences of the reference sequence of the human PNPLA3 gene may be found in, for example, GenBank Accession No. GI:17196625 (RefSeq Accession No. NM_025225.2; SEQ ID NO:1; SEQ ID NO:2). Mammalian orthologs of the human PNPLA3 gene may be found in, for example, GenBank Accession Nos. GI: 544461323 (RefSeq Accession No. XM_005567051.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8); GI: 544461325 (RefSeq Accession No. XM_005567052.1, cynomolgus monkey; SEQ ID NO:11 and SEQ ID NO:12); GI:297261270 (RefSeq Accession No. XM_001109144.2, rhesus monkey, SEQ ID NO:9 and SEQ ID NO:10); GI:144226244 (RefSeq Accession No. NM_054088.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:537361027 (RefSeq Accession No. NM_001282324.1, rat; SEQ ID NO:5 and SEQ ID NO:6).

Additional examples of PNPLA3 mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an PNPLA3 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an PNPLA3 gene. In one embodiment, the target sequence is within the protein coding region of PNPLA3.

The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of a PNPLA3 gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a PNPLA3 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an PNPLA3 gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, and/or modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 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, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., an PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAi agent is a dsRNA that is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a PNPLA3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an PNPLA3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5′- and/or 3′-terminus of the iRNA. In one embodiment, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the antisense strand. In another embodiment, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotides from the 3′-terminus of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a PNPLA3 gene). For example, a polynucleotide is complementary to at least a part of an PNPLA3 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a PNPLA3 gene.

Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target PNPLA3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target PNPLA3 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:2, or, or a fragment of SEQ ID NO:2, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In another embodiment, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target PNPLA3 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of the sense strands in any one of Tables 3-5, 7, and 8, or a fragment of any one of the sense strands in any one of Tables 3-5, 7, and 8, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression and/or replication; a human at risk for a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression; a human having a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in PNPLA3 gene expression, as described herein. In one embodiment, the subject is a female human. In another embodiment, the subject is a male human.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms associated with PNPLA3 gene expression and/or PNPLA3 protein production, e.g., the presence of increased protein activity in the hedgehog (Hh) signaling pathway, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of PNPLA3 gene expression and/or PNPLA3 protein production in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more and is preferably down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an PNPLA3 gene and/or production of PNPLA3 protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of PNPLA3 gene expression, such as the presence of elevated levels of proteins in the hedgehog signaling pathway, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “Patatin-Like Phospholipase Domain Containing 3-associated disease” or “PNPLA3-associated disease,” is a disease or disorder that is caused by, or associated with PNPLA3 gene expression or PNPLA3 protein production. The term “PNPLA3-associated disease” includes a disease, disorder or condition that would benefit from a decrease in PNPLA3 gene expression, replication, or protein activity. Non-limiting examples of PNPLA3-associated diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). In another embodiment, the PNPLA3-associated disease is nonalcoholic steatohepatitis (NASH). In another embodiment, the PNPLA3-associated disease is liver cirrhosis. In another embodiment, the PNPLA3-associated disease is insulin resistance. In another embodiment, the PNPLA3-associated disease is not insulin resistance. In one embodiment, the PNPLA3-associated disease is obesity.

In one embodiment, an PNPLA3-associated disease is nonalcoholic fatty liver disease (NAFLD). As used herein, “nonalcoholic fatty liver disease,” used interchangeably with the term “NAFLD,” refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol ingestion per day. NAFLD is the most common liver disease in the United States, and is commonly associated with insulin resistance/type 2 diabetes mellitus and obesity. NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153-1163 the entire contents of which are incorporated herein by reference.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a patient for treating a subject having PNPLA3-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by PNPLA3 gene expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject who does not yet experience or display symptoms of a PNPLA3-associated disease, but who may be predisposed, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. RNAi agents employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes), the retina or parts of the retina (e.g., retinal pigment epithelium), the central nervous system or parts of the central nervous system (e.g., ventricles or choroid plexus), or the pancreas or certain cells or parts of the pancreas. In some embodiments, a “sample derived from a subject” refers tocerebrospinal fluid obtained from the subject. In preferred embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject. In further embodiments, a “sample derived from a subject” refers to liver tissue (or subcomponents thereof) or retinal tissue (or subcomponents thereof) derived from the subject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of a PNPLA3 gene. In one embodiment, the iRNA agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a PNPLA3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having an PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD). The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an PNPLA3 gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the PNPLA3 gene, the iRNA inhibits the expression of the PNPLA3 gene (e.g., a human, a primate, a non-primate, or a bird PNPLA3 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an PNPLA3 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 23 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target PNPLA3 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3-5, 7, and 8, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 3-5, 7, and 8. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an PNPLA3 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 3-5, 7, and 8, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 3-5, 7, and 8. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in any one of Tables 3, 4, and 7 are not described as modified and/or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 3-5, 7, and 8, or the sequences of any one of Tables 3-5, 7, and 8 that are modified, or the sequences of any one of Tables 3-5, 7, and 8 that are conjugated. In other words, the invention encompasses dsRNA of any one of Tables 3-5, 7, and 8 which are unmodified, un-conjugated, modified, and/or conjugated, as described herein.

In another aspect, a double stranded ribonucleic acid (dsRNA) of the invention for inhibiting expression of PNPLA3 comprises, consists essentially of, or consists of a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of a sense strand in any one of Tables 3-5, 7, and 8 and the antisense strand comprises the nucleotide sequence of the corresponding antisense strand in any one of Tables 3-5, 7, and 8.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Table 3-5, 7, and 8, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of any one of Tables 3-5, 7, and 8 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of any one of Tables 3-5, 7, and 8, and differing in their ability to inhibit the expression of a PNPLA3 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in any one of Tables 3-5, 7, and 8 identify a site(s) in a PNPLA3 transcript that is susceptible to RISC-mediated cleavage (see, e.g., Table 9). As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in any one of Tables 3-5, 7, and 8 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a PNPLA3 gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. Thus, while the sequences identified, for example, in any one of Tables 3-5, 7, and 8 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in any one of Tables 3-5, 7, and 8, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an PNPLA3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an PNPLA3 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an PNPLA3 gene is important, especially if the particular region of complementarity in an PNPLA3 gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA, is unmodified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified iRNAs of the invention in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. 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; 5,177,195; 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; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms 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; 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; 5,64,562; 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; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA 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 compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: 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 can be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Exemplary suitable modifications 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. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, 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 iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can 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; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 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 uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal 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, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA 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 featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 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; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof, see e.g., U.S. Pat. No. 8,278,283); 4′-CH2 N(OCH3)-2′ (and analogs thereof, see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. patents and US patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and 3-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may also include a hydroxymethyl substituted nucleotide. A “hydroxymethyl substituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, also referred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents of the invention include agents with chemical modifications as disclosed, for example, in U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011, or in PCT/US2012/065691, filed on Nov. 16, 2012, the entire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710 or PCT Application No. PCT/US2012/065691, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand and/or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense and/or antisense strand. The RNAi agent may be optionally conjugated with a GalNAc derivative ligand, for instance on the sense strand. The resulting RNAi agents present superior gene silencing activity.

More specifically, it has been surprisingly discovered that when the sense strand and antisense strand of the double stranded RNAi agent are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of an RNAi agent, the gene silencing activity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., PNPLA3 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may range from 12-30 nucleotides in length. For example, each strand may be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhang regions and/or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3-terminal end of the sense strand or, alternatively, at the 3-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^stnucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^stpaired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_aand/or N_bcomprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ” “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In one embodiment, the RNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′-3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 5′-3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.

In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif For example, the portion of the sequence containing the motif is “ . . . N_aYYYN_b. . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_a” and “N_b” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_aand N_bcan be the same or different modifications. Alternatively, N_aand/or N_bmay be present or absent when there is a wing modification present.

The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and/or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-terminus or the 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the RNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented by formula (I):

(I)

5′ n_p-N_a-(X X X )_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_aindependently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_bindependently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_pand n_qindependently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_aand/or N_bcomprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sense strand, the count starting from the 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

(Ib)

5′ n_p-N_a-YYY-N_b-ZZZ-N_a-n_q 3′;

(Ic)

5′ n_p-N_a-XXX-N_b-YYY-N_a-n_q 3′;

or

(Id)

5′ n_p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q 3′.

When the sense strand is represented by formula (Ib), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_brepresents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_bindependently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6 Each N_acan independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

(Ia)

5′ n_p-N_a-YYY-N_a-n_q 3′.

When the sense strand is represented by formula (Ia), each N_aindependently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

(II)

5′ n_q′-N_a′-(Z′Z′Z′)_k-N_b′-Y′Y′Y′-N_b′-(X′X′X′)_i-N′_a-n_p′ 3′

wherein:

- k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_p′ and n_q′ independently represent an overhang nucleotide;

wherein N_b′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, the N_a′ and/or N_b′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^stnucleotide, from the 5′-end; or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

(Ib)

5′ n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_a′-n_p′ 3′;

(Ic)

5′ n_q′-N_a′-Y′Y′Y′-N_b′-X′X′X′-n_p′-3′;

or

(Id)

5′ n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_b′-X′X′X′-N_a′-n_p′ 3′.

When the antisense strand is represented by formula (IIb), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_b′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_bis 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may be represented by the formula:

(Ia)

5′ n_p′-N_a′-Y′Y′Y′-N_a′-n_q′ 3′.

When the antisense strand is represented as formula (IIa), each N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^stnucleotide from the 5′-end, or optionally, the count starting at the 1^stpaired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the RNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

(III)

sense:

5′ n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_l-N_a-n_q 3′

antisense:

3′ n_p′-N_a′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_aand N_a′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_band N_b′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein each n_p′, n_p, n_q′, and n_q, each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

(IIIa)

5′ n_p-N_a-Y Y Y-N_a-n_q 3′

3′ n_p′-N_a′-Y′Y′Y′-N_a′n_q′ 5′

(IIIb)

5′ n_p-N_a-Y Y Y-N_b-Z Z Z-N_a-n_q 3′

3′ n_p′-N_a′-Y′Y′Y′-N_b-Z′Z′Z′-N_a′n_q′ 5′

(IIIc)

5′ n_p-N_a-X X X-N_b-Y Y Y-N_a-n_q 3′

3′ n_p′-N_a-X′X′X′-N_b-Y′Y′Y′-N_a′-n_q′ 5′

(IIId)

5′ n_p-N_a-XXX-N_b-Y Y Y-N_b-Z Z Z-N_a-n_q 3′

3′ n_p′-N_a′-X′X′X′-N_b-Y′Y′Y′-N_b-Z′Z′Z′-N_a-n_q′ 5′

When the RNAi agent is represented by formula (IIIa), each N_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_bindependently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_b, N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_b, N_b′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_a, N_a′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_a, N_a′, N_band N_b′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications and n_p′ >0 and at least one n_p′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the N_amodifications are 2′-O-methyl or 2′-fluoro modifications, n_p′>0 and at least one n_p′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 3-5, 7, and 8. These agents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. 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 triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-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-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present 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. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 14) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 15) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 16) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include HBV and above (e.g., HBV, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., HBV, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

[Image Omitted]

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

[Image Omitted]

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

[Image Omitted]

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

[Image Omitted]

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII)-(XXXV):

[Image Omitted]

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^2A, P^2B, P^3A, P^3B, P^4A, P^4B, P^5A, P^5B, P^5C, T^2A, T^2B, T^3A, T^3B, T^4A, T^4B, T^4A, T^5B, T^5Care each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^2A, Q^2B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^5B, Q^5Care independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^N), C(R′)═C(R″), C≡C or C(O);

R^2A, R^2B, R^3A, R^3B, R^4A, R^4B, R^5A, R^5B, R^5Care each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), —C(O)—CH(R^a)—NH—, CO, CH═N—O,

[Image Omitted]

or heterocyclyl;

L^2A, L^2B, L^3A, L^3B, L^4A, L^4B, L^5A, L^5Band L^5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^ais H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

[Image Omitted]

wherein L^5A, L^5Band L^5Crepresent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA 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; 5,591,584; 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; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which 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 dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs 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.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), 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; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disease, disorder or condition associated with PNPLA3 gene expression) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention iRNA targeting the PNPLA3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an PNPLA3 gene.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular, (IM), or intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of an PNPLA3 gene.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a PNPLA3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months). After an initial treatment regimen, the treatments can be administered on a less frequent basis.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as a disorder that would benefit from reduction in the expression of PNPLA3. Such models can be used for in vivo testing of an agent, as well as for determining a therapeutically effective dose. Suitable dietary and genetic mouse models are reviewed in Kanuri and Bergheim (Int. J. Mol. Sci. (2013) 14:11963-11980).

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), 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; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administration can 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 can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable 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). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C_1-20alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate iRNA. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” 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 (A) comprises one or more glycolipids, such as monosialoganglioside G_M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_Mi, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_Mi or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNA agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin. In some implementations, liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the penetration of iRNA agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA agent can be delivered, for example, subcutaneously by infection in order to deliver iRNA agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈to C₂₂alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs, of in the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle. The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the lipidoid ND98-4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[Image Omitted]

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1

cationic lipid/non-cationic

lipid/cholesterol/PEG-lipid conjugate

Ionizable/Cationic Lipid
Lipid:siRNA ratio

SNALP-
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-

1
dimethylaminopropane
cDMA

(DLinDMA)
(57.1/7.1/34.4/1.4)

lipid:siRNA~7:1

2-XTC
2,2-Dilinoleyl-4-
XTC/DPPC/Cholesterol/PEG-cDMA

dimethylaminoethyl-
57.1/7.1/34.4/1.4

[1,3]-dioxolane (XTC)
lipid:siRNA~7:1

LNP05
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG

dimethylaminoethyl-
57.5/7.5/31.5/3.5

[1,3]-dioxolane (XTC)
lipid:siRNA~6:1

LNP06
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG

dimethylaminoethyl-
57.5/7.5/31.5/3.5

[1,3]-dioxolane (XTC)
lipid:siRNA~11:1

LNP07
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG

dimethylaminoethyl-
60/7.5/31/1.5,

[1,3]-dioxolane (XTC)
lipid:siRNA~6:1

LNP08
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG

dimethylaminoethyl-
60/7.5/31/1.5,

[1,3]-dioxolane (XTC)
lipid:siRNA~11:1

LNP09
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG

dimethylaminoethyl-
50/10/38.5/1.5

[1,3]-dioxolane (XTC)
Lipid:siRNA 10:1

LNP10
(3aR,5s,6aS)-N,N-
ALN100/DSPC/Cholesterol/

dimethyl-2,2-
PEG-DMG

di((9Z,12Z)-octadeca-
50/10/38.5/1.5

9,12-dienyl)tetrahydro-
Lipid:siRNA 10:1

3aH-cyclo-

penta[d][1,3]dioxol-

5-amine (ALN100)

LNP11
(6Z,9Z,28Z,31Z)-
MC-3/DSPC/Cholesterol/

heptatriaconta-
PEG-DMG

6,9,28,31-tetraen-
50/10/38.5/1.5

19-yl 4-(dimethyl-
Lipid:siRNA 10:1

amino)butanoate

(MC3)

LNP12
1,1′-(2-(4-(2-((2-
Tech G1/DSPC/Cholesterol/

(bis(2-hydroxy-
PEG-DMG

dodecyl)amino)-
50/10/38.5/1.5

ethyl)(2-hydroxy
Lipid:siRNA 10:1

dodecyl)-amino)-

ethyl)piperazin-

1-yl)ethylazanediyl)-

didodecan-2-ol

(Tech G1)

LNP13
XTC
XTC/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 33:1

LNP14
MC3
MC3/DSPC/Chol/PEG-DMG

40/15/40/5

Lipid:siRNA: 11:1

LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/

GalNAc-PEG-DSG

50/10/35/4.5/0.5

Lipid:siRNA: 11:1

LNP16
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 7:1

LNP17
MC3
MC3/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 10:1

LNP18
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/38.5/1.5

Lipid:siRNA: 12:1

LNP19
MC3
MC3/DSPC/Chol/PEG-DMG

50/10/35/5

Lipid:siRNA: 8:1

LNP20
MC3
MC3/DSPC/Chol/PEG-DPG

50/10/38.5/1.5

Lipid:siRNA: 10:1

LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 7:1

LNP22
XTC
XTC/DSPC/Chol/PEG-DSG

50/10/38.5/1.5

Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. W02009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

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 can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can 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.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can 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 compositions of the present invention can 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 can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can 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). Each of these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) 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 iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetration enhancers 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, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts 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 (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; 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, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards 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, 315-339). Suitable chelating agents 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)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, 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, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can 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), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), iRNAMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^aD1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can 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 a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized 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, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the 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 formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-iRNA mechanism and which are useful in treating a hemolytic disorder. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent.

In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PNPLA3 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VII. Methods For Inhibiting PNPLA3 Expression

The present invention also provides methods of inhibiting expression of a PNPLA3 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of PNPLA3 in the cell, thereby inhibiting expression of PNPLA3 in the cell.

Contacting of a cell with an RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ligand, or any other ligand that directs the RNAi agent to a site of interest.

In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a PNPLA3” is intended to refer to inhibition of expression of any PNPLA3 gene (such as, e.g., a mouse PNPLA3 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene) as well as variants or mutants of a PNPLA3 gene. Thus, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (such as a mutant PNPLA3 gene giving rise to amyloid deposition), or a transgenic PNPLA3 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a PNPLA3 gene” includes any level of inhibition of a PNPLA3 gene, e.g., at least partial suppression of the expression of a PNPLA3 gene. The expression of the PNPLA3 gene may be assessed based on the level, or the change in the level, of any variable associated with PNPLA3 gene expression, e.g., PNPLA3 mRNA level, PNPLA3 protein level, or the number or extent of amyloid deposits. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with PNPLA3 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of a PNPLA3 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

Inhibition of the expression of a PNPLA3 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a PNPLA3 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of a PNPLA3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)). In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$

Alternatively, inhibition of the expression of a PNPLA3 gene may be assessed in terms of a reduction of a parameter that is functionally linked to PNPLA3 gene expression, e.g., PNPLA3 protein expression or Hedgehog pathway protein activities. PNPLA3 gene silencing may be determined in any cell expressing PNPLA3, either constitutively or by genomic engineering, and by any assay known in the art.

Inhibition of the expression of a PNPLA3 protein may be manifested by a reduction in the level of the PNPLA3 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of a PNPLA3 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

The level of PNPLA3 mRNA that is expressed by a cell or group of cells, or the level of circulating PNPLA3 mRNA, may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of PNPLA3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the PNPLA3 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating PNPLA3 mRNA may be detected using methods the described in PCT/US2012/043584, the entire contents of which are hereby incorporated herein by reference.

In one embodiment, the level of expression of PNPLA3 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific PNPLA3. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PNPLA3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of PNPLA3 mRNA.

An alternative method for determining the level of expression of PNPLA3 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of PNPLA3 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

The expression levels of PNPLA3 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of PNPLA3 expression level may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

The level of PNPLA3 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention can be monitored by detecting or monitoring a reduction in a symptom of a PNPLA3 disease, such as reduction in edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; or abdominal pain. These symptoms may be assessed in vitro or in vivo using any method known in the art.

In some embodiments of the methods of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of PNPLA3 may be assessed using measurements of the level or change in the level of PNPLA3 mRNA or PNPLA3 protein in a sample derived from fluid or tissue from the specific site within the subject. In preferred embodiments, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites. The site may also include cells that express a particular type of receptor.

VIII. Methods of Treating or Preventing PNPLA3-Associated Diseases

The present invention provides therapeutic and prophylactic methods which include administering to a subject with a PNPLA3-associated disease, disorder, and/or condition, or prone to developing, a PNPLA3-associated disease, disorder, and/or condition, compositions comprising an iRNA agent, or pharmaceutical compositions comprising an iRNA agent, or vectors comprising an iRNA of the invention. Non-limiting examples of PNPLA3-associated diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In one embodiment, the PNPLA3-associated disease is NAFLD. In another embodiment, the PNPLA3-associated disease is NASH. In another embodiment, the PNPLA3-associated disease is fatty liver (steatosis). In another embodiment, the PNPLA3-associated disease is insulin resistance. In another embodiment, the PNPLA3-associated disease is not insulin resistance.

The methods of the invention are useful for treating a subject having a PNPLA3-associated disease, e.g., a subject that would benefit from reduction in PNPLA3 gene expression and/or PNPLA3 protein production. In one aspect, the present invention provides methods of reducing the level of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene expression in a subject having nonalcoholic fatty liver disease (NAFLD). In another aspect, the present invention provides methods of reducing the level of PNPLA3 protein in a subject with NAFLD. The present invention also provides methods of reducing the level of activity of the hedgehog pathway in a subject with NAFLD.

In another aspect, the present invention provides methods of treating a subject having an NAFLD. In one aspect, the present invention provides methods of treating a subject having an PNPLA3-associated disease, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The treatment methods (and uses) of the invention include administering to the subject, e.g., a human, a therapeutically effective amount of an iRNA agent of the invention targeting a PNPLA3 gene or a pharmaceutical composition comprising an iRNA agent of the invention targeting a PNPLA3 gene or a vector of the invention comprising an iRNA agent targeting an PNPLA3 gene.

In one aspect, the invention provides methods of preventing at least one symptom in a subject having NAFLD, e.g., the presence of elevated hedgehog signaling pathways, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider-like blood vessels, yellowing of the skin and eyes (jaundice), itching, fluid build up and swelling of the legs (edema), abdomen swelling (ascites), and mental confusion. The methods include administering to the subject a therapeutically effective amount of the iRNA agent, e.g. dsRNA, pharmaceutical compositions, or vectors of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in PNPLA3 gene expression.

In another aspect, the present invention provides uses of a therapeutically effective amount of an iRNA agent of the invention for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression.

In a further aspect, the present invention provides uses of an iRNA agent, e.g., a dsRNA, of the invention targeting an PNPLA3 gene or pharmaceutical composition comprising an iRNA agent targeting an PNPLA3 gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production, such as a subject having a disorder that would benefit from reduction in PNPLA3 gene expression, e.g., a PNPLA3-associated disease.

In another aspect, the invention provides uses of an iRNA, e.g., a dsRNA, of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production.

In a further aspect, the present invention provides uses of an iRNA agent of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or SCAP protein production, such as a PNPLA3-associated disease.

In one embodiment, an iRNA agent targeting PNPLA3 is administered to a subject having a PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD), such that the expression of a PNPLA3 gene, e.g., in a cell, tissue, blood or other tissue or fluid of the subject are reduced by at least about 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%, 62%, 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%, or at least about 99% or more when the dsRNA agent is administered to the subject.

The methods and uses of the invention include administering a composition described herein such that expression of the target PNPLA3 gene is decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target PNPLA3 gene is decreased for an extended duration, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or longer.

Administration of the dsRNA according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD). By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 4%5% 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of NAFLD may be assessed, for example, by periodic monitoring of NAFLD symptoms, liver fat levels, or expression of downstream genes. Comparison of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting PNPLA3 or pharmaceutical composition thereof, “effective against” an PNPLA3-associated disease indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating NAFLD and/or an PNPLA3-associated disease and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg dsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kg dsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA. In one embodiment, subjects can be administered 0.5 mg/kg of the dsRNA. Values and ranges intermediate to the recited values are also intended to be part of this invention.

Administration of the iRNA can reduce the presence of PNPLA3 protein levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6%, 7%, 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%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on PNPLA3 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, where the modified or unmodified iRNA agent is directly suspended in aqueous or suitable buffer solvent, as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The free iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of PNPLA3 gene expression are those having nonalcoholic fatty liver disease (NAFLD) and/or an PNPLA3-associated disease or disorder as described herein.

Treatment of a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression includes therapeutic and prophylactic treatment.

The invention further provides methods and uses of an iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of PNPLA3 gene expression, e.g., a subject having a PNPLA3-associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.

For example, in certain embodiments, an iRNA targeting a PNPLA3 gene is administered in combination with, e.g., an agent useful in treating an PNPLA3-associated disease as described elsewhere herein. For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in PNPLA3 expression, e.g., a subject having a PNPLA3-associated disease, include an iRNA agent targeting a different portion of the PNPLA3 gene, a therapeutic agent, and/or procedures for treating a PNPLA3-associated disease or a combination of any of the foregoing.

In certain embodiments, a first iRNA agent targeting a PNPLA3 gene is administered in combination with a second iRNA agent targeting a different portion of the PNPLA3 gene. For example, the first RNAi agent comprises a first sense strand and a first antisense strand forming a double stranded region, wherein substantially all of the nucleotides of said first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, wherein said first sense strand is conjugated to a ligand attached at the 3′-terminus, and wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker; and the second RNAi agent comprises a second sense strand and a second antisense strand forming a double stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand attached at the 3′-terminus, and wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, all of the nucleotides of the first and second sense strand and/or all of the nucleotides of the first and second antisense strand comprise a modification.

In one embodiment, the at least one of the modified nucleotides is selected from the group consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, and a nucleotide comprising a 5′-phosphate mimic.

In certain embodiments, a first iRNA agent targeting a PNPLA3 gene is administered in combination with a second iRNA agent targeting a gene that is different from the PNPLA3 gene. For example, the iRNA agent targeting the PNPLA3 gene may be administered in combination with an iRNA agent targeting the SCAP gene. The first iRNA agent targeting a PNPLA3 gene and the second iRNA agent targeting a gene different from the PNPLA3 gene, e.g., the SCAP gene, may be administered as parts of the same pharmaceutical composition. Alternatively, the first iRNA agent targeting a PNPLA3 gene and the second iRNA agent targeting a gene different from the PNPLA3 gene, e.g., the SCAP gene, may be administered as parts of different pharmaceutical compositions.

The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

The present invention also provides methods of using an iRNA agent of the invention and/or a composition containing an iRNA agent of the invention to reduce and/or inhibit PNPLA3 expression in a cell. In other aspects, the present invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting PNPLA3 gene expression in a cell. In yet other aspects, use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament for reducing and/or inhibiting PNPLA3 gene expression in a cell are provided. In still other aspects, the present invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting PNPLA3 protein production in a cell. In yet other aspects, use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament for reducing and/or inhibiting PNPLA3 protein production in a cell are provided. The methods and uses include contacting the cell with an iRNA, e.g., a dsRNA, of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene or inhibiting PNPLA3 protein production in the cell.

Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of PNPLA3 may be determined by determining the mRNA expression level of PNPLA3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of PNPLA3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, flow cytometry methods, ELISA, and/or by determining a biological activity of PNPLA3.

In the methods and uses of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an PNPLA3 gene, e.g., a cell from a subject having NAFLD or a cell comprising an expression vector comprising a PNPLA3 gene or portion of a PNPLA3 gene. A cell suitable for use in the methods and uses of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell.

PNPLA3 gene expression may be inhibited in the cell by at least about 5%, 6%, 7%, 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%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

PNPLA3 protein production may be inhibited in the cell by at least about 5%, 6%, 7%, 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%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PNPLA3 gene of the mammal to be treated. When the organism to be treated is a human, the composition can be administered by any means known in the art including, but not limited to subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection. In one embodiment, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of PNPLA3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the subject.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an PNPLA3 gene in a mammal, e.g., a human. The present invention also provides a composition comprising an iRNA, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of a mammal for use in inhibiting expression of the PNPLA3 gene in the mammal. In another aspect, the present invention provides use of an iRNA, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of a mammal in the manufacture of a medicament for inhibiting expression of the PNPLA3 gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human, a composition comprising an iRNA, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sample of the iRNA-administered subject by any methods known it the art, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g., ELISA or Western blotting, described herein. In one embodiment, a tissue sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression. In another embodiment, a blood sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target in vivo following administration of iRNA agent is done by performing 5′-RACE or modifications of the protocol as known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al. (2006) Nature 441: 111-4).

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES
Example 1. iRNA Synthesis
Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts

siRNA Design

A set of iRNAs targeting human PNPLA3, “Patatin-Like Phospholipase Domain Containing 3” (RefSeq Accession No. NM_025225, GI:17196625; SEQ ID NO:1 and SEQ ID NO:2) and PNPLA3 orthologs from toxicology species (for example, GenBank Accession Nos. GI: 544461323 (REFSEQ Accession No. XM_005567051.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8); GI: 544461325 (RefSeq Accession No. XM_005567052.1, cynomolgus monkey; SEQ ID NO:11 and SEQ ID NO:12); GI:297261270 (RefSeq Accession No. XM_001109144.2, rhesus monkey, SEQ ID NO:9 and SEQ ID NO:10); GI:144226244 (RefSeq Accession No. NM_054088.3, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:537361027 (RefSeq Accession No. NM_001282324.1, rat; SEQ ID NO:5 and SEQ ID NO: 6)) were designed using custom R and Python scripts.

The human PNPLA3 RefSeq mRNA has a length of 2805 bases. The rationale and method for the set of iRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 1 through position 2805 of human PNPLA3 mRNA (containing the coding region) was determined using a linear model that predicted the direct measure of mRNA knockdown based on the data of more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the PNPLA3 iRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PNPLA3 orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the iRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weights for the mismatches were 2.8 for seed mismatches, 1.2 for cleavage site mismatches, and 1 for mismatches in other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was greater than or equal to 3.0 and predicted efficacy was greater than or equal to 70% knockdown of the PNPLA3 transcript. One set of iRNAs containing structure-activity modifications, including various 2′-O-methyl and 2′-fluoro substitution patterns, were also designed, synthesized and screened.

A detailed list of the unmodified PNPLA3 sense and antisense strand sequences is shown in Table 3.

siRNA Synthesis

PNPLA3 iRNA sequences were synthesized at 1 μmol scale on a Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support is controlled pore glass (500 A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee, Wis.) and Hongene (China). 2′F 2′-O-Methyl, GNA (glycol nucleic acids), 5′phosphate and other modifications were introduced using the corresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 minutes employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. All sequences were synthesized with final removal of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides were cleaved from the solid support and deprotected in sealed 96 deep well plates using 200 μL Aqueous Methylamine reagents at 60° C. for 20 minutes. For sequences containing 2′ ribo residues (2′-OH) that were protected with a tert-butyl dimethyl silyl (TBDMS) group, a second step deprotection was performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200 uL of dimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent were added and the solution was incubated for additional 20 minutes at 60° C. At the end of cleavage and deprotection step, the synthesis plate was allowed to come to room temperature and was precipitated by addition of 1 mL of acetontile:ethanol mixture (9:1). The plates were cooled at −80 C for 2 hours, supernatant was decanted carefully with the aid of a multi channel pipette. The oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and was desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in 96-well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to determine purity.

Annealing of PNPLA3 single strands was performed on a Tecan liquid handling robot. Equimolar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combining the complementary single strands, the 96-well plate was sealed tightly and heated in an oven at 100° C. for 10 minutes and allowed to come slowly to room temperature over a period 2-3 hours. The concentration of each duplex was normalized to 10 μM in 1×PBS.

TABLE 2

Abbreviations of nucleotide monomers used in nucleic acid

sequence representation. It will be understood that these monomers,

when present in an oligonucleotide, are mutually linked by

5′-3′-phosphodiester bonds.

Abbreviation
Nucleotide(s)

A
Adenosine-3′-phosphate

Af
2′-fluoroadenosine-3′-phosphate

Afs
2′-fluoroadenosine-3′-phosphorothioate

As
adenosine-3′-phosphorothioate

C
cytidine-3′-phosphate

Cf
2′-fluorocytidine-3′-phosphate

Cfs
2′-fluorocytidine-3′-phosphorothioate

Cs
cytidine-3′-phosphorothioate

G
guanosine-3′-phosphate

Gf
2′-fluoroguanosine-3′-phosphate

Gfs
2′-fluoroguanosine-3′-phosphorothioate

Gs
guanosine-3′-phosphorothioate

T
5′-methyluridine-3′-phosphate

Tf
2′-fluoro-5-methyluridine-3′-phosphate

Tfs
2′-fluoro-5-methyluridine-3′-phosphorothioate

Ts
5-methyluridine-3′-phosphorothioate

U
Uridine-3′-phosphate

Uf
2′-fluorouridine-3′-phosphate

Ufs
2′-fluorouridine -3′-phosphorothioate

Us
uridine-3′-phosphorothioate

N
any nucleotide (G, A, C, T or U)

a
2′-O-methyladenosine-3′-phosphate

as
2′-O-methyladenosine-3′-phosphorothioate

c
2′-O-methylcytidine-3′-phosphate

cs
2′-O-methylcytidine-3′-phosphorothioate

g
2′-O-methylguanosine-3′-phosphate

gs
2′-O-methylguanosine-3′-phosphorothioate

t
2′-O-methyl-5-methyluridine-3′-phosphate

ts
2′-O-methyl-5-methyluridine-3′-phosphorothioate

u
2′-O-methyluridine-3′-phosphate

us
2′-O-methyluridine-3′-phosphorothioate

s
phosphorothioate linkage

L96
N[tris(GalNAc-alkyl)-amidodecanoyl)]-4-

hydroxyprolinol Hyp-(GalNAc-alkyl)3

(dt)
2′-deoxythymidine-3′-phosphate

Y34
2-hydroxymethyl-tetrahydrofurane-4-methoxy-

3-phosphate (abasic 2′-OMe furanose)

Y44
2-hydroxymethyl-tetrahydrofurane-5-phosphate

(Agn)
Adenosine-glycol nucleic acid (GNA)

(Tgn)
Thymidine-glycol nucleic acid (GNA) S-Isomer

(Cgn)
Cytidine-glycol nucleic acid (GNA)

P
Phosphate

VP
Vinyl-phosphate

TABLE 3

Unmodified Sense and Antisense Strand Sequences of PNPLA3 RNAi agents

Sense
SEQ
Anti-

Target

Se-

sense
SEQ

Site in

Start
quence
ID
Sequence
ID
Other
Gen Bank

Oligo Name
Position
(5′-3′)
NO
(5′-3′)
NO
Set
Ref. No.

NM_025225.2_219-
217
GGCUU
19
UUGGU
111
hcmr
217-239

240_C21_Asense

CCUGG

AGAAG

GCUUC

CCCAG

UACCA

GAAGC

A

CGC

NM_054088.3_250-
248
UAUAA
20
UCCAU
112
mr
248-270

271_sense

UGGAG

GAGGA

AUCCU

UCUCC

CAUGG

AUUAU

A

ACG

NM_025225.2_388-
386
UUGUG
21
UACUC
113
hc
386-408

409_E21A_sense

CGGAA

CUGGC

GGCCA

CUUCC

GGAGU

GCACA

A

AGA

NM_025225.2_396-
394
AAGGC
22
AAUGU
114
hc
394-416

417_sense

CAGGA

UCCGA

GUCGG

CUCCU

AACAU

GGCCU

U

UCC

NM_025225.2_397-
395
AGGCC
23
UAAUG
115
hc
395-417

418_G21_Asenseense

AGGAG

UUCCG

UCGGA

ACUCC

ACAUU

UGGCC

A

UUC

NM_054088.3_443_-
441
GUGUC
24
UUUGG
116
mr
441-463

464_senseense

UGAGU

AAUGG

UCCAU

AACUC

UCCAA

AGACA

A

CCA

NM_054088.3_469-
467
AGUCG
25
UACAC
117
mr
467-489

490_G21_Asenseense

UGGAU

CAGGG

GCCCU

CAUCC

GGUGU

ACGAC

A

UUC

NM_025225.2_549-
547
AACGU
26
AAAGU
118
hc
547-569

570_sense

UCUGG

CAGAC

UGUCU

ACCAG

GACUU

AACGU

U

UUU

NM_025225.2_562-
560
CUGAC
27
UGUCU
119
he
560-582

583_G21A_sense

UUUCG

UUGGA

GUCCA

CCGAA

AAGAC

AGUCA

A

GAC

NM_025225.2_569-
567
UCGGU
28
ACGAC
120
hc
567-589

590_sense

CCAAA

UUCGU

GACGA

CUUUG

AGUCG

GACCG

U

AAA

NM_025225.2_570-
568
CGGUC
29
UACGA
121
he
568-590

591_G21A_sense

CAAAG

CUUCG

ACGAA

UCUUU

GUCGU

GGACC

A

GAA

NM_025225.2_579-
577
GACGA
30
UAAGG
122
he
577-599

600_G21_Asense

AGUCG

CAUCC

UGGAU

ACGAC

GCCUU

UUCGU

A

CUU

NM_025225.2_596-
594
CUUGG
31
AUGAA
123
hemr
594-616

617_sense

UAUGU

GCAGG

UCCUG

AACAU

CUUCA

ACCAA

U

GGC

NM_025225.2_630-
628
GGCCU
32
UAAGG
124
hemr
628-650

651C21_Asense

UAUCC

AAGGA

CUCCU

GGGAU

UCCUU

AAGGC

A

CAC

NM_025225.2_674-
672
AGGAG
33
UGUAC
125
he
672-694

695_C21_Asense

UGAGU

GUUGU

GACAA

CACUC

CGUAC

ACUCC

A

UCC

NM_025225.2_678-
676
GUGAG
34
UAAGG
126
he
676-698

699_C21A_sense

UGACA

GUACG

ACGUA

UUGUC

CCCUU

ACUCA

A

CUC

NM_025225.2_701-
699
UGAUG
35
UUGAU
127
he
699-721

722_C21_Asense

CCAAA

GGUUG

ACAAC

UUUUG

CAUCA

GCAUC

A

AAU

NM_025225.2_746-
744
CGACA
36
UUGAC
128
he
744-766

767_sense

UCUGC

UUUAG

CCUAA

GGCAG

AGUCA

AUGUC

A

GUA

NM_054088.3_770-
768
UGCUA
37
UUCCA
129
mr
768-790

791C21_Asense

UCAAG

GGUAC

GGUAC

CCUUG

CUGGA

AUAGC

A

ACA

NM_025225.2_771-
769
ACGAA
38
UUCCA
130
he
769-791

792_C21_Asense

CUUUC

CAUGA

UUCAU

AGAAA

GUGGA

GUUCG

A

UGG

NM_025225.2_817-
815
GCACA
39
UAAGG
131
he
815-837

838_C21_Asense

GGGAA

UAGAG

CCUCU

GUUCC

ACCUU

CUGUG

A

CAG

NM_025225.2_871-
869
UGCUG
40
UAAGG
132
he
869-891

892_C21_Asense

GGAGA

CAUAU

GAUAU

CUCUC

GCCUU

CCAGC

A

ACC

NM_025225.2_874-
872
UGGGA
41
UUCGA
133
he
872-894

895_G21A_sense

GAGAU

AGGCA

AUGCC

UAUCU

UUCGA

CUCCC

A

AGC

NM_025225.2_878-
876
AGAGA
42
UAUCC
134
he
876-898

899_sense

UAUGC

UCGAA

CUUCG

GGCAU

AGGAU

AUCUC

A

UCC

NM_025225.2_882-
880
AUAUG
43
UAAAU
135
he
880-902

903_G21A_sense

CCUUC

AUCCU

GAGGA

CGAAG

UAUUU

GCAUA

A

UCU

NM_025225.2_885-
883
UGCCU
44
AUCCA
136
he
883-905

906_sense

UCGAG

AAUAU

GAUAU

CCUCG

UUGGA

AAGGC

U

AUA

NM_025225.2_908-
906
AUUCA
45
UUCUC
137
he
906-928

929_sense

GGUUC

UUCCA

UUGGA

AGAAC

AGAGA

CUGAA

A

UGC

NM_025225.2_964-
962
CAUCC
46
UAUCC
138
he
962-984

985_C21A_sense

UCAGA

AUCCC

AGGGA

UUCUG

UGGAU

AGGAU

A

GAC

NM_025225.2_1100-
1098
CCUGC
47
AUGCU
139
he
1098-1120

1121_sense

CCUGG

CUCAU

GAUGA

CCCAG

GAGCA

GGCAG

U

GAU

NM_054088.3_1163-
1161
UCCCA
48
UAUUC
140
mr
1161-1183

1184_G21A_sense

GGUUU

GGGCA

GUGCC

CAAAC

CGAAU

CUGGG

A

AUG

NM_054088.3_1165-
1163
CCAGG
49
UUCAU
141
mr
1163-1185

1186_C21_Asense

UUUGU

UCGGG

GCCCG

CACAA

AAUGA

ACCUG

A

GGA

NM_025225.2_1173-
1171
GACAA
50
UCUCA
142
he
1171-1193

1194_C21_Asense

AGGUG

UGUAU

GAUAC

CCACC

AUGAG

UUUGU

A

CUU

NM_025225.2_1176-
1174
AAAGG
51
UUUGC
143
he
1174-1196

1197_G21_Asense

UGGAU

UCAUG

ACAUG

UAUCC

AGCAA

ACCUU

A

UGU

NM_025225.2_1180-
1178
GUGGA
52
AAAUC
144
he
1178-1200

1201_se_use

UACAU

UUGCU

GAGCA

CAUGU

AGAUU

AUCCA

U

CCU

NM_025225.2_1181-
1179
UGGAU
53
UAAAU
145
he
1179-1201

1202_G21_Asense

ACAUG

CUUGC

AGCAA

UCAUG

GAUUU

UAUCC

A

ACC

NM_025225.2_1184-
1182
AUACA
54
UUGCA
146
he
1182-1204

1205_sense

UGAGC

AAUCU

AAGAU

UGCUC

UUGCA

AUGUA

A

UCC

NM_025225.2_1191-
1189
AGCAA
55
UAGCA
147
he
1189-1211

1212_sense

GAUUU

AGUUG

GCAAC

CAAAU

UUGCU

CUUGC

A

UCA

NM_025225.2_1193-
1191
CAAGA
56
UGUAG
148
he
1191-1213

1214C21_Asense

UUUGC

CAAGU

AACUU

UGCAA

GCUAC

AUCUU

A

GCU

NM_025225.2_1196-
1194
GAUUU
57
AUGGG
149
he
1194-1216

1217_sense

GCAAC

UAGCA

UUGCU

AGUUG

ACCCA

CAAAU

U

CUU

NM_025225.2_1200-
1198
UGCAA
58
UCUAA
150
he
1198-1220

1221_G21A_sense

CUUGC

UGGGU

UACCC

AGCAA

AUUAG

GUUGC

A

AAA

NM_025225.2_1203-
1201
AACUU
59
UAUCC
151
he
1201-1223

1224_sense

GCUAC

UAAUG

CCAUU

GGUAG

AGGAU

CAAGU

A

UGC

NM_025225.2_1266-
1264
GCCAU
60
UCUCU
152
he
1264-1286

1287_sense

UGCGA

GGACA

UUGUC

AUCGC

CAGAG

AAUGG

A

CAG

NM_025225.2_1274-
1272
GAUUG
61
UUCAC
153
he
1272-1294

1295_C21_A_sense

UCCAG

CAGUC

AGACU

UCUGG

GGUGA

ACAAU

A

CGC

NM_025225.2_1288-
1286
UGGUG
62
UAUCU
154
he
1286-1308

1309_sense

ACAUG

GGAAG

GCUUC

CCAUG

CAGAU

UCACC

A

AGU

NM_025225.2_1302-
1300
CCAGA
63
UACAU
155
he
1300-1322

1323_C21A_sense

UAUGC

CGUCG

CCGAC

GGCAU

GAUGU

AUCUG

A

GAA

NM_025225.2_1325-
1323
GUGGU
64
UAGGU
156
he
1323-1345

1346_C21_Asense

UGCAG

CACCC

UGGGU

ACUGC

GACCU

AACCA

A

CAG

NM_025225.2_1389-
1387
AGGUC
65
UCUCA
157
he
1387-1409

1410_C21A_sense

CCAAA

CUGGC

UGCCA

AUUUG

GUGAG

GGACC

A

UGG

NM_025225.2_1621-
1619
UCACU
66
UAGAC
158
he
1619-1641

1642_sense

UGAGG

UCGCC

AGGCG

UCCUC

AGUCU

AAGUG

A

ACU

NM_025225.2__1636-
1634
AGUCU
67
UCUGA
159
he
1634-1656

1657_sense

AGCAG

AAGAA

AUUCU

UCUGC

UUCAG

UAGAC

A

UCG

NM_025225.2_1646-
1644
AUUCU
68
UUUUA
160
he
1644-1666

1667_G21_Asense

UUCAG

GCACC

AGGUG

UCUGA

CUAAA

AAGAA

A

UCU

NM_025225.2_1647-
1645
UUCUU
69
ACUUU
161
he
1645-1667

1668_sense

UCAGA

AGCAC

GGUGC

CUCUG

UAAAG

AAAGA

U

AUC

NM_025225.2__1658-
1656
GUGCU
70
AAAGA
162
he
1656-1678

1679_sense

AAAGU

UGGGA

UUCCC

AACUU

AUCUU

UAGCA

U

CCU

NM_025225.2_1669-
1667
UCCCA
71
UGUAG
163
he
1667-1689

1690_C21A_sense

UCUUU

CUGCA

GUGCA

CAAAG

GCUAC

AUGGG

A

AAA

NM_025225.2_1713-
1711
CUGCC
72
UAUCC
164
he
1711-1733

1734_C21A_sense

UGUGA

UCCAC

CGUGG

GUCAC

AGGAU

AGGCA

A

GGG

NM_025225.2_1718-
1716
UGUGA
73
UCUGG
165
he
1716-1738

1739_C21_A_sense

CGUGG

GAUCC

AGGAU

UCCAC

CCCAG

GUCAC

A

AGG

NM_025225.2_1740-
1738
UCUGA
74
AUAAA
166
he
1738-1760

1761_sense

GCUGA

ACCAA

GUUGG

CUCAG

UUUUA

CUCAG

U

AGG

NM_025225.2__1741_-
1739
CUGAG
75
UAUAA
167
he
1739-1761

1762_G21_A_sense

CUGAG

AACCA

UUGGU

ACUCA

UUUAU

GCUCA

A

GAG

NM_025225.2_1749-
1747
AGUUG
76
UAGCU
168
he
1747-1769

1770_sense

GUUUU

UUUCA

AUGAA

UAAAA

AAGCU

CCAAC

A

UCA

NM_025225.2_1751-
1749
UUGGU
77
UCUAG
169
he
1749-1771

1772_G21_Asense

UUUAU

CUUUU

GAAAA

CAUAA

GCUAG

AACCA

A

ACU

NM_025225.2_1753-
1751
GGUUU
78
UUCCU
170
he
1751-1773

1774_sense

UAUGA

AGCUU

AAAGC

UUCAU

UAGGA

AAAAC

A

CAA

NM_025225.2_1754-
1752
GUUUU
79
UUUCC
171
he
1752-1774

1775_G21_Asense

AUGAA

UAGCU

AAGCU

UUUCA

AGGAA

UAAAA

A

CCA

NM_025225.2_1755-
1753
UUUUA
80
UCUUC
172
he
1753-1775

1776_C21_Asense

UGAAA

CUAGC

AGCUA

UUUUC

GGAAG

AUAAA

A

ACC

NM_025225.2_1758-
1756
UAUGA
81
UUUGC
173
he
1756-1778

1779_C21_Asense

AAAGC

UUCCU

UAGGA

AGCUU

AGCAA

UUCAU

A

AAA

NM_025225.2_1827-
1825
CGUUA
82
UCCCA
174
he
1825-1847

1848_sense

AUUCA

ACCAG

GCUGG

CUGAA

UUGGG

UUAAC

A

GCA

NM_025225.2_1828-
1826
GUUAA
83
UUCCC
175
he
1826-1848

1849_sense

UUCAG

AACCA

CUGGU

GCUGA

UGGGA

AUUAA

A

CGC

NM_025225.2_1836-
1834
AGCUG
84
UGUGU
176
he
1834-1856

1857_C21A_sense

GUUGG

CAUUU

GAAAU

CCCAA

GACAC

CCAGC

A

UGA

NM_025225.2_1900-
1898
CCUAU
85
AACAG
177
he
1898-1920

1921_sense

UAAUG

UCUGA

GUCAG

CCAUU

ACUGU

AAUAG

U

GGC

NM_025225.2_1901_-
1899
CUAUU
86
UAACA
178
he
1899-1921

1922_C21A_sense

AAUGG

GUCUG

UCAGA

ACCAU

CUGUU

UAAUA

A

GGG

NM_025225.2_1984-
1982
GCUGG
87
UAAGA
179
he
1982-2004

2005_G21_Asense

CCCAU

UCACA

GUGUG

CAUGG

AUCUU

GCCAG

A

CCU

NM_025225.2_1986-
1984
UGGCC
88
UACAA
180
he
1984-2006

2007_G21_Asense

CAUGU

GAUCA

GUGAU

CACAU

CUUGU

GGGCC

A

AGC

NM_025225.2_2190-
2188
CCUAA
89
UUAAA
181
he
2188-2210

2211_sense

CUAAA

CAUUA

AUAAU

UUUUA

GUUUA

GUUAG

A

GUG

NM_025225.2_2243-
2241
UUACC
90
AAUAC
182
he
2241-2263

2264_sense

UGUUG

AAAAU

AAUUU

UCAAC

UGUAU

AGGUA

U

ACA

NM_025225.2_2245-
2243
ACCUG
91
AUAAU
183
he
2243-2265

2266_sense

UUGAA

ACAAA

UUUUG

AUUCA

UAUUA

ACAGG

U

UAA

NM_025225.2_2258-
2256
UGUAU
92
UUCAC
184
he
2256-2278

2279_G21_Asense

UAUGU

UGAUU

GAAUC

CACAU

AGUGA

AAUAC

A

AAA

NM_025225.2_2263-
2261
UAUGU
93
AACAU
185
he
2261-2283

2284_sense

GAAUC

CUCAC

AGUGA

UGAUU

GAUGU

CACAU

U

AAU

NM_025225.2_2278-
2276
GAUGU
94
AAGGC
186
he
2276-2298

2299_sense

UAGUA

UUAUU

GAAUA

CUACU

AGCCU

AACAU

U

CUC

NM_025225.2_2279-
2277
AUGUU
95
UAAGG
187
he
2277-2299

2300_sense

AGUAG

CUUAU

AAUAA

UCUAC

GCCUU

UAACA

A

UCU

NM_054088.3_3032-
3030
UGGAG
96
UAUCU
188
mr
3030-3052

3053_G21A_sense

CAACA

AGACA

GUGUC

CUGUU

UAGAU

GCUCC

A

AGA

NM_054088.3_3106-
3104
CUUUU
97
UUUCC
189
mr
3104-3126

3127_G21_Asense

GGAGG

UAGCU

CAGCU

GCCUC

AGGAA

CAAAA

A

GUA

NM_054088.3_3226-
3224
AAGAC
98
UAAAC
190
mr
3224-3246

3247_sense

AAUGA

ACCAA

UUUGG

AUCAU

UGUUU

UGUCU

A

UUG

NM_054088.3_3228-
3226
GACAA
99
UCUAA
191
mr
3226-3248

3249_sense

UGAUU

ACACC

UGGUG

AAAUC

UUUAG

AUUGU

A

CUU

NM_054088.3_3230-
3228
CAAUG
100
UUUCU
192
mr
3228-3250

3251_sense

AUUUG

AAACA

GUGUU

CCAAA

UAGAA

UCAUU

A

GUC

NM_054088.3_3447-
3445
UGCCA
101
AAAGU
193
mr
3445-3467

3468_sense

GAUAA

AAUAA

CUUAU

GUUAU

UACUU

CUGGC

U

AGG

NM_054088.3_3473-
3471
ACACC
102
AUUAG
194
mr
3471-3493

3494_sense

UUUGG

UAAGA

CUCUU

GCCAA

ACUAA

AGGUG

U

UCC

NM_054088.3_3629-
3627
CUGGC
103
UAUAC
195
mr
3627-3649

3650_sense

UCCAA

AAAGA

AUCUU

UUUGG

UGUAU

AGCCA

A

GUG

NM_054088.3_3630-
3628
UGGCU
104
UUAUA
196
mr
3628-3650

3651_G21A_sense

CCAAA

CAAAG

UCUUU

AUUUG

GUAUA

GAGCC

A

AGU

NM_054088.3_3635-
3633
CCAAA
105
UAUGA
197
mr
3633-3655

3656_C21_Asense

UCUUU

CUAUA

GUAUA

CAAAG

GUCAU

AUUUG

A

GAG

NM_054088.3_3986-
3984
AGAGA
106
UAGCC
198
mr
39844006

4007_sense

CAAAG

UAGAC

UGUCU

ACUUU

AGGCU

GUCUC

A

UAG

NM_054088.3_3993-
3991
AAGUG
107
UUCUG
199
mr
3991-4013

4014_sense

UCUAG

UGUAG

GCUAC

CCUAG

ACAGA

ACACU

A

UUG

NM_054088.3_4283-
4281
AGAAA
108
UAAAG
200
mr
4281-4303

4304_G21_Asense

CUUCU

CAAGG

GCCUU

CAGAA

GCUUU

GUUUC

A

UAC

NM_054088.3_4540-
4538
GAAGG
109
UGUGU
201
mr
4538-4560

4561_C21_Asense

AUUGA

AUCCA

AUGGA

UUCAA

UACAC

UCCUU

A

CUG

NM_054088.3_4543-
4541
GGAUU
110
UUUGG
202
mr
4541-4563

4564_sense

GAAUG

UGUAU

GAUAC

CCAUU

ACCAA

CAAUC

A

CUU

Example 2. iRNA Synthesis
Source of Reagents

Transcripts

siRNA Design

A set of iRNAs targeting the human PNPLA3 (human: NCBI refseqID NM_025225; NCBI GeneID: 80339), as well as toxicology-species PNPLA3 orthologs (cynomolgus monkey: XM_005567051; mouse: NM_054088; rat: XM_006242109) were designed using custom R and Python scripts. The human PNPLA3 REFSEQ mRNA has a length of 2805 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 174 through position 2805 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the PNPLA3 iRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PNPLA3 orthologs. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey, mouse, and rat PNPLA3 orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, e.g., positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the siRNA, e.g., positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8, 1.2 and 1 for seed mismatches, cleavage site, and other positions up through antisense position 19, respectively. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was >=3.0 and predicted efficacy was >=70% knockdown of the PNPLA3 transcript.

A detailed list of the unmodified PNPLA3 sense and antisense strand sequences is shown in Table 4. A detailed list of the modified PNPLA3 sense and antisense strand sequences is shown in Table 5.

In Vitro Screening

Cell Culture and Transfections

Hep3b cells were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of iRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Fifty μl of EMEM containing ˜5×10³cells were then added to the iRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 20 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl of Lysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 150 μl Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150 μl Elution Buffer, re-captured and supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 h 37° C.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Hs99999905), 0.5 μl PNPLA3 probe (Hs00228747_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 20 nM AD-1955, or mock transfected cells. The results from the assays are shown in Table 6.

TABLE 4

Unmodified Sense and Antisense Strand Sequences of PNPLA3 RNAi Agents

Start
Nucleotide

Site
Range

Sense
Sense
SEQ
Antisense
Antisense
SEQ
in
in

Lex
Oligo
Sequence
ID
Oligo
Sequence
ID
NM_
NM_

Name
Name
(5′-3′)
NO
Name
(5′-3′)
NO
025225.2
025225.2

AD-68792.1
A-138374.1
GGGGGCGGGGCUGACGUCA
203
A-138375.1
UGACGUCAGCCCCGCCCCC
454
11
11-29

AD-68793.1
A-138376.1
ACGUCGCGCUGGGAAUGCA
204
A-138377.1
UGCAUUCCCAGCGCGACGU
455
24
24-42

AD-68794.1
A-138378.1
GGAAUGCCCUGGCCGAGAA
205
A-138379.1
UUCUCGGCCAGGGCAUUCC
456
35
35-53

AD-68795.1
A-138380.1
UGGCCGAGACACUGAGGCA
206
A-138381.1
UGCCUCAGUGUCUCGGCCA
457
44
44-62

AD-68796.1
A-138382.1
UGAGGCAGGGUAGAGAGCA
207
A-138383.1
UGCUCUCUACCCUGCCUCA
458
56
56-74

AD-68797.1
A-138384.1
AGAGAGCGCUUGCGGGCGA
208
A-138385.1
UCGCCCGCAAGCGCUCUCU
459
67
67-85

AD-68798.1
A-138386.1
CGGGCGCCGGGCGGAGCUA
209
A-138387.1
UAGCUCCGCCCGGCGCCCG
460
79
79-97

AD-68799.1
A-138388.1
GCGGAGCUGCUGCGGAUCA
210
A-138389.1
UGAUCCGCAGCAGCUCCGC
461
89
89-107

AD-68800.1
A-138390.1
UGCGGAUCAGGACCCGAGA
211
A-138391.1
UCUCGGGUCCUGAUCCGCA
462
99
99-117

AD-68801.1
A-138392.1
ACCCGAGCCGAUUCCCGAU
212
A-138393.1
AUCGGGAAUCGGCUCGGGU
463
110
110-128

AD-68802.1
A-138394.1
UUCCCGAUCCCGACCCAGA
213
A-138395.1
UCUGGGUCGGGAUCGGGAA
464
121
121-139

AD-68803.1
A-138396.1
ACCCAGAUCCUAACCCGCA
214
A-138397.1
UGCGGGUUAGGAUCUGGGU
465
133
133-151

AD-68804.1
A-138398.1
UAACCCGCGCCCCCGCCCA
215
A-138399.1
UGGGCGGGGGCGCGGGUUA
466
143
143-161

AD-68805.1
A-138400.1
CCGCCCCGCCGCCGCCGCA
216
A-138401.1
UGCGGCGGCGGCGGGGCGG
467
155
155-173

AD-68806.1
A-138402.1
CGCCGCCAUGUACGACGCA
217
A-138403.1
UGCGUCGUACAUGGCGGCG
468
167
167-185

AD-68807.1
A-138404.1
UACGACGCAGAGCGCGGCU
218
A-138405.1
AGCCGCGCUCUGCGUCGUA
469
177
177-195

AD-68808.1
A-138406.1
CGCGGCUGGAGCUUGUCCU
219
A-138407.1
AGGACAAGCUCCAGCCGCG
470
189
189-207

AD-68809.1
A-138408.1
AGCUUGUCCUUCGCGGGCU
220
A-138409.1
AGCCCGCGAAGGACAAGCU
471
198
198-216

AD-68810.1
A-138410.1
CGCGGGCUGCGGCUUCCUA
221
A-138411.1
UAGGAAGCCGCAGCCCGCG
472
209
209-227

AD-68811.1
A-138412.1
UUCCUGGGCUUCUACCACA
222
A-138413.1
UGUGGUAGAAGCCCAGGAA
473
222
222-240

AD-68812.1
A-138414.1
UUCUACCACGUCGGGGCGA
223
A-138415.1
UCGCCCCGACGUGGUAGAA
474
231
231-249

AD-68813.1
A-138416.1
CGGGGCGACCCGCUGCCUA
224
A-138417.1
UAGGCAGCGGGUCGCCCCG
475
242
242-260

AD-68814.1
A-138418.1
UGCCUGAGCGAGCACGCCA
225
A-138419.1
UGGCGUGCUCGCUCAGGCA
476
255
255-273

AD-68815.1
A-138420.1
AGCACGCCCCGCACCUCCU
226
A-138421.1
AGGAGGUGCGGGGCGUGCU
477
265
265-283

AD-68816.1
A-138422.1
ACCUCCUCCGCGACGCGCA
227
A-138423.1
UGCGCGUCGCGGAGGAGGU
478
277
277-295

AD-68817.1
A-138424.1
GCGACGCGCGCAUGUUGUU
228
A-138425.1
AACAACAUGCGCGCGUCGC
479
286
286-304

AD-68818.1
A-138426.1
UGUUGUUCGGCGCUUCGGA
229
A-138427.1
UCCGAAGCGCCGAACAACA
480
298
298-316

AD-68819.1
A-138428.1
CUUCGGCCGGGGCGUUGCA
230
A-138429.1
UGCAACGCCCCGGCCGAAG
481
310
310-328

AD-68820.1
A-138430.1
GGCGUUGCACUGCGUCGGA
231
A-138431.1
UCCGACGCAGUGCAACGCC
482
320
320-338

AD-68821.1
A-138432.1
UGCGUCGGCGUCCUCUCCA
232
A-138433.1
UGGAGAGGACGCCGACGCA
483
330
330-348

AD-68822.1
A-138434.1
UCUCCGGUAUCCCGCUGGA
233
A-138435.1
UCCAGCGGGAUACCGGAGA
484
343
343-361

AD-68823.1
A-138436.1
UCCCGCUGGAGCAGACUCU
234
A-138437.1
AGAGUCUGCUCCAGCGGGA
485
352
352-370

AD-68824.1
A-138438.1
CAGACUCUGCAGGUCCUCU
235
A-138439.1
AGAGGACCUGCAGAGUCUG
486
363
363-381

AD-68825.1
A-138440.1
UCCUCUCAGAUCUUGUGCA
236
A-138441.1
UGCACAAGAUCUGAGAGGA
487
376
376-394

AD-68826.1
A-138442.1
UCUUGUGCGGAAGGCCAGA
237
A-138443.1
UCUGGCCUUCCGCACAAGA
488
386
386-404

AD-68827.1
A-138444.1
AAGGCCAGGAGUCGGAACA
238
A-138445.1
UGUUCCGACUCCUGGCCUU
489
396
396-414

AD-68828.1
A-138446.1
CGGAACAUUGGCAUCUUCA
239
A-138447.1
UGAAGAUGCCAAUGUUCCG
490
408
408-426

AD-68829.1
A-138448.1
GCAUCUUCCAUCCAUCCUU
240
A-138449.1
AAGGAUGGAUGGAAGAUGC
491
418
418-436

AD-68830.1
A-138450.1
CCAUCCUUCAACUUAAGCA
241
A-138451.1
UGCUUAAGUUGAAGGAUGG
492
429
429-447

AD-68831.1
A-138452.1
UUAAGCAAGUUCCUCCGAA
242
A-138453.1
UUCGGAGGAACUUGCUUAA
493
441
441-459

AD-68832.1
A-138454.1
CCUCCGACAGGGUCUCUGA
243
A-138455.1
UCAGAGACCCUGUCGGAGG
494
452
452-470

AD-68833.1
A-138456.1
UCUCUGCAAAUGCCUCCCA
244
A-138457.1
UGGGAGGCAUUUGCAGAGA
495
464
464-482

AD-68834.1
A-138458.1
UGCCUCCCGGCCAAUGUCA
245
A-138459.1
UGACAUUGGCCGGGAGGCA
496
474
474-492

AD-68835.1
A-138460.1
AAUGUCCACCAGCUCAUCU
246
A-138461.1
AGAUGAGCUGGUGGACAUU
497
486
486-504

AD-68836.1
A-138462.1
AGCUCAUCUCCGGCAAAAU
247
A-138463.1
AUUUUGCCGGAGAUGAGCU
498
496
496-514

AD-68837.1
A-138464.1
CGGCAAAAUAGGCAUCUCU
248
A-138465.1
AGAGAUGCCUAUUUUGCCG
499
506
506-524

AD-68838.1
A-138466.1
AUCUCUCUUACCAGAGUGU
249
A-138467.1
ACACUCUGGUAAGAGAGAU
500
519
519-537

AD-68839.1
A-138468.1
ACCAGAGUGUCUGAUGGGA
250
A-138469.1
UCCCAUCAGACACUCUGGU
501
528
528-546

AD-68840.1
A-138470.1
AUGGGGAAAACGUUCUGGU
251
A-138471.1
ACCAGAACGUUUUCCCCAU
502
541
541-559

AD-68841.1
A-138472.1
ACGUUCUGGUGUCUGACUU
252
A-138473.1
AAGUCAGACACCAGAACGU
503
550
550-568

AD-68842.1
A-138474.1
UCUGACUUUCGGUCCAAAG
253
A-138475.1
CUUUGGACCGAAAGUCAGA
504
561
561-579

AD-68843.1
A-138476.1
UCCAAAGACGAAGUCGUGA
254
A-138477.1
UCACGACUUCGUCUUUGGA
505
573
573-591

AD-68844.1
A-138478.1
AAGUCGUGGAUGCCUUGGU
255
A-138479.1
ACCAAGGCAUCCACGACUU
506
583
583-601

AD-68845.1
A-138480.1
CCUUGGUAUGUUCCUGCUU
256
A-138481.1
AAGCAGGAACAUACCAAGG
507
595
595-613

AD-68846.1
A-138482.1
UCCUGCUUCAUCCCCUUCU
257
A-138483.1
AGAAGGGGAUGAAGCAGGA
508
606
606-624

AD-68847.1
A-138484.1
UCCCCUUCUACAGUGGCCU
258
A-138485.1
AGGCCACUGUAGAAGGGGA
509
616
616-634

AD-68848.1
A-138486.1
AGUGGCCUUAUCCCUCCUU
259
A-138487.1
AAGGAGGGAUAAGGCCACU
510
627
627-645

AD-68849.1
A-138488.1
CCUCCUUCCUUCAGAGGCA
260
A-138489.1
UGCCUCUGAAGGAAGGAGG
511
639
639-657

AD-68850.1
A-138490.1
UCAGAGGCGUGCGAUAUGU
261
A-138491.1
ACAUAUCGCACGCCUCUGA
512
649
649-667

AD-68851.1
A-138492.1
GAUAUGUGGAUGGAGGAGU
262
A-138493.1
ACUCCUCCAUCCACAUAUC
513
661
661-679

AD-68852.1
A-138494.1
GAGGAGUGAGUGACAACGU
263
A-138495.1
ACGUUGUCACUCACUCCUC
514
673
673-691

AD-68853.1
A-138496.1
UGACAACGUACCCUUCAUU
264
A-138497.1
AAUGAAGGGUACGUUGUCA
515
683
683-701

AD-68854.1
A-138498.1
CCUUCAUUGAUGCCAAAAC
265
A-138499.1
GUUUUGGCAUCAAUGAAGG
516
694
694-712

AD-68855.1
A-138500.1
UGCCAAAACAACCAUCACA
266
A-138501.1
UGUGAUGGUUGUUUUGGCA
517
704
704-722

AD-68856.1
A-138502.1
AUCACCGUGUCCCCCUUCU
267
A-138503.1
AGAAGGGGGACACGGUGAU
518
717
717-735

AD-68857.1
A-138504.1
UCCCCCUUCUAUGGGGAGU
268
A-138505.1
ACUCCCCAUAGAAGGGGGA
519
726
726-744

AD-68858.1
A-138506.1
UGGGGAGUACGACAUCUGA
269
A-138507.1
UCAGAUGUCGUACUCCCCA
520
737
737-755

AD-68859.1
A-138508.1
AUCUGCCCUAAAGUCAAGU
270
A-138509.1
ACUUGACUUUAGGGCAGAU
521
750
750-768

AD-68860.1
A-138510.1
AGUCAAGUCCACGAACUUU
271
A-138511.1
AAAGUUCGUGGACUUGACU
522
761
761-779

AD-68861.1
A-138512.1
ACGAACUUUCUUCAUGUGA
272
A-138513.1
UCACAUGAAGAAAGUUCGU
523
771
771-789

AD-68862.1
A-138514.1
UUCAUGUGGACAUCACCAA
273
A-138515.1
UUGGUGAUGUCCACAUGAA
524
781
781-799

AD-68863.1
A-138516.1
UCACCAAGCUCAGUCUACA
274
A-138517.1
UGUAGACUGAGCUUGGUGA
525
793
793-811

AD-68864.1
A-138518.1
AGUCUACGCCUCUGCACAA
275
A-138519.1
UUGUGCAGAGGCGUAGACU
526
804
804-822

AD-68865.1
A-138520.1
CUGCACAGGGAACCUCUAA
276
A-138521.1
UUAGAGGUUCCCUGUGCAG
527
815
815-833

AD-68866.1
A-138522.1
AACCUCUACCUUCUCUCGA
277
A-138523.1
UCGAGAGAAGGUAGAGGUU
528
825
825-843

AD-68867.1
A-138524.1
UCUCGAGAGCUUUUGUCCA
278
A-138525.1
UGGACAAAAGCUCUCGAGA
529
838
838-856

AD-68868.1
A-138526.1
UUUGUCCCCCCGGAUCUCA
279
A-138527.1
UGAGAUCCGGGGGGACAAA
530
849
849-867

AD-68869.1
A-138528.1
CCGGAUCUCAAGGUGCUGA
280
A-138529.1
UCAGCACCUUGAGAUCCGG
531
858
858-876

AD-68870.1
A-138530.1
UGCUGGGAGAGAUAUGCCU
281
A-138531.1
AGGCAUAUCUCUCCCAGCA
532
871
871-889

AD-68871.1
A-138532.1
AGAUAUGCCUUCGAGGAUA
282
A-138533.1
UAUCCUCGAAGGCAUAUCU
533
880
880-898

AD-68872.1
A-138534.1
AGGAUAUUUGGAUGCAUUA
283
A-138535.1
UAAUGCAUCCAAAUAUCCU
534
893
893-911

AD-68873.1
A-138536.1
AUGCAUUCAGGUUCUUGGA
284
A-138537.1
UCCAAGAACCUGAAUGCAU
535
904
904-922

AD-68874.1
A-138538.1
UUCUUGGAAGAGAAGGGCA
285
A-138539.1
UGCCCUUCUCUUCCAAGAA
536
915
915-933

AD-68875.1
A-138540.1
GAGAAGGGCAUCUGCAACA
286
A-138541.1
UGUUGCAGAUGCCCUUCUC
537
924
924-942

AD-68876.1
A-138542.1
UGCAACAGGCCCCAGCCAA
287
A-138543.1
UUGGCUGGGGCCUGUUGCA
538
936
936-954

AD-68877.1
A-138544.1
CAGCCAGGCCUGAAGUCAU
288
A-138545.1
AUGACUUCAGGCCUGGCUG
539
948
948-966

AD-68878.1
A-138546.1
GAAGUCAUCCUCAGAAGGA
289
A-138547.1
UCCUUCUGAGGAUGACUUC
540
959
959-977

AD-68879.1
A-138548.1
UCAGAAGGGAUGGAUCCUA
290
A-138549.1
UAGGAUCCAUCCCUUCUGA
541
969
969-987

AD-68880.1
A-138550.1
UGGAUCCUGAGGUCGCCAU
291
A-138551.1
AUGGCGACCUCAGGAUCCA
542
979
979-997

AD-68881.1
A-138552.1
CGCCAUGCCCAGCUGGGCA
292
A-138553.1
UGCCCAGCUGGGCAUGGCG
543
992
992-1010

AD-68882.1
A-138554.1
CAGCUGGGCAAACAUGAGU
293
A-138555.1
ACUCAUGUUUGCCCAGCUG
544
1001
1001-1019

AD-68883.1
A-138556.1
CAUGAGUCUGGAUUCUUCA
294
A-138557.1
UGAAGAAUCCAGACUCAUG
545
1013
1013-1031

AD-68884.1
A-138558.1
UUCUUCCCCGGAGUCGGCU
295
A-138559.1
AGCCGACUCCGGGGAAGAA
546
1025
1025-1043

AD-68885.1
A-138560.1
AGUCGGCUGCCUUGGCUGU
296
A-138561.1
ACAGCCAAGGCAGCCGACU
547
1036
1036-1054

AD-68905.1
A-138562.1
UUGGCUGUGAGGCUGGAGA
297
A-138563.1
UCUCCAGCCUCACAGCCAA
548
1047
1047-1065

AD-68906.1
A-138564.1
AGGCUGGAGGGAGAUGAGA
298
A-138565.1
UCUCAUCUCCCUCCAGCCU
549
1056
1056-1074

AD-68907.1
A-138566.1
AUGAGCUGCUAGACCACCU
299
A-138567.1
AGGUGGUCUAGCAGCUCAU
550
1069
1069-1087

AD-68908.1
A-138568.1
UAGACCACCUGCGUCUCAA
300
A-138569.1
UUGAGACGCAGGUGGUCUA
551
1078
1078-1096

AD-68909.1
A-138570.1
CGUCUCAGCAUCCUGCCCU
301
A-138571.1
AGGGCAGGAUGCUGAGACG
552
1089
1089-1107

AD-68910.1
A-138572.1
CCUGCCCUGGGAUGAGAGA
302
A-138573.1
UCUCUCAUCCCAGGGCAGG
553
1100
1100-1118

AD-68911.1
A-138574.1
AUGAGAGCAUCCUGGACAA
303
A-138575.1
UUGUCCAGGAUGCUCUCAU
554
1111
1111-1129

AD-68912.1
A-138576.1
UGGACACCCUCUCGCCCAA
304
A-138577.1
UUGGGCGAGAGGGUGUCCA
555
1123
1123-1141

AD-68913.1
A-138578.1
UCGCCCAGGCUCGCUACAA
305
A-138579.1
UUGUAGCGAGCCUGGGCGA
556
1134
1134-1152

AD-68914.1
A-138580.1
UCGCUACAGCACUGAGUGA
306
A-138581.1
UCACUCAGUGCUGUAGCGA
557
1144
1144-1162

AD-68915.1
A-138582.1
CUGAGUGAAGAAAUGAAAG
307
A-138583.1
CUUUCAUUUCUUCACUCAG
558
1155
1155-1173

AD-68916.1
A-138584.1
AUGAAAGACAAAGGUGGAU
308
A-138585.1
AUCCACCUUUGUCUUUCAU
559
1167
1167-1185

AD-68917.1
A-138586.1
AAGGUGGAUACAUGAGCAA
309
A-138587.1
UUGCUCAUGUAUCCACCUU
560
1177
1177-1195

AD-68918.1
A-138588.1
AUGAGCAAGAUUUGCAACU
310
A-138589.1
AGUUGCAAAUCUUGCUCAU
561
1188
1188-1206

AD-68919.1
A-138590.1
UUGCAACUUGCUACCCAUU
311
A-138591.1
AAUGGGUAGCAAGUUGCAA
562
1199
1199-1217

AD-68920.1
A-138592.1
ACCCAUUAGGAUAAUGUCU
312
A-138593.1
AGACAUUAUCCUAAUGGGU
563
1211
1211-1229

AD-68921.1
A-138594.1
UAAUGUCUUAUGUAAUGCU
313
A-138595.1
AGCAUUACAUAAGACAUUA
564
1222
1222-1240

AD-68922.1
A-138596.1
UAAUGCUGCCCUGUACCCU
314
A-138597.1
AGGGUACAGGGCAGCAUUA
565
1234
1234-1252

AD-68923.1
A-138598.1
UGUACCCUGCCUGUGGAAU
315
A-138599.1
AUUCCACAGGCAGGGUACA
566
1245
1245-1263

AD-68924.1
A-138600.1
UGUGGAAUCUGCCAUUGCA
316
A-138601.1
UGCAAUGGCAGAUUCCACA
567
1256
1256-1274

AD-68925.1
A-138602.1
UGCCAUUGCGAUUGUCCAA
317
A-138603.1
UUGGACAAUCGCAAUGGCA
568
1265
1265-1283

AD-68926.1
A-138604.1
UUGUCCAGAGACUGGUGAA
318
A-138605.1
UUCACCAGUCUCUGGACAA
569
1276
1276-1294

AD-68927.1
A-138606.1
GGUGACAUGGCUUCCAGAU
319
A-138607.1
AUCUGGAAGCCAUGUCACC
570
1289
1289-1307

AD-68928.1
A-138608.1
UUCCAGAUAUGCCCGACGA
320
A-138609.1
UCGUCGGGCAUAUCUGGAA
571
1300
1300-1318

AD-68929.1
A-138610.1
UGCCCGACGAUGUCCUGUA
321
A-138611.1
UACAGGACAUCGUCGGGCA
572
1309
1309-1327

AD-68930.1
A-138612.1
UCCUGUGGUUGCAGUGGGU
322
A-138613.1
ACCCACUGCAACCACAGGA
573
1321
1321-1339

AD-68931.1
A-138614.1
AGUGGGUGACCUCACAGGU
323
A-138615.1
ACCUGUGAGGUCACCCACU
574
1333
1333-1351

AD-68932.1
A-138616.1
UCACAGGUGUUCACUCGAA
324
A-138617.1
UUCGAGUGAACACCUGUGA
575
1344
1344-1362

AD-68933.1
A-138618.1
UUCACUCGAGUGCUGAUGU
325
A-138619.1
ACAUCAGCACUCGAGUGAA
576
1353
1353-1371

AD-68934.1
A-138620.1
UGAUGUGUCUGCUCCCCGA
326
A-138621.1
UCGGGGAGCAGACACAUCA
577
1366
1366-1384

AD-68935.1
A-138622.1
UGCUCCCCGCCUCCAGGUA
327
A-138623.1
UACCUGGAGGCGGGGAGCA
578
1375
1375-1393

AD-68936.1
A-138624.1
CCAGGUCCCAAAUGCCAGU
328
A-138625.1
ACUGGCAUUUGGGACCUGG
579
1387
1387-1405

AD-68937.1
A-138626.1
AAUGCCAGUGAGCAGCCAA
329
A-138627.1
UUGGCUGCUCACUGGCAUU
580
1397
1397-1415

AD-68938.1
A-138628.1
AGCCAACAGGCCUCCCCAU
330
A-138629.1
AUGGGGAGGCCUGUUGGCU
581
1410
1410-1428

AD-68939.1
A-138630.1
CCUCCCCAUGCACACCUGA
331
A-138631.1
UCAGGUGUGCAUGGGGAGG
582
1420
1420-1438

AD-68940.1
A-138632.1
CACCUGAGCAGGACUGGCA
332
A-138633.1
UGCCAGUCCUGCUCAGGUG
583
1432
1432-1450

AD-68941.1
A-138634.1
GACUGGCCCUGCUGGACUA
333
A-138635.1
UAGUCCAGCAGGGCCAGUC
584
1443
1443-1461

AD-68942.1
A-138636.1
UGCUGGACUCCCUGCUCCA
334
A-138637.1
UGGAGCAGGGAGUCCAGCA
585
1452
1452-1470

AD-68943.1
A-138638.1
CUGCUCCCCCAAGGGCUGU
335
A-138639.1
ACAGCCCUUGGGGGAGCAG
586
1463
1463-1481

AD-68944.1
A-138640.1
AGGGCUGUCCAGCAGAGAA
336
A-138641.1
UUCUCUGCUGGACAGCCCU
587
1474
1474-1492

AD-68945.1
A-138642.1
CAGAGACCAAAGCAGAGGA
337
A-138643.1
UCCUCUGCUUUGGUCUCUG
588
1486
1486-1504

AD-68946.1
A-138644.1
AGCAGAGGCCACCCCGCGA
338
A-138645.1
UCGCGGGGUGGCCUCUGCU
589
1496
1496-1514

AD-68947.1
A-138646.1
CCGCGGUCCAUCCUCAGGU
339
A-138647.1
ACCUGAGGAUGGACCGCGG
590
1509
1509-1527

AD-68948.1
A-138648.1
CCUCAGGUCCAGCCUGAAA
340
A-138649.1
UUUCAGGCUGGACCUGAGG
591
1520
1520-1538

AD-68949.1
A-138650.1
AGCCUGAACUUCUUCUUGA
341
A-138651.1
UCAAGAAGAAGUUCAGGCU
592
1530
1530-1548

AD-68950.1
A-138652.1
UUCUUGGGCAAUAAAGUAA
342
A-138653.1
UUACUUUAUUGCCCAAGAA
593
1542
1542-1560

AD-68951.1
A-138654.1
UAAAGUACCUGCUGGUGCU
343
A-138655.1
AGCACCAGCAGGUACUUUA
594
1553
1553-1571

AD-68952.1
A-138656.1
CUGGUGCUGAGGGGCUCUA
344
A-138657.1
UAGAGCCCCUCAGCACCAG
595
1564
1564-1582

AD-68953.1
A-138658.1
AGGGGCUCUCCACCUUUCA
345
A-138659.1
UGAAAGGUGGAGAGCCCCU
596
1573
1573-1591

AD-68954.1
A-138660.1
CCUUUCCCAGUUUUUCACU
346
A-138661.1
AGUGAAAAACUGGGAAAGG
597
1585
1585-1603

AD-68955.1
A-138662.1
UUUUUCACUAGAGAAGAGU
347
A-138663.1
ACUCUUCUCUAGUGAAAAA
598
1595
1595-1613

AD-68956.1
A-138664.1
AAGAGUCUGUGAGUCACUU
348
A-138665.1
AAGUGACUCACAGACUCUU
599
1608
1608-1626

AD-68957.1
A-138666.1
AGUCACUUGAGGAGGCGAA
349
A-138667.1
UUCGCCUCCUCAAGUGACU
600
1619
1619-1637

AD-68958.1
A-138668.1
AGGAGGCGAGUCUAGCAGA
350
A-138669.1
UCUGCUAGACUCGCCUCCU
601
1628
1628-1646

AD-68959.1
A-138670.1
AGCAGAUUCUUUCAGAGGU
351
A-138671.1
ACCUCUGAAAGAAUCUGCU
602
1641
1641-1659

AD-68960.1
A-138672.1
UUCAGAGGUGCUAAAGUUU
352
A-138673.1
AAACUUUAGCACCUCUGAA
603
1651
1651-1669

AD-68961.1
A-138674.1
UAAAGUUUCCCAUCUUUGU
353
A-138675.1
ACAAAGAUGGGAAACUUUA
604
1662
1662-1680

AD-68962.1
A-138676.1
UCUUUGUGCAGCUACCUCA
354
A-138677.1
UGAGGUAGCUGCACAAAGA
605
1674
1674-1692

AD-68963.1
A-138678.1
AGCUACCUCCGCAUUGCUA
355
A-138679.1
UAGCAAUGCGGAGGUAGCU
606
1683
1683-1701

AD-68964.1
A-138680.1
UUGCUGUGUAGUGACCCCU
356
A-138681.1
AGGGGUCACUACACAGCAA
607
1696
1696-1714

AD-68965.1
A-138682.1
UGACCCCUGCCUGUGACGU
357
A-138683.1
ACGUCACAGGCAGGGGUCA
608
1707
1707-1725

AD-68966.1
A-138684.1
UGUGACGUGGAGGAUCCCA
358
A-138685.1
UGGGAUCCUCCACGUCACA
609
1718
1718-1736

AD-68967.1
A-138686.1
AGGAUCCCAGCCUCUGAGA
359
A-138687.1
UCUCAGAGGCUGGGAUCCU
610
1728
1728-1746

AD-68968.1
A-138688.1
CUCUGAGCUGAGUUGGUUU
360
A-138689.1
AAACCAACUCAGCUCAGAG
611
1739
1739-1757

AD-68969.1
A-138690.1
UUGGUUUUAUGAAAAGCUA
361
A-138691.1
UAGCUUUUCAUAAAACCAA
612
1751
1751-1769

AD-68970.1
A-138692.1
AAAAGCUAGGAAGCAACCU
362
A-138693.1
AGGUUGCUUCCUAGCUUUU
613
1762
1762-1780

AD-68971.1
A-138694.1
GAAGCAACCUUUCGCCUGU
363
A-138695.1
ACAGGCGAAAGGUUGCUUC
614
1771
1771-1789

AD-68972.1
A-138696.1
UCGCCUGUGCAGCGGUCCA
364
A-138697.1
UGGACCGCUGCACAGGCGA
615
1782
1782-1800

AD-68973.1
A-138698.1
CGGUCCAGCACUUAACUCU
365
A-138699.1
AGAGUUAAGUGCUGGACCG
616
1794
1794-1812

AD-68974.1
A-138700.1
UUAACUCUAAUACAUCAGA
366
A-138701.1
UCUGAUGUAUUAGAGUUAA
617
1805
1805-1823

AD-68975.1
A-138702.1
UACAUCAGCAUGCGUUAAU
367
A-138703.1
AUUAACGCAUGCUGAUGUA
618
1815
1815-1833

AD-68976.1
A-138704.1
CGUUAAUUCAGCUGGUUGA
368
A-138705.1
UCAACCAGCUGAAUUAACG
619
1827
1827-1845

AD-68977.1
A-138706.1
CUGGUUGGGAAAUGACACA
369
A-138707.1
UGUGUCAUUUCCCAACCAG
620
1838
1838-1856

AD-68978.1
A-138708.1
AAUGACACCAGGAAGCCCA
370
A-138709.1
UGGGCUUCCUGGUGUCAUU
621
1848
1848-1866

AD-68979.1
A-138710.1
AAGCCCAGUGCAGAGGGUA
371
A-138711.1
UACCCUCUGCACUGGGCUU
622
1860
1860-1878

AD-68980.1
A-138712.1
AGAGGGUCCCUUACUGACU
372
A-138713.1
AGUCAGUAAGGGACCCUCU
623
1871
1871-1889

AD-68981.1
A-138714.1
UUACUGACUGUUUCGUGGA
373
A-138715.1
UCCACGAAACAGUCAGUAA
624
1881
1881-1899

AD-68982.1
A-138716.1
UUCGUGGCCCUAUUAAUGA
374
A-138717.1
UCAUUAAUAGGGCCACGAA
625
1892
1892-1910

AD-68983.1
A-138718.1
UUAAUGGUCAGACUGUUCA
375
A-138719.1
UGAACAGUCUGACCAUUAA
626
1904
1904-1922

AD-68984.1
A-138720.1
GACUGUUCCAGCAUGAGGU
376
A-138721.1
ACCUCAUGCUGGAACAGUC
627
1914
1914-1932

AD-68985.1
A-138722.1
UGAGGUUCUUAGAAUGACA
377
A-138723.1
UGUCAUUCUAAGAACCUCA
628
1927
1927-1945

AD-68986.1
A-138724.1
UAGAAUGACAGGUGUUUGA
378
A-138725.1
UCAAACACCUGUCAUUCUA
629
1936
1936-1954

AD-68987.1
A-138726.1
UGUUUGGAUGGGUGGGGGA
379
A-138727.1
UCCCCCACCCAUCCAAACA
630
1948
1948-1966

AD-68988.1
A-138728.1
UGGGGGCCUUGUGAUGGGA
380
A-138729.1
UCCCAUCACAAGGCCCCCA
631
1960
1960-1978

AD-68989.1
A-138730.1
UGUGAUGGGGGGUAGGCUA
381
A-138731.1
UAGCCUACCCCCCAUCACA
632
1969
1969-1987

AD-68990.1
A-138732.1
UAGGCUGGCCCAUGUGUGA
382
A-138733.1
UCACACAUGGGCCAGCCUA
633
1981
1981-1999

AD-68991.1
A-138734.1
UGUGUGAUCUUGUGGGGUA
383
A-138735.1
UACCCCACAAGAUCACACA
634
1993
1993-2011

AD-68992.1
A-138736.1
UUGUGGGGUGGAGGGAAGA
384
A-138737.1
UCUUCCCUCCACCCCACAA
635
2002
2002-2020

AD-68993.1
A-138738.1
AGGGAAGAGAAUAGCAUGA
385
A-138739.1
UCAUGCUAUUCUCUUCCCU
636
2013
2013-2031

AD-68994.1
A-138740.1
UAGCAUGAUCCCACUUCCA
386
A-138741.1
UGGAAGUGGGAUCAUGCUA
637
2024
2024-2042

AD-68995.1
A-138742.1
ACUUCCCCAUGCUGUGGGA
387
A-138743.1
UCCCACAGCAUGGGGAAGU
638
2036
2036-2054

AD-68996.1
A-138744.1
CUGUGGGAAGGGGUGCAGU
388
A-138745.1
ACUGCACCCCUUCCCACAG
639
2047
2047-2065

AD-68997.1
A-138746.1
GUGCAGUUCGUCCCCAAGA
389
A-138747.1
UCUUGGGGACGAACUGCAC
640
2059
2059-2077

AD-68998.1
A-138748.1
UCCCCAAGAACGACACUGA
390
A-138749.1
UCAGUGUCGUUCUUGGGGA
641
2069
2069-2087

AD-69014.1
A-138750.1
ACACUGCCUGUCAGGUGGU
391
A-138751.1
ACCACCUGACAGGCAGUGU
642
2081
2081-2099

AD-69015.1
A-138752.1
UCAGGUGGUCUGCAAAGAU
392
A-138753.1
AUCUUUGCAGACCACCUGA
643
2091
2091-2109

AD-69016.1
A-138754.1
UGCAAAGAUGAUAACCUUA
393
A-138755.1
UAAGGUUAUCAUCUUUGCA
644
2101
2101-2119

AD-69017.1
A-138756.1
AACCUUGACUACUAAAAAC
394
A-138757.1
GUUUUUAGUAGUCAAGGUU
645
2113
2113-2131

AD-69018.1
A-138758.1
UAAAAACGUCUCCAUGGCA
395
A-138759.1
UGCCAUGGAGACGUUUUUA
646
2125
2125-2143

AD-69019.1
A-138760.1
CCAUGGCGGGGGUAACAAA
396
A-138761.1
UUUGUUACCCCCGCCAUGG
647
2136
2136-2154

AD-69020.1
A-138762.1
GGUAACAAGAUGAUAAUCU
397
A-138763.1
AGAUUAUCAUCUUGUUACC
648
2146
2146-2164

AD-69021.1
A-138764.1
UGAUAAUCUACUUAAUUUU
398
A-138765.1
AAAAUUAAGUAGAUUAUCA
649
2156
2156-2174

AD-69022.1
A-138766.1
UUAAUUUUAGAACACCUUU
399
A-138767.1
AAAGGUGUUCUAAAAUUAA
650
2167
2167-2185

AD-69023.1
A-138768.1
ACACCUUUUUCACCUAACU
400
A-138769.1
AGUUAGGUGAAAAAGGUGU
651
2178
2178-2196

AD-69024.1
A-138770.1
CCUAACUAAAAUAAUGUUU
401
A-138771.1
AAACAUUAUUUUAGUUAGG
652
2190
2190-2208

AD-69025.1
A-138772.1
AUAAUGUUUAAAGAGUUUU
402
A-138773.1
AAAACUCUUUAAACAUUAU
653
2200
2200-2218

AD-69026.1
A-138774.1
GAGUUUUGUAUAAAAAUGU
403
A-138775.1
ACAUUUUUAUACAAAACUC
654
2212
2212-2230

AD-69027.1
A-138776.1
AAAAAUGUAAGGAAGCGUU
404
A-138777.1
AACGCUUCCUUACAUUUUU
655
2223
2223-2241

AD-69028.1
A-138778.1
GGAAGCGUUGUUACCUGUU
405
A-138779.1
AACAGGUAACAACGCUUCC
656
2233
2233-2251

AD-69029.1
A-138780.1
UACCUGUUGAAUUUUGUAU
406
A-138781.1
AUACAAAAUUCAACAGGUA
657
2244
2244-2262

AD-69030.1
A-138782.1
UUGUAUUAUGUGAAUCAGU
407
A-138783.1
ACUGAUUCACAUAAUACAA
658
2257
2257-2275

AD-69031.1
A-138784.1
GAAUCAGUGAGAUGUUAGU
408
A-138785.1
ACUAACAUCUCACUGAUUC
659
2268
2268-2286

AD-69032.1
A-138786.1
AUGUUAGUAGAAUAAGCCU
409
A-138787.1
AGGCUUAUUCUACUAACAU
660
2279
2279-2297

AD-69033.1
A-138788.1
AUAAGCCUUAAAAAAAAAA
410
A-138789.1
UUUUUUUUUUAAGGCUUAU
661
2290
2290-2308

AD-69034.1
A-138790.1
AAAAAAAAAAAAAUCGGUU
411
A-138791.1
AACCGAUUUUUUUUUUUUU
662
2299
2299-2317

AD-69035.1
A-138792.1
AAUCGGUUGGGUGCAGUGA
412
A-138793.1
UCACUGCACCCAACCGAUU
663
2310
2310-2328

AD-69036.1
A-138794.1
UGCAGUGGCACACGGCUGU
413
A-138795.1
ACAGCCGUGUGCCACUGCA
664
2321
2321-2339

AD-69037.1
A-138796.1
GGCUGUAAUCCCAGCACUU
414
A-138797.1
AAGUGCUGGGAUUACAGCC
665
2334
2334-2352

AD-69038.1
A-138798.1
CAGCACUUUGGGAGGCCAA
415
A-138799.1
UUGGCCUCCCAAAGUGCUG
666
2345
2345-2363

AD-69039.1
A-138800.1
GAGGCCAAGGUUGGCAGAU
416
A-138801.1
AUCUGCCAACCUUGGCCUC
667
2356
2356-2374

AD-69040.1
A-138802.1
UUGGCAGAUCACCUGAGGU
417
A-138803.1
ACCUCAGGUGAUCUGCCAA
668
2366
2366-2384

AD-69041.1
A-138804.1
CUGAGGUCAGGAGUUCAAA
418
A-138805.1
UUUGAACUCCUGACCUCAG
669
2378
2378-2396

AD-69042.1
A-138806.1
GAGUUCAAGACCAGUCUGA
419
A-138807.1
UCAGACUGGUCUUGAACUC
670
2388
2388-2406

AD-69043.1
A-138808.1
CAGUCUGGCCAACAUAGCA
420
A-138809.1
UGCUAUGUUGGCCAGACUG
671
2399
2399-2417

AD-69044.1
A-138810.1
AACAUAGCAAAACCCUGUA
421
A-138811.1
UACAGGGUUUUGCUAUGUU
672
2409
2409-2427

AD-69045.1
A-138812.1
CCCUGUCUCUACUAAAAAU
422
A-138813.1
AUUUUUAGUAGAGACAGGG
673
2421
2421-2439

AD-69046.1
A-138814.1
CUAAAAAUACAAAAAUUAU
423
A-138815.1
AUAAUUUUUGUAUUUUUAG
674
2432
2432-2450

AD-69047.1
A-138816.1
AAAAUUAUCUGGGCAUGGU
424
A-138817.1
ACCAUGCCCAGAUAAUUUU
675
2443
2443-2461

AD-69048.1
A-138818.1
GGCAUGGUGGUGCAUGCCU
425
A-138819.1
AGGCAUGCACCACCAUGCC
676
2454
2454-2472

AD-69049.1
A-138820.1
CAUGCCUGUAAUCCCAGCU
426
A-138821.1
AGCUGGGAUUACAGGCAUG
677
2466
2466-2484

AD-69050.1
A-138822.1
AAUCCCAGCUAUUCGGAAA
427
A-138823.1
UUUCCGAAUAGCUGGGAUU
678
2475
2475-2493

AD-69051.1
A-138824.1
UUCGGAAGGCUGAGGCAGA
428
A-138825.1
UCUGCCUCAGCCUUCCGAA
679
2486
2486-2504

AD-69052.1
A-138826.1
AGGCAGGAGAAUCACUUGA
429
A-138827.1
UCAAGUGAUUCUCCUGCCU
680
2498
2498-2516

AD-69053.1
A-138828.1
AUCACUUGAACCCAGGAGA
430
A-138829.1
UCUCCUGGGUUCAAGUGAU
681
2508
2508-2526

AD-69054.1
A-138830.1
CAGGAGGCGGAGGUUGCGA
431
A-138831.1
UCGCAACCUCCGCCUCCUG
682
2520
2520-2538

AD-69055.1
A-138832.1
GUUGCGGUGAGCUGAGAUU
432
A-138833.1
AAUCUCAGCUCACCGCAAC
683
2532
2532-2550

AD-69056.1
A-138834.1
CUGAGAUUGCACCAUUUCA
433
A-138835.1
UGAAAUGGUGCAAUCUCAG
684
2543
2543-2561

AD-69057.1
A-138836.1
CACCAUUUCAUUCCAGCCU
434
A-138837.1
AGGCUGGAAUGAAAUGGUG
685
2552
2552-2570

AD-69058.1
A-138838.1
CAGCCUGGGCAACAUGAGU
435
A-138839.1
ACUCAUGUUGCCCAGGCUG
686
2565
2565-2583

AD-69059.1
A-138840.1
AACAUGAGUGAAAGUCUGA
436
A-138841.1
UCAGACUUUCACUCAUGUU
687
2575
2575-2593

AD-69060.1
A-138842.1
AGUCUGACUCAAAAAAAAA
437
A-138843.1
UUUUUUUUUGAGUCAGACU
688
2587
2587-2605

AD-69061.1
A-138844.1
AAAAAAAAAAAAUUUAAAA
438
A-138845.1
UUUUAAAUUUUUUUUUUUU
689
2597
2597-2615

AD-69062.1
A-138846.1
UUUAAAAAACAAAAUAAUA
439
A-138847.1
UAUUAUUUUGUUUUUUAAA
690
2609
2609-2627

AD-69063.1
A-138848.1
AAAAUAAUCUAGUGUGCAA
440
A-138849.1
UUGCACACUAGAUUAUUUU
691
2619
2619-2637

AD-69064.1
A-138850.1
GUGUGCAGGGCAUUCACCU
441
A-138851.1
AGGUGAAUGCCCUGCACAC
692
2630
2630-2648

AD-69065.1
A-138852.1
CAUUCACCUCAGCCCCCCA
442
A-138853.1
UGGGGGGCUGAGGUGAAUG
693
2640
2640-2658

AD-69066.1
A-138854.1
CCCCCAGGCAGGAGCCAAA
443
A-138855.1
UUUGGCUCCUGCCUGGGGG
694
2653
2653-2671

AD-69067.1
A-138856.1
AGGAGCCAAGCACAGCAGA
444
A-138857.1
UCUGCUGUGCUUGGCUCCU
695
2662
2662-2680

AD-69068.1
A-138858.1
ACAGCAGGAGCUUCCGCCU
445
A-138859.1
AGGCGGAAGCUCCUGCUGU
696
2673
2673-2691

AD-69069.1
A-138860.1
UUCCGCCUCCUCUCCACUA
446
A-138861.1
UAGUGGAGAGGAGGCGGAA
697
2684
2684-2702

AD-69070.1
A-138862.1
UCCACUGGAGCACACAACU
447
A-138863.1
AGUUGUGUGCUCCAGUGGA
698
2696
2696-2714

AD-69071.1
A-138864.1
ACACAACUUGAACCUGGCU
448
A-138865.1
AGCCAGGUUCAAGUUGUGU
699
2707
2707-2725

AD-69072.1
A-138866.1
AACCUGGCUUAUUUUCUGA
449
A-138867.1
UCAGAAAAUAAGCCAGGUU
700
2717
2717-2735

AD-69073.1
A-138868.1
UUCUGCAGGGACCAGCCCA
450
A-138869.1
UGGGCUGGUCCCUGCAGAA
701
2730
2730-2748

AD-69074.1
A-138870.1
CCAGCCCCACAUGGUCAGU
451
A-138871.1
ACUGACCAUGUGGGGCUGG
702
2741
2741-2759

AD-69076.1
A-138874.1
UUUCUCCCCAUGUGUGGCA
452
A-138875.1
UGCCACACAUGGGGAGAAA
703
2763
2763-2781

AD-69077.1
A-138878.1
AGAGAGUGUAGAAAUAAAG
453
A-138879.1
CUUUAUUUCUACACUCUCU
704
2785
2785-2803

TABLE 5

Modified Sense and Antisense Strand Sequences of PNPLA3 RNAi Agents

SEQ

SEQ

Sense Oligo

ID

Antisense
ID

Duplex Name
Name
Sense Sequence (5′-3′)
NO
Oligo Name
Sequence (5′-3′)
NO

AD-68792.1
A-138374.1
GGGGGCGGGGCUGACGUCAdTdT
705
A-138375.1
UGACGUCAGCCCCGCCCCCdTdT
956

AD-68793.1
A-138376.1
ACGUCGCGCUGGGAAUGCAdTdT
706
A-138377.1
UGCAUUCCCAGCGCGACGUdTdT
957

AD-68794.1
A-138378.1
GGAAUGCCCUGGCCGAGAAdTdT
707
A-138379.1
UUCUCGGCCAGGGCAUUCCdTdT
958

AD-68795.1
A-138380.1
UGGCCGAGACACUGAGGCAdTdT
708
A-138381.1
UGCCUCAGUGUCUCGGCCAdTdT
959

AD-68796.1
A-138382.1
UGAGGCAGGGUAGAGAGCAdTdT
709
A-138383.1
UGCUCUCUACCCUGCCUCAdTdT
960

AD-68797.1
A-138384.1
AGAGAGCGCUUGCGGGCGAdTdT
710
A-138385.1
UCGCCCGCAAGCGCUCUCUdTdT
961

AD-68798.1
A-138386.1
CGGGCGCCGGGCGGAGCUAdTdT
711
A-138387.1
UAGCUCCGCCCGGCGCCCGdTdT
962

AD-68799.1
A-138388.1
GCGGAGCUGCUGCGGAUCAdTdT
712
A-138389.1
UGAUCCGCAGCAGCUCCGCdTdT
963

AD-68800.1
A-138390.1
UGCGGAUCAGGACCCGAGAdTdT
713
A-138391.1
UCUCGGGUCCUGAUCCGCAdTdT
964

AD-68801.1
A-138392.1
ACCCGAGCCGAUUCCCGAUdTdT
714
A-138393.1
AUCGGGAAUCGGCUCGGGUdTdT
965

AD-68802.1
A-138394.1
UUCCCGAUCCCGACCCAGAdTdT
715
A-138395.1
UCUGGGUCGGGAUCGGGAAdTdT
966

AD-68803.1
A-138396.1
ACCCAGAUCCUAACCCGCAdTdT
716
A-138397.1
UGCGGGUUAGGAUCUGGGUdTdT
967

AD-68804.1
A-138398.1
UAACCCGCGCCCCCGCCCAdTdT
717
A-138399.1
UGGGCGGGGGCGCGGGUUAdTdT
968

AD-68805.1
A-138400.1
CCGCCCCGCCGCCGCCGCAdTdT
718
A-138401.1
UGCGGCGGCGGCGGGGCGGdTdT
969

AD-68806.1
A-138402.1
CGCCGCCAUGUACGACGCAdTdT
719
A-138403.1
UGCGUCGUACAUGGCGGCGdTdT
970

AD-68807.1
A-138404.1
UACGACGCAGAGCGCGGCUdTdT
720
A-138405.1
AGCCGCGCUCUGCGUCGUAdTdT
971

AD-68808.1
A-138406.1
CGCGGCUGGAGCUUGUCCUdTdT
721
A-138407.1
AGGACAAGCUCCAGCCGCGdTdT
972

AD-68809.1
A-138408.1
AGCUUGUCCUUCGCGGGCUdTdT
722
A-138409.1
AGCCCGCGAAGGACAAGCUdTdT
973

AD-68810.1
A-138410.1
CGCGGGCUGCGGCUUCCUAdTdT
723
A-138411.1
UAGGAAGCCGCAGCCCGCGdTdT
974

AD-68811.1
A-138412.1
UUCCUGGGCUUCUACCACAdTdT
724
A-138413.1
UGUGGUAGAAGCCCAGGAAdTdT
975

AD-68812.1
A-138414.1
UUCUACCACGUCGGGGCGAdTdT
725
A-138415.1
UCGCCCCGACGUGGUAGAAdTdT
976

AD-68813.1
A-138416.1
CGGGGCGACCCGCUGCCUAdTdT
726
A-138417.1
UAGGCAGCGGGUCGCCCCGdTdT
977

AD-68814.1
A-138418.1
UGCCUGAGCGAGCACGCCAdTdT
727
A-138419.1
UGGCGUGCUCGCUCAGGCAdTdT
978

AD-68815.1
A-138420.1
AGCACGCCCCGCACCUCCUdTdT
728
A-138421.1
AGGAGGUGCGGGGCGUGCUdTdT
979

AD-68816.1
A-138422.1
ACCUCCUCCGCGACGCGCAdTdT
729
A-138423.1
UGCGCGUCGCGGAGGAGGUdTdT
980

AD-68817.1
A-138424.1
GCGACGCGCGCAUGUUGUUdTdT
730
A-138425.1
AACAACAUGCGCGCGUCGCdTdT
981

AD-68818.1
A-138426.1
UGUUGUUCGGCGCUUCGGAdTdT
731
A-138427.1
UCCGAAGCGCCGAACAACAdTdT
982

AD-68819.1
A-138428.1
CUUCGGCCGGGGCGUUGCAdTdT
732
A-138429.1
UGCAACGCCCCGGCCGAAGdTdT
983

AD-68820.1
A-138430.1
GGCGUUGCACUGCGUCGGAdTdT
733
A-138431.1
UCCGACGCAGUGCAACGCCdTdT
984

AD-68821.1
A-138432.1
UGCGUCGGCGUCCUCUCCAdTdT
734
A-138433.1
UGGAGAGGACGCCGACGCAdTdT
985

AD-68822.1
A-138434.1
UCUCCGGUAUCCCGCUGGAdTdT
735
A-138435.1
UCCAGCGGGAUACCGGAGAdTdT
986

AD-68823.1
A-138436.1
UCCCGCUGGAGCAGACUCUdTdT
736
A-138437.1
AGAGUCUGCUCCAGCGGGAdTdT
987

AD-68824.1
A-138438.1
CAGACUCUGCAGGUCCUCUdTdT
737
A-138439.1
AGAGGACCUGCAGAGUCUGdTdT
988

AD-68825.1
A-138440.1
UCCUCUCAGAUCUUGUGCAdTdT
738
A-138441.1
UGCACAAGAUCUGAGAGGAdTdT
989

AD-68826.1
A-138442.1
UCUUGUGCGGAAGGCCAGAdTdT
739
A-138443.1
UCUGGCCUUCCGCACAAGAdTdT
990

AD-68827.1
A-138444.1
AAGGCCAGGAGUCGGAACAdTdT
740
A-138445.1
UGUUCCGACUCCUGGCCUUdTdT
991

AD-68828.1
A-138446.1
CGGAACAUUGGCAUCUUCAdTdT
741
A-138447.1
UGAAGAUGCCAAUGUUCCGdTdT
992

AD-68829.1
A-138448.1
GCAUCUUCCAUCCAUCCUUdTdT
742
A-138449.1
AAGGAUGGAUGGAAGAUGCdTdT
993

AD-68830.1
A-138450.1
CCAUCCUUCAACUUAAGCAdTdT
743
A-138451.1
UGCUUAAGUUGAAGGAUGGdTdT
994

AD-68831.1
A-138452.1
UUAAGCAAGUUCCUCCGAAdTdT
744
A-138453.1
UUCGGAGGAACUUGCUUAAdTdT
995

AD-68832.1
A-138454.1
CCUCCGACAGGGUCUCUGAdTdT
745
A-138455.1
UCAGAGACCCUGUCGGAGGdTdT
996

AD-68833.1
A-138456.1
UCUCUGCAAAUGCCUCCCAdTdT
746
A-138457.1
UGGGAGGCAUUUGCAGAGAdTdT
997

AD-68834.1
A-138458.1
UGCCUCCCGGCCAAUGUCAdTdT
747
A-138459.1
UGACAUUGGCCGGGAGGCAdTdT
998

AD-68835.1
A-138460.1
AAUGUCCACCAGCUCAUCUdTdT
748
A-138461.1
AGAUGAGCUGGUGGACAUUdTdT
999

AD-68836.1
A-138462.1
AGCUCAUCUCCGGCAAAAUdTdT
749
A-138463.1
AUUUUGCCGGAGAUGAGCUdTdT
1000

AD-68837.1
A-138464.1
CGGCAAAAUAGGCAUCUCUdTdT
750
A-138465.1
AGAGAUGCCUAUUUUGCCGdTdT
1001

AD-68838.1
A-138466.1
AUCUCUCUUACCAGAGUGUdTdT
751
A-138467.1
ACACUCUGGUAAGAGAGAUdTdT
1002

AD-68839.1
A-138468.1
ACCAGAGUGUCUGAUGGGAdTdT
752
A-138469.1
UCCCAUCAGACACUCUGGUdTdT
1003

AD-68840.1
A-138470.1
AUGGGGAAAACGUUCUGGUdTdT
753
A-138471.1
ACCAGAACGUUUUCCCCAUdTdT
1004

AD-68841.1
A-138472.1
ACGUUCUGGUGUCUGACUUdTdT
754
A-138473.1
AAGUCAGACACCAGAACGUdTdT
1005

AD-68842.1
A-138474.1
UCUGACUUUCGGUCCAAAGdTdT
755
A-138475.1
CUUUGGACCGAAAGUCAGAdTdT
1006

AD-68843.1
A-138476.1
UCCAAAGACGAAGUCGUGAdTdT
756
A-138477.1
UCACGACUUCGUCUUUGGAdTdT
1007

AD-68844.1
A-138478.1
AAGUCGUGGAUGCCUUGGUdTdT
757
A-138479.1
ACCAAGGCAUCCACGACUUdTdT
1008

AD-68845.1
A-138480.1
CCUUGGUAUGUUCCUGCUUdTdT
758
A-138481.1
AAGCAGGAACAUACCAAGGdTdT
1009

AD-68846.1
A-138482.1
UCCUGCUUCAUCCCCUUCUdTdT
759
A-138483.1
AGAAGGGGAUGAAGCAGGAdTdT
1010

AD-68847.1
A-138484.1
UCCCCUUCUACAGUGGCCUdTdT
760
A-138485.1
AGGCCACUGUAGAAGGGGAdTdT
1011

AD-68848.1
A-138486.1
AGUGGCCUUAUCCCUCCUUdTdT
761
A-138487.1
AAGGAGGGAUAAGGCCACUdTdT
1012

AD-68849.1
A-138488.1
CCUCCUUCCUUCAGAGGCAdTdT
762
A-138489.1
UGCCUCUGAAGGAAGGAGGdTdT
1013

AD-68850.1
A-138490.1
UCAGAGGCGUGCGAUAUGUdTdT
763
A-138491.1
ACAUAUCGCACGCCUCUGAdTdT
1014

AD-68851.1
A-138492.1
GAUAUGUGGAUGGAGGAGUdTdT
764
A-138493.1
ACUCCUCCAUCCACAUAUCdTdT
1015

AD-68852.1
A-138494.1
GAGGAGUGAGUGACAACGUdTdT
765
A-138495.1
ACGUUGUCACUCACUCCUCdTdT
1016

AD-68853.1
A-138496.1
UGACAACGUACCCUUCAUUdTdT
766
A-138497.1
AAUGAAGGGUACGUUGUCAdTdT
1017

AD-68854.1
A-138498.1
CCUUCAUUGAUGCCAAAACdTdT
767
A-138499.1
GUUUUGGCAUCAAUGAAGGdTdT
1018

AD-68855.1
A-138500.1
UGCCAAAACAACCAUCACAdTdT
768
A-138501.1
UGUGAUGGUUGUUUUGGCAdTdT
1019

AD-68856.1
A-138502.1
AUCACCGUGUCCCCCUUCUdTdT
769
A-138503.1
AGAAGGGGGACACGGUGAUdTdT
1020

AD-68857.1
A-138504.1
UCCCCCUUCUAUGGGGAGUdTdT
770
A-138505.1
ACUCCCCAUAGAAGGGGGAdTdT
1021

AD-68858.1
A-138506.1
UGGGGAGUACGACAUCUGAdTdT
771
A-138507.1
UCAGAUGUCGUACUCCCCAdTdT
1022

AD-68859.1
A-138508.1
AUCUGCCCUAAAGUCAAGUdTdT
772
A-138509.1
ACUUGACUUUAGGGCAGAUdTdT
1023

AD-68860.1
A-138510.1
AGUCAAGUCCACGAACUUUdTdT
773
A-138511.1
AAAGUUCGUGGACUUGACUdTdT
1024

AD-68861.1
A-138512.1
ACGAACUUUCUUCAUGUGAdTdT
774
A-138513.1
UCACAUGAAGAAAGUUCGUdTdT
1025

AD-68862.1
A-138514.1
UUCAUGUGGACAUCACCAAdTdT
775
A-138515.1
UUGGUGAUGUCCACAUGAAdTdT
1026

AD-68863.1
A-138516.1
UCACCAAGCUCAGUCUACAdTdT
776
A-138517.1
UGUAGACUGAGCUUGGUGAdTdT
1027

AD-68864.1
A-138518.1
AGUCUACGCCUCUGCACAAdTdT
777
A-138519.1
UUGUGCAGAGGCGUAGACUdTdT
1028

AD-68865.1
A-138520.1
CUGCACAGGGAACCUCUAAdTdT
778
A-138521.1
UUAGAGGUUCCCUGUGCAGdTdT
1029

AD-68866.1
A-138522.1
AACCUCUACCUUCUCUCGAdTdT
779
A-138523.1
UCGAGAGAAGGUAGAGGUUdTdT
1030

AD-68867.1
A-138524.1
UCUCGAGAGCUUUUGUCCAdTdT
780
A-138525.1
UGGACAAAAGCUCUCGAGAdTdT
1031

AD-68868.1
A-138526.1
UUUGUCCCCCCGGAUCUCAdTdT
781
A-138527.1
UGAGAUCCGGGGGGACAAAdTdT
1032

AD-68869.1
A-138528.1
CCGGAUCUCAAGGUGCUGAdTdT
782
A-138529.1
UCAGCACCUUGAGAUCCGGdTdT
1033

AD-68870.1
A-138530.1
UGCUGGGAGAGAUAUGCCUdTdT
783
A-138531.1
AGGCAUAUCUCUCCCAGCAdTdT
1034

AD-68871.1
A-138532.1
AGAUAUGCCUUCGAGGAUAdTdT
784
A-138533.1
UAUCCUCGAAGGCAUAUCUdTdT
1035

AD-68872.1
A-138534.1
AGGAUAUUUGGAUGCAUUAdTdT
785
A-138535.1
UAAUGCAUCCAAAUAUCCUdTdT
1036

AD-68873.1
A-138536.1
AUGCAUUCAGGUUCUUGGAdTdT
786
A-138537.1
UCCAAGAACCUGAAUGCAUdTdT
1037

AD-68874.1
A-138538.1
UUCUUGGAAGAGAAGGGCAdTdT
787
A-138539.1
UGCCCUUCUCUUCCAAGAAdTdT
1038

AD-68875.1
A-138540.1
GAGAAGGGCAUCUGCAACAdTdT
788
A-138541.1
UGUUGCAGAUGCCCUUCUCdTdT
1039

AD-68876.1
A-138542.1
UGCAACAGGCCCCAGCCAAdTdT
789
A-138543.1
UUGGCUGGGGCCUGUUGCAdTdT
1040

AD-68877.1
A-138544.1
CAGCCAGGCCUGAAGUCAUdTdT
790
A-138545.1
AUGACUUCAGGCCUGGCUGdTdT
1041

AD-68878.1
A-138546.1
GAAGUCAUCCUCAGAAGGAdTdT
791
A-138547.1
UCCUUCUGAGGAUGACUUCdTdT
1042

AD-68879.1
A-138548.1
UCAGAAGGGAUGGAUCCUAdTdT
792
A-138549.1
UAGGAUCCAUCCCUUCUGAdTdT
1043

AD-68880.1
A-138550.1
UGGAUCCUGAGGUCGCCAUdTdT
793
A-138551.1
AUGGCGACCUCAGGAUCCAdTdT
1044

AD-68881.1
A-138552.1
CGCCAUGCCCAGCUGGGCAdTdT
794
A-138553.1
UGCCCAGCUGGGCAUGGCGdTdT
1045

AD-68882.1
A-138554.1
CAGCUGGGCAAACAUGAGUdTdT
795
A-138555.1
ACUCAUGUUUGCCCAGCUGdTdT
1046

AD-68883.1
A-138556.1
CAUGAGUCUGGAUUCUUCAdTdT
796
A-138557.1
UGAAGAAUCCAGACUCAUGdTdT
1047

AD-68884.1
A-138558.1
UUCUUCCCCGGAGUCGGCUdTdT
797
A-138559.1
AGCCGACUCCGGGGAAGAAdTdT
1048

AD-68885.1
A-138560.1
AGUCGGCUGCCUUGGCUGUdTdT
798
A-138561.1
ACAGCCAAGGCAGCCGACUdTdT
1049

AD-68905.1
A-138562.1
UUGGCUGUGAGGCUGGAGAdTdT
799
A-138563.1
UCUCCAGCCUCACAGCCAAdTdT
1050

AD-68906.1
A-138564.1
AGGCUGGAGGGAGAUGAGAdTdT
800
A-138565.1
UCUCAUCUCCCUCCAGCCUdTdT
1051

AD-68907.1
A-138566.1
AUGAGCUGCUAGACCACCUdTdT
801
A-138567.1
AGGUGGUCUAGCAGCUCAUdTdT
1052

AD-68908.1
A-138568.1
UAGACCACCUGCGUCUCAAdTdT
802
A-138569.1
UUGAGACGCAGGUGGUCUAdTdT
1053

AD-68909.1
A-138570.1
CGUCUCAGCAUCCUGCCCUdTdT
803
A-138571.1
AGGGCAGGAUGCUGAGACGdTdT
1054

AD-68910.1
A-138572.1
CCUGCCCUGGGAUGAGAGAdTdT
804
A-138573.1
UCUCUCAUCCCAGGGCAGGdTdT
1055

AD-68911.1
A-138574.1
AUGAGAGCAUCCUGGACAAdTdT
805
A-138575.1
UUGUCCAGGAUGCUCUCAUdTdT
1056

AD-68912.1
A-138576.1
UGGACACCCUCUCGCCCAAdTdT
806
A-138577.1
UUGGGCGAGAGGGUGUCCAdTdT
1057

AD-68913.1
A-138578.1
UCGCCCAGGCUCGCUACAAdTdT
807
A-138579.1
UUGUAGCGAGCCUGGGCGAdTdT
1058

AD-68914.1
A-138580.1
UCGCUACAGCACUGAGUGAdTdT
808
A-138581.1
UCACUCAGUGCUGUAGCGAdTdT
1059

AD-68915.1
A-138582.1
CUGAGUGAAGAAAUGAAAGdTdT
809
A-138583.1
CUUUCAUUUCUUCACUCAGdTdT
1060

AD-68916.1
A-138584.1
AUGAAAGACAAAGGUGGAUdTdT
810
A-138585.1
AUCCACCUUUGUCUUUCAUdTdT
1061

AD-68917.1
A-138586.1
AAGGUGGAUACAUGAGCAAdTdT
811
A-138587.1
UUGCUCAUGUAUCCACCUUdTdT
1062

AD-68918.1
A-138588.1
AUGAGCAAGAUUUGCAACUdTdT
812
A-138589.1
AGUUGCAAAUCUUGCUCAUdTdT
1063

AD-68919.1
A-138590.1
UUGCAACUUGCUACCCAUUdTdT
813
A-138591.1
AAUGGGUAGCAAGUUGCAAdTdT
1064

AD-68920.1
A-138592.1
ACCCAUUAGGAUAAUGUCUdTdT
814
A-138593.1
AGACAUUAUCCUAAUGGGUdTdT
1065

AD-68921.1
A-138594.1
UAAUGUCUUAUGUAAUGCUdTdT
815
A-138595.1
AGCAUUACAUAAGACAUUAdTdT
1066

AD-68922.1
A-138596.1
UAAUGCUGCCCUGUACCCUdTdT
816
A-138597.1
AGGGUACAGGGCAGCAUUAdTdT
1067

AD-68923.1
A-138598.1
UGUACCCUGCCUGUGGAAUdTdT
817
A-138599.1
AUUCCACAGGCAGGGUACAdTdT
1068

AD-68924.1
A-138600.1
UGUGGAAUCUGCCAUUGCAdTdT
818
A-138601.1
UGCAAUGGCAGAUUCCACAdTdT
1069

AD-68925.1
A-138602.1
UGCCAUUGCGAUUGUCCAAdTdT
819
A-138603.1
UUGGACAAUCGCAAUGGCAdTdT
1070

AD-68926.1
A-138604.1
UUGUCCAGAGACUGGUGAAdTdT
820
A-138605.1
UUCACCAGUCUCUGGACAAdTdT
1071

AD-68927.1
A-138606.1
GGUGACAUGGCUUCCAGAUdTdT
821
A-138607.1
AUCUGGAAGCCAUGUCACCdTdT
1072

AD-68928.1
A-138608.1
UUCCAGAUAUGCCCGACGAdTdT
822
A-138609.1
UCGUCGGGCAUAUCUGGAAdTdT
1073

AD-68929.1
A-138610.1
UGCCCGACGAUGUCCUGUAdTdT
823
A-138611.1
UACAGGACAUCGUCGGGCAdTdT
1074

AD-68930.1
A-138612.1
UCCUGUGGUUGCAGUGGGUdTdT
824
A-138613.1
ACCCACUGCAACCACAGGAdTdT
1075

AD-68931.1
A-138614.1
AGUGGGUGACCUCACAGGUdTdT
825
A-138615.1
ACCUGUGAGGUCACCCACUdTdT
1076

AD-68932.1
A-138616.1
UCACAGGUGUUCACUCGAAdTdT
826
A-138617.1
UUCGAGUGAACACCUGUGAdTdT
1077

AD-68933.1
A-138618.1
UUCACUCGAGUGCUGAUGUdTdT
827
A-138619.1
ACAUCAGCACUCGAGUGAAdTdT
1078

AD-68934.1
A-138620.1
UGAUGUGUCUGCUCCCCGAdTdT
828
A-138621.1
UCGGGGAGCAGACACAUCAdTdT
1079

AD-68935.1
A-138622.1
UGCUCCCCGCCUCCAGGUAdTdT
829
A-138623.1
UACCUGGAGGCGGGGAGCAdTdT
1080

AD-68936.1
A-138624.1
CCAGGUCCCAAAUGCCAGUdTdT
830
A-138625.1
ACUGGCAUUUGGGACCUGGdTdT
1081

AD-68937.1
A-138626.1
AAUGCCAGUGAGCAGCCAAdTdT
831
A-138627.1
UUGGCUGCUCACUGGCAUUdTdT
1082

AD-68938.1
A-138628.1
AGCCAACAGGCCUCCCCAUdTdT
832
A-138629.1
AUGGGGAGGCCUGUUGGCUdTdT
1083

AD-68939.1
A-138630.1
CCUCCCCAUGCACACCUGAdTdT
833
A-138631.1
UCAGGUGUGCAUGGGGAGGdTdT
1084

AD-68940.1
A-138632.1
CACCUGAGCAGGACUGGCAdTdT
834
A-138633.1
UGCCAGUCCUGCUCAGGUGdTdT
1085

AD-68941.1
A-138634.1
GACUGGCCCUGCUGGACUAdTdT
835
A-138635.1
UAGUCCAGCAGGGCCAGUCdTdT
1086

AD-68942.1
A-138636.1
UGCUGGACUCCCUGCUCCAdTdT
836
A-138637.1
UGGAGCAGGGAGUCCAGCAdTdT
1087

AD-68943.1
A-138638.1
CUGCUCCCCCAAGGGCUGUdTdT
837
A-138639.1
ACAGCCCUUGGGGGAGCAGdTdT
1088

AD-68944.1
A-138640.1
AGGGCUGUCCAGCAGAGAAdTdT
838
A-138641.1
UUCUCUGCUGGACAGCCCUdTdT
1089

AD-68945.1
A-138642.1
CAGAGACCAAAGCAGAGGAdTdT
839
A-138643.1
UCCUCUGCUUUGGUCUCUGdTdT
1090

AD-68946.1
A-138644.1
AGCAGAGGCCACCCCGCGAdTdT
840
A-138645.1
UCGCGGGGUGGCCUCUGCUdTdT
1091

AD-68947.1
A-138646.1
CCGCGGUCCAUCCUCAGGUdTdT
841
A-138647.1
ACCUGAGGAUGGACCGCGGdTdT
1092

AD-68948.1
A-138648.1
CCUCAGGUCCAGCCUGAAAdTdT
842
A-138649.1
UUUCAGGCUGGACCUGAGGdTdT
1093

AD-68949.1
A-138650.1
AGCCUGAACUUCUUCUUGAdTdT
843
A-138651.1
UCAAGAAGAAGUUCAGGCUdTdT
1094

AD-68950.1
A-138652.1
UUCUUGGGCAAUAAAGUAAdTdT
844
A-138653.1
UUACUUUAUUGCCCAAGAAdTdT
1095

AD-68951.1
A-138654.1
UAAAGUACCUGCUGGUGCUdTdT
845
A-138655.1
AGCACCAGCAGGUACUUUAdTdT
1096

AD-68952.1
A-138656.1
CUGGUGCUGAGGGGCUCUAdTdT
846
A-138657.1
UAGAGCCCCUCAGCACCAGdTdT
1097

AD-68953.1
A-138658.1
AGGGGCUCUCCACCUUUCAdTdT
847
A-138659.1
UGAAAGGUGGAGAGCCCCUdTdT
1098

AD-68954.1
A-138660.1
CCUUUCCCAGUUUUUCACUdTdT
848
A-138661.1
AGUGAAAAACUGGGAAAGGdTdT
1099

AD-68955.1
A-138662.1
UUUUUCACUAGAGAAGAGUdTdT
849
A-138663.1
ACUCUUCUCUAGUGAAAAAdTdT
1100

AD-68956.1
A-138664.1
AAGAGUCUGUGAGUCACUUdTdT
850
A-138665.1
AAGUGACUCACAGACUCUUdTdT
1101

AD-68957.1
A-138666.1
AGUCACUUGAGGAGGCGAAdTdT
851
A-138667.1
UUCGCCUCCUCAAGUGACUdTdT
1102

AD-68958.1
A-138668.1
AGGAGGCGAGUCUAGCAGAdTdT
852
A-138669.1
UCUGCUAGACUCGCCUCCUdTdT
1103

AD-68959.1
A-138670.1
AGCAGAUUCUUUCAGAGGUdTdT
853
A-138671.1
ACCUCUGAAAGAAUCUGCUdTdT
1104

AD-68960.1
A-138672.1
UUCAGAGGUGCUAAAGUUUdTdT
854
A-138673.1
AAACUUUAGCACCUCUGAAdTdT
1105

AD-68961.1
A-138674.1
UAAAGUUUCCCAUCUUUGUdTdT
855
A-138675.1
ACAAAGAUGGGAAACUUUAdTdT
1106

AD-68962.1
A-138676.1
UCUUUGUGCAGCUACCUCAdTdT
856
A-138677.1
UGAGGUAGCUGCACAAAGAdTdT
1107

AD-68963.1
A-138678.1
AGCUACCUCCGCAUUGCUAdTdT
857
A-138679.1
UAGCAAUGCGGAGGUAGCUdTdT
1108

AD-68964.1
A-138680.1
UUGCUGUGUAGUGACCCCUdTdT
858
A-138681.1
AGGGGUCACUACACAGCAAdTdT
1109

AD-68965.1
A-138682.1
UGACCCCUGCCUGUGACGUdTdT
859
A-138683.1
ACGUCACAGGCAGGGGUCAdTdT
1110

AD-68966.1
A-138684.1
UGUGACGUGGAGGAUCCCAdTdT
860
A-138685.1
UGGGAUCCUCCACGUCACAdTdT
1111

AD-68967.1
A-138686.1
AGGAUCCCAGCCUCUGAGAdTdT
861
A-138687.1
UCUCAGAGGCUGGGAUCCUdTdT
1112

AD-68968.1
A-138688.1
CUCUGAGCUGAGUUGGUUUdTdT
862
A-138689.1
AAACCAACUCAGCUCAGAGdTdT
1113

AD-68969.1
A-138690.1
UUGGUUUUAUGAAAAGCUAdTdT
863
A-138691.1
UAGCUUUUCAUAAAACCAAdTdT
1114

AD-68970.1
A-138692.1
AAAAGCUAGGAAGCAACCUdTdT
864
A-138693.1
AGGUUGCUUCCUAGCUUUUdTdT
1115

AD-68971.1
A-138694.1
GAAGCAACCUUUCGCCUGUdTdT
865
A-138695.1
ACAGGCGAAAGGUUGCUUCdTdT
1116

AD-68972.1
A-138696.1
UCGCCUGUGCAGCGGUCCAdTdT
866
A-138697.1
UGGACCGCUGCACAGGCGAdTdT
1117

AD-68973.1
A-138698.1
CGGUCCAGCACUUAACUCUdTdT
867
A-138699.1
AGAGUUAAGUGCUGGACCGdTdT
1118

AD-68974.1
A-138700.1
UUAACUCUAAUACAUCAGAdTdT
868
A-138701.1
UCUGAUGUAUUAGAGUUAAdTdT
1119

AD-68975.1
A-138702.1
UACAUCAGCAUGCGUUAAUdTdT
869
A-138703.1
AUUAACGCAUGCUGAUGUAdTdT
1120

AD-68976.1
A-138704.1
CGUUAAUUCAGCUGGUUGAdTdT
870
A-138705.1
UCAACCAGCUGAAUUAACGdTdT
1121

AD-68977.1
A-138706.1
CUGGUUGGGAAAUGACACAdTdT
871
A-138707.1
UGUGUCAUUUCCCAACCAGdTdT
1122

AD-68978.1
A-138708.1
AAUGACACCAGGAAGCCCAdTdT
872
A-138709.1
UGGGCUUCCUGGUGUCAUUdTdT
1123

AD-68979.1
A-138710.1
AAGCCCAGUGCAGAGGGUAdTdT
873
A-138711.1
UACCCUCUGCACUGGGCUUdTdT
1124

AD-68980.1
A-138712.1
AGAGGGUCCCUUACUGACUdTdT
874
A-138713.1
AGUCAGUAAGGGACCCUCUdTdT
1125

AD-68981.1
A-138714.1
UUACUGACUGUUUCGUGGAdTdT
875
A-138715.1
UCCACGAAACAGUCAGUAAdTdT
1126

AD-68982.1
A-138716.1
UUCGUGGCCCUAUUAAUGAdTdT
876
A-138717.1
UCAUUAAUAGGGCCACGAAdTdT
1127

AD-68983.1
A-138718.1
UUAAUGGUCAGACUGUUCAdTdT
877
A-138719.1
UGAACAGUCUGACCAUUAAdTdT
1128

AD-68984.1
A-138720.1
GACUGUUCCAGCAUGAGGUdTdT
878
A-138721.1
ACCUCAUGCUGGAACAGUCdTdT
1129

AD-68985.1
A-138722.1
UGAGGUUCUUAGAAUGACAdTdT
879
A-138723.1
UGUCAUUCUAAGAACCUCAdTdT
1130

AD-68986.1
A-138724.1
UAGAAUGACAGGUGUUUGAdTdT
880
A-138725.1
UCAAACACCUGUCAUUCUAdTdT
1131

AD-68987.1
A-138726.1
UGUUUGGAUGGGUGGGGGAdTdT
881
A-138727.1
UCCCCCACCCAUCCAAACAdTdT
1132

AD-68988.1
A-138728.1
UGGGGGCCUUGUGAUGGGAdTdT
882
A-138729.1
UCCCAUCACAAGGCCCCCAdTdT
1133

AD-68989.1
A-138730.1
UGUGAUGGGGGGUAGGCUAdTdT
883
A-138731.1
UAGCCUACCCCCCAUCACAdTdT
1134

AD-68990.1
A-138732.1
UAGGCUGGCCCAUGUGUGAdTdT
884
A-138733.1
UCACACAUGGGCCAGCCUAdTdT
1135

AD-68991.1
A-138734.1
UGUGUGAUCUUGUGGGGUAdTdT
885
A-138735.1
UACCCCACAAGAUCACACAdTdT
1136

AD-68992.1
A-138736.1
UUGUGGGGUGGAGGGAAGAdTdT
886
A-138737.1
UCUUCCCUCCACCCCACAAdTdT
1137

AD-68993.1
A-138738.1
AGGGAAGAGAAUAGCAUGAdTdT
887
A-138739.1
UCAUGCUAUUCUCUUCCCUdTdT
1138

AD-68994.1
A-138740.1
UAGCAUGAUCCCACUUCCAdTdT
888
A-138741.1
UGGAAGUGGGAUCAUGCUAdTdT
1139

AD-68995.1
A-138742.1
ACUUCCCCAUGCUGUGGGAdTdT
889
A-138743.1
UCCCACAGCAUGGGGAAGUdTdT
1140

AD-68996.1
A-138744.1
CUGUGGGAAGGGGUGCAGUdTdT
890
A-138745.1
ACUGCACCCCUUCCCACAGdTdT
1141

AD-68997.1
A-138746.1
GUGCAGUUCGUCCCCAAGAdTdT
891
A-138747.1
UCUUGGGGACGAACUGCACdTdT
1142

AD-68998.1
A-138748.1
UCCCCAAGAACGACACUGAdTdT
892
A-138749.1
UCAGUGUCGUUCUUGGGGAdTdT
1143

AD-69014.1
A-138750.1
ACACUGCCUGUCAGGUGGUdTdT
893
A-138751.1
ACCACCUGACAGGCAGUGUdTdT
1144

AD-69015.1
A-138752.1
UCAGGUGGUCUGCAAAGAUdTdT
894
A-138753.1
AUCUUUGCAGACCACCUGAdTdT
1145

AD-69016.1
A-138754.1
UGCAAAGAUGAUAACCUUAdTdT
895
A-138755.1
UAAGGUUAUCAUCUUUGCAdTdT
1146

AD-69017.1
A-138756.1
AACCUUGACUACUAAAAACdTdT
896
A-138757.1
GUUUUUAGUAGUCAAGGUUdTdT
1147

AD-69018.1
A-138758.1
UAAAAACGUCUCCAUGGCAdTdT
897
A-138759.1
UGCCAUGGAGACGUUUUUAdTdT
1148

AD-69019.1
A-138760.1
CCAUGGCGGGGGUAACAAAdTdT
898
A-138761.1
UUUGUUACCCCCGCCAUGGdTdT
1149

AD-69020.1
A-138762.1
GGUAACAAGAUGAUAAUCUdTdT
899
A-138763.1
AGAUUAUCAUCUUGUUACCdTdT
1150

AD-69021.1
A-138764.1
UGAUAAUCUACUUAAUUUUdTdT
900
A-138765.1
AAAAUUAAGUAGAUUAUCAdTdT
1151

AD-69022.1
A-138766.1
UUAAUUUUAGAACACCUUUdTdT
901
A-138767.1
AAAGGUGUUCUAAAAUUAAdTdT
1152

AD-69023.1
A-138768.1
ACACCUUUUUCACCUAACUdTdT
902
A-138769.1
AGUUAGGUGAAAAAGGUGUdTdT
1153

AD-69024.1
A-138770.1
CCUAACUAAAAUAAUGUUUdTdT
903
A-138771.1
AAACAUUAUUUUAGUUAGGdTdT
1154

AD-69025.1
A-138772.1
AUAAUGUUUAAAGAGUUUUdTdT
904
A-138773.1
AAAACUCUUUAAACAUUAUdTdT
1155

AD-69026.1
A-138774.1
GAGUUUUGUAUAAAAAUGUdTdT
905
A-138775.1
ACAUUUUUAUACAAAACUCdTdT
1156

AD-69027.1
A-138776.1
AAAAAUGUAAGGAAGCGUUdTdT
906
A-138777.1
AACGCUUCCUUACAUUUUUdTdT
1157

AD-69028.1
A-138778.1
GGAAGCGUUGUUACCUGUUdTdT
907
A-138779.1
AACAGGUAACAACGCUUCCdTdT
1158

AD-69029.1
A-138780.1
UACCUGUUGAAUUUUGUAUdTdT
908
A-138781.1
AUACAAAAUUCAACAGGUAdTdT
1159

AD-69030.1
A-138782.1
UUGUAUUAUGUGAAUCAGUdTdT
909
A-138783.1
ACUGAUUCACAUAAUACAAdTdT
1160

AD-69031.1
A-138784.1
GAAUCAGUGAGAUGUUAGUdTdT
910
A-138785.1
ACUAACAUCUCACUGAUUCdTdT
1161

AD-69032.1
A-138786.1
AUGUUAGUAGAAUAAGCCUdTdT
911
A-138787.1
AGGCUUAUUCUACUAACAUdTdT
1162

AD-69033.1
A-138788.1
AUAAGCCUUAAAAAAAAAAdTdT
912
A-138789.1
UUUUUUUUUUAAGGCUUAUdTdT
1163

AD-69034.1
A-138790.1
AAAAAAAAAAAAAUCGGUUdTdT
913
A-138791.1
AACCGAUUUUUUUUUUUUUdTdT
1164

AD-69035.1
A-138792.1
AAUCGGUUGGGUGCAGUGAdTdT
914
A-138793.1
UCACUGCACCCAACCGAUUdTdT
1165

AD-69036.1
A-138794.1
UGCAGUGGCACACGGCUGUdTdT
915
A-138795.1
ACAGCCGUGUGCCACUGCAdTdT
1166

AD-69037.1
A-138796.1
GGCUGUAAUCCCAGCACUUdTdT
916
A-138797.1
AAGUGCUGGGAUUACAGCCdTdT
1167

AD-69038.1
A-138798.1
CAGCACUUUGGGAGGCCAAdTdT
917
A-138799.1
UUGGCCUCCCAAAGUGCUGdTdT
1168

AD-69039.1
A-138800.1
GAGGCCAAGGUUGGCAGAUdTdT
918
A-138801.1
AUCUGCCAACCUUGGCCUCdTdT
1169

AD-69040.1
A-138802.1
UUGGCAGAUCACCUGAGGUdTdT
919
A-138803.1
ACCUCAGGUGAUCUGCCAAdTdT
1170

AD-69041.1
A-138804.1
CUGAGGUCAGGAGUUCAAAdTdT
920
A-138805.1
UUUGAACUCCUGACCUCAGdTdT
1171

AD-69042.1
A-138806.1
GAGUUCAAGACCAGUCUGAdTdT
921
A-138807.1
UCAGACUGGUCUUGAACUCdTdT
1172

AD-69043.1
A-138808.1
CAGUCUGGCCAACAUAGCAdTdT
922
A-138809.1
UGCUAUGUUGGCCAGACUGdTdT
1173

AD-69044.1
A-138810.1
AACAUAGCAAAACCCUGUAdTdT
923
A-138811.1
UACAGGGUUUUGCUAUGUUdTdT
1174

AD-69045.1
A-138812.1
CCCUGUCUCUACUAAAAAUdTdT
924
A-138813.1
AUUUUUAGUAGAGACAGGGdTdT
1175

AD-69046.1
A-138814.1
CUAAAAAUACAAAAAUUAUdTdT
925
A-138815.1
AUAAUUUUUGUAUUUUUAGdTdT
1176

AD-69047.1
A-138816.1
AAAAUUAUCUGGGCAUGGUdTdT
926
A-138817.1
ACCAUGCCCAGAUAAUUUUdTdT
1177

AD-69048.1
A-138818.1
GGCAUGGUGGUGCAUGCCUdTdT
927
A-138819.1
AGGCAUGCACCACCAUGCCdTdT
1178

AD-69049.1
A-138820.1
CAUGCCUGUAAUCCCAGCUdTdT
928
A-138821.1
AGCUGGGAUUACAGGCAUGdTdT
1179

AD-69050.1
A-138822.1
AAUCCCAGCUAUUCGGAAAdTdT
929
A-138823.1
UUUCCGAAUAGCUGGGAUUdTdT
1180

AD-69051.1
A-138824.1
UUCGGAAGGCUGAGGCAGAdTdT
930
A-138825.1
UCUGCCUCAGCCUUCCGAAdTdT
1181

AD-69052.1
A-138826.1
AGGCAGGAGAAUCACUUGAdTdT
931
A-138827.1
UCAAGUGAUUCUCCUGCCUdTdT
1182

AD-69053.1
A-138828.1
AUCACUUGAACCCAGGAGAdTdT
932
A-138829.1
UCUCCUGGGUUCAAGUGAUdTdT
1183

AD-69054.1
A-138830.1
CAGGAGGCGGAGGUUGCGAdTdT
933
A-138831.1
UCGCAACCUCCGCCUCCUGdTdT
1184

AD-69055.1
A-138832.1
GUUGCGGUGAGCUGAGAUUdTdT
934
A-138833.1
AAUCUCAGCUCACCGCAACdTdT
1185

AD-69056.1
A-138834.1
CUGAGAUUGCACCAUUUCAdTdT
935
A-138835.1
UGAAAUGGUGCAAUCUCAGdTdT
1186

AD-69057.1
A-138836.1
CACCAUUUCAUUCCAGCCUdTdT
936
A-138837.1
AGGCUGGAAUGAAAUGGUGdTdT
1187

AD-69058.1
A-138838.1
CAGCCUGGGCAACAUGAGUdTdT
937
A-138839.1
ACUCAUGUUGCCCAGGCUGdTdT
1188

AD-69059.1
A-138840.1
AACAUGAGUGAAAGUCUGAdTdT
938
A-138841.1
UCAGACUUUCACUCAUGUUdTdT
1189

AD-69060.1
A-138842.1
AGUCUGACUCAAAAAAAAAdTdT
939
A-138843.1
UUUUUUUUUGAGUCAGACUdTdT
1190

AD-69061.1
A-138844.1
AAAAAAAAAAAAUUUAAAAdTdT
940
A-138845.1
UUUUAAAUUUUUUUUUUUUdTdT
1191

AD-69062.1
A-138846.1
UUUAAAAAACAAAAUAAUAdTdT
941
A-138847.1
UAUUAUUUUGUUUUUUAAAdTdT
1192

AD-69063.1
A-138848.1
AAAAUAAUCUAGUGUGCAAdTdT
942
A-138849.1
UUGCACACUAGAUUAUUUUdTdT
1193

AD-69064.1
A-138850.1
GUGUGCAGGGCAUUCACCUdTdT
943
A-138851.1
AGGUGAAUGCCCUGCACACdTdT
1194

AD-69065.1
A-138852.1
CAUUCACCUCAGCCCCCCAdTdT
944
A-138853.1
UGGGGGGCUGAGGUGAAUGdTdT
1195

AD-69066.1
A-138854.1
CCCCCAGGCAGGAGCCAAAdTdT
945
A-138855.1
UUUGGCUCCUGCCUGGGGGdTdT
1196

AD-69067.1
A-138856.1
AGGAGCCAAGCACAGCAGAdTdT
946
A-138857.1
UCUGCUGUGCUUGGCUCCUdTdT
1197

AD-69068.1
A-138858.1
ACAGCAGGAGCUUCCGCCUdTdT
947
A-138859.1
AGGCGGAAGCUCCUGCUGUdTdT
1198

AD-69069.1
A-138860.1
UUCCGCCUCCUCUCCACUAdTdT
948
A-138861.1
UAGUGGAGAGGAGGCGGAAdTdT
1199

AD-69070.1
A-138862.1
UCCACUGGAGCACACAACUdTdT
949
A-138863.1
AGUUGUGUGCUCCAGUGGAdTdT
1200

AD-69071.1
A-138864.1
ACACAACUUGAACCUGGCUdTdT
950
A-138865.1
AGCCAGGUUCAAGUUGUGUdTdT
1201

AD-69072.1
A-138866.1
AACCUGGCUUAUUUUCUGAdTdT
951
A-138867.1
UCAGAAAAUAAGCCAGGUUdTdT
1202

AD-69073.1
A-138868.1
UUCUGCAGGGACCAGCCCAdTdT
952
A-138869.1
UGGGCUGGUCCCUGCAGAAdTdT
1203

AD-69074.1
A-138870.1
CCAGCCCCACAUGGUCAGUdTdT
953
A-138871.1
ACUGACCAUGUGGGGCUGGdTdT
1204

AD-69076.1
A-138874.1
UUUCUCCCCAUGUGUGGCAdTdT
954
A-138875.1
UGCCACACAUGGGGAGAAAdTdT
1205

AD-69077.1
A-138878.1
AGAGAGUGUAGAAAUAAAGdTdT
955
A-138879.1
CUUUAUUUCUACACUCUCUdTdT
1206

TABLE 6

PNPLA3 Single Dose Screen in Hep3B Cells

Data are expressed as percent message remaining

relative to AD-1955, a non-targeting control duplex.

Duplex Name
20 nM_AVG
20 nM_STDEV

AD-68792.1
106.53
9.20

AD-68793.1
90.00
15.49

AD-68794.1
55.08
11.00

AD-68795.1
77.11
20.01

AD-68796.1
71.27
7.67

AD-68797.1
53.86
1.23

AD-68798.1
76.58
29.01

AD-68799.1
61.71
33.05

AD-68800.1
84.27
23.89

AD-68801.1
58.51
23.74

AD-68802.1
48.71
3.47

AD-68803.1
52.69
8.91

AD-68804.1
56.10
9.15

AD-68805.1
56.10
29.42

AD-68806.1
52.09
4.59

AD-68807.1
69.70
8.99

AD-68808.1
83.88
7.42

AD-68809.1
67.95
17.68

AD-68810.1
52.56
22.52

AD-68811.1
73.72
12.31

AD-68812.1
70.61
22.53

AD-68813.1
63.84
17.87

AD-68814.1
56.57
4.47

AD-68815.1
50.13
8.52

AD-68816.1
91.97
18.35

AD-68817.1
49.93
3.88

AD-68818.1
74.08
23.36

AD-68819.1
74.87
26.63

AD-68820.1
59.47
18.45

AD-68821.1
81.26
37.48

AD-68822.1
63.53
8.85

AD-68823.1
49.54
9.87

AD-68824.1
87.65
12.09

AD-68825.1
107.35
28.04

AD-68826.1
100.30
41.14

AD-68827.1
62.87
13.83

AD-68828.1
63.50
18.27

AD-68829.1
40.09
7.84

AD-68830.1
32.34
4.08

AD-68831.1
46.76
7.68

AD-68832.1
78.43
16.54

AD-68833.1
125.50
3.95

AD-68834.1
112.62
6.58

AD-68835.1
97.95
2.75

AD-68836.1
117.74
52.61

AD-68837.1
40.88
4.78

AD-68838.1
91.56
20.60

AD-68839.1
59.94
8.72

AD-68840.1
79.60
5.47

AD-68841.1
39.27
7.63

AD-68842.1
88.01
18.52

AD-68843.1
56.54
5.00

AD-68844.1
51.39
10.45

AD-68845.1
59.74
4.73

AD-68846.1
54.54
14.99

AD-68847.1
94.59
4.92

AD-68848.1
92.93
14.62

AD-68849.1
74.04
7.30

AD-68850.1
110.43
16.00

AD-68851.1
61.74
5.05

AD-68852.1
63.66
21.55

AD-68853.1
49.87
6.96

AD-68854.1
47.59
6.65

AD-68855.1
73.32
11.72

AD-68856.1
106.96
18.30

AD-68857.1
123.97
37.64

AD-68858.1
60.42
4.02

AD-68859.1
81.29
14.80

AD-68860.1
68.06
17.18

AD-68861.1
89.36
8.04

AD-68862.1
62.20
19.06

AD-68863.1
78.73
13.90

AD-68864.1
71.54
10.06

AD-68865.1
79.83
18.10

AD-68866.1
90.56
9.37

AD-68867.1
76.38
25.29

AD-68868.1
106.98
9.34

AD-68869.1
80.37
23.99

AD-68870.1
62.13
19.67

AD-68871.1
82.72
12.73

AD-68872.1
78.95
8.19

AD-68873.1
71.57
3.92

AD-68874.1
118.98
25.63

AD-68875.1
82.64
10.49

AD-68876.1
106.02
17.93

AD-68877.1
47.83
10.91

AD-68878.1
68.77
9.18

AD-68879.1
92.72
13.76

AD-68880.1
121.48
13.92

AD-68881.1
99.48
5.55

AD-68882.1
90.81
8.65

AD-68883.1
88.48
16.79

AD-68884.1
126.40
27.97

AD-68885.1
79.31
13.00

AD-68905.1
59.11
11.11

AD-68906.1
62.09
23.14

AD-68907.1
91.47
18.05

AD-68908.1
69.14
6.98

AD-68909.1
57.61
0.00

AD-68910.1
53.43
6.58

AD-68911.1
49.21
4.14

AD-68912.1
55.29
11.49

AD-68913.1
60.30
3.64

AD-68914.1
64.75
6.02

AD-68915.1
77.72
5.80

AD-68916.1
51.18
6.74

AD-68917.1
61.47
5.86

AD-68918.1
63.11
5.98

AD-68919.1
58.34
10.77

AD-68920.1
50.34
15.08

AD-68921.1
82.27
16.34

AD-68922.1
76.90
14.57

AD-68923.1
73.35
4.56

AD-68924.1
54.86
10.39

AD-68925.1
79.75
12.87

AD-68926.1
67.63
6.30

AD-68927.1
70.30
11.39

AD-68928.1
71.51
12.69

AD-68929.1
66.30
18.72

AD-68930.1
71.14
21.97

AD-68931.1
71.05
8.92

AD-68932.1
77.92
4.34

AD-68933.1
101.43
16.21

AD-68934.1
53.20
9.90

AD-68935.1
99.51
9.41

AD-68936.1
49.46
8.03

AD-68937.1
57.51
13.53

AD-68938.1
88.20
15.56

AD-68939.1
74.32
14.17

AD-68940.1
77.38
17.70

AD-68941.1
76.90
11.02

AD-68942.1
86.39
14.95

AD-68943.1
110.51
36.72

AD-68944.1
66.71
10.77

AD-68945.1
70.73
19.44

Example 3. iRNA Synthesis
Source of Reagents

Transcripts

siRNA Design

A set of iRNAs targeting the human PNPLA3 (human: NCBI refseqID NM_025225; NCBI GeneID: 80339), as well as toxicology-species PNPLA3 orthologs (cynomolgus monkey: XM_005567051; mouse: NM_054088; rat: XM_006242109) were designed using custom R and Python scripts. The human PNPLA3 REFSEQ mRNA has a length of 2805 bases. The rationale and method for the set of iRNA designs is as follows: the predicted efficacy for every potential 19mer iRNA from position 174 through position 2805 (the coding region and 3′ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct iRNA designs targeting a large number of vertebrate genes. Subsets of the PNPLA3 iRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PNPLA3 orthologs. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey, mouse, and rat PNPLA3 orthologs. For each strand of the iRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the iRNA and all potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed region, defined here as positions 2-9 of the antisense oligonucleotide, as well the cleavage site of the iRNA, defined here as positions 10-11 of the antisense oligonucleotide. The relative weight of the mismatches was 2.8, 1.2 and 1 for seed mismatches, cleavage site, and other positions up through antisense position 19, respectively. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to iRNAs whose antisense score in human and cynomolgus monkey was >=3.0 and predicted efficacy was >=70% knockdown of the PNPLA3 transcript.

A detailed list of the unmodified PNPLA3 sense and antisense strand sequences is shown in Table 7. A detailed list of the modified PNPLA3 sense and antisense strand sequences is shown in Table 8. Table 9 provides the mRNA target sequences of the modified PNPLA3 agents provided in Table 8

In Vitro Screening

Cell Culture and Transfections

Hep3b cells, mouse and cynomolgus monkey primary hepatocytes were transfected, independently, by adding 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of iRNA duplexes per well into a 384-well plate and incubated at room temperature for 15 minutes. Forty μl of EMEM containing about 5×10³Hep3b cells, or 40 μl of William's media containing about 5×10³primary mouse hepatocytes or primary cynomolgus monkey hepatocytes were then added to the iRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Two single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations and dose response experiments were performed over a range of doses from 10 nM to 36 fM final duplex concentration over 8, 6-fold dilutions.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl 10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 6.6 μl of H2O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours 37° C.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Hs99999905), 0.5 μl PNPLA3 probe and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Hep3b qPCR was probed with GAPDH TaqMan Probe (Hs99999905) and PNPLA3 probe (Hs00228747_m1). Mouse primary hepatocytes qPCR was probed with Mouse GAPDH TaqMan Probe (Mm03302249_g1) and Mouse PNPLA3 Taqman Probe (Mm00504420_m1). Cynomolgus monkey primary hepatocytes qPCR was probed with custom Cynomolgus GAPDH probe and custom Cynomolgus PNPLA3 probe (5′-AGCGGGGUCUGAAGUCAU-3′(SEQ ID NO: 1207)). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 20 nM AD-1955, a non-targeting control iRNA, or mock transfected cells. The sense and antisense sequences of AD-1955 are:

SENSE:

(SEQ ID NO: 1208)

cuuAcGcuGAGuAcuucGAdTsdT;

ANTISENSE:

(SEQ ID NO: 1209)

UCGAAGuACUcAGCGuAAGdTsdT.

In Vitro Dual-Glo® Screening

Cell Culture and Transfections

Cos 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Dual-Glo® Luciferase constructs were generated in the psiCHECK2 plasmid and contained approximately 2.8 kb (human) PNPLA3 sequences (SEQ ID NO:18). Dual-luciferase plasmids were co-transfected with siRNA into 15×10³cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat #13778-150). For each well of a 96 well plate, 0.2 μl of Lipofectamine were added to 10 ng of plasmid vector and iRNA in 15 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells resuspended in 80 μl of fresh complete media. Cells were incubated for 48 hours before luciferase was measured. Two single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations and dose response experiments were performed over a range of doses from 10 nM to 36 fM final duplex concentration over 8, 6-fold dilutions.

Dual-Glo® Luciferase Assay

Fifty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to PNPLA3 target sequence in 3′ UTR) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 75 μl of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mix. The mixture was incubated at room temperature for 30 minutes before luminescence (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 75 μl of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent, quenches the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. iRNA activity was determined by normalizing the Renilla (PNPLA3) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with iRNA or were treated with a non-targeting iRNA. All transfections were done in triplicate.

Table 10 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in Hep3B cells transfected with the indicated modified RNAi agents. Data are expressed as percent of message remaining relative to untreated cells.

Table 11 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in Cynomolgus monkey primary hepatocytes transfected with the indicated modified RNAi agents. Data are expressed as percent of message remaining relative to untreated cells. Table 12 shows the dose response in primary Cynomolgus monkey hepatocytes transfected with the indicated modified RNAi agents. The indicated IC₅₀values represent the IC₅₀values relative to untreated cells.

Table 13 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in mouse primary hepatocytes transfected with the indicated modified RNAi agents. Data are expressed as percent of message remaining relative to untreated cells.

Table 14 shows the dose response in primary mouse hepatocytes transfected with the indicated modified RNAi agents. The indicated IC₅₀values represent the IC₅₀values relative to untreated cells.

Table 15 shows the results of a single 10 nM dose screen and a single 0.1 nM dose screen in Cos 7 cells transfected with the indicated modified PNPLA3 RNAi agents. Data are expressed as percent of mRNA remaining relative to negative control.

Table 16 shows the dose response in Cos 7 cells transfected with the indicated modified RNAi agents. The indicated IC₅₀values represent the IC₅₀values relative to untreated cells.

TABLE 7

PNPLA3 Unmodified Sequences

Start

Start

in

in

Sense
Sense
SEQ
SEQ
Antisense
Antisense
SEQ
SEQ

Duplex
Oligo
Sequence
ID
ID
Oligo
Sequence
ID
ID

Name
Name
5′ to 3′
NO
NO: 1
Name
5′ to 3′
NO
NO: 1
Range

AD-67524.1
A-135231.1
GGCU
1210
219
A-135232.1
UUGG
1300
217
217-239

UCCU

UAGA

GGGC

AGCC

UUCU

CAGG

ACCA

AAGC

A

CGC

AD-67611.1
A-135409.1
UUGU
1211
388
A-135410.1
UACU
1301
386
386-408

GCGG

CCUG

AAGG

GCCU

CCAG

UCCG

GAGU

CACA

A

AGA

AD-67601.1
A-135389.1
AAGG
1212
396
A-135390.1
AAUG
1302
394
394-416

CCAG

UUCC

GAGU

GACU

CGGA

CCUG

ACAU

GCCU

U

UCC

AD-67579.1
A-135341.1
AGGC
1213
397
A-135342.1
UAAU
1303
395
395-417

CAGG

GUUC

AGUC

CGAC

GGAA

UCCU

CAUU

GGCC

A

UUC

AD-67588.1
A-135361.1
AACG
1214
549
A-135362.1
AAAG
1304
547
547-569

UUCU

UCAG

GGUG

ACAC

UCUG

CAGA

ACUU

ACGU

U

UUU

AD-67602.1
A-135391.1
CUGA
1215
562
A-135392.1
UGUC
1305
560
560-582

CUUU

UUUG

CGGU

GACC

CCAA

GAAA

AGAC

GUCA

A

GAC

AD-67570.1
A-135323.1
UCGG
1216
569
A-135324.1
ACGA
1306
567
567-589

UCCA

CUUC

AAGA

GUCU

CGAA

UUGG

GUCG

ACCG

U

AAA

AD-67553.1
A-135289.1
CGGU
1217
570
A-135290.1
UACG
1307
568
568-590

CCAA

ACUU

AGAC

CGUC

GAAG

UUUG

UCGU

GACC

A

GAA

AD-67612.1
A-135411.1
GACG
1218
579
A-135412.1
UAAG
1308
577
577-599

AAGU

GCAU

CGUG

CCAC

GAUG

GACU

CCUU

UCGU

A

CUU

AD-67525.1
A-135233.1
CUUG
1219
596
A-135234.1
AUGA
1309
594
594-616

GUAU

AGCA

GUUC

GGAA

CUGC

CAUA

UUCA

CCAA

U

GGC

AD-67526.1
A-135235.1
GGCC
1220
630
A-135236.1
UAAG
1310
628
628-650

UUAU

GAAG

CCCU

GAGG

CCUU

GAUA

CCUU

AGGC

A

CAC

AD-67592.1
A-135371.1
AGGA
1221
674
A-135372.1
UGUA
1311
672
672-694

GUGA

CGUU

GUGA

GUCA

CAAC

CUCA

GUAC

CUCC

A

UCC

AD-67578.1
A-135339.1
GUGA
1222
678
A-135340.1
UAAG
1312
676
676-698

GUGA

GGUA

CAAC

CGUU

GUAC

GUCA

CCUU

CUCA

A

CUC

AD-67555.1
A-135293.1
UGAU
1223
701
A-135294.1
UUGA
1313
699
699-721

GCCA

UGGU

AAAC

UGUU

AACC

UUGG

AUCA

CAUC

A

AAU

AD-67577.1
A-135337.1
CGAC
1224
746
A-135338.1
UUGA
1314
744
744-766

AUCU

CUUU

GCCC

AGGG

UAAA

CAGA

GUCA

UGUC

A

GUA

AD-67594.1
A-135375.1
ACGA
1225
771
A-135376.1
UUCC
1315
769
769-791

ACUU

ACAU

UCUU

GAAG

CAUG

AAAG

UGGA

UUCG

A

UGG

AD-67568.1
A-135319.1
GCAC
1226
817
A-135320.1
UAAG
1316
815
815-837

AGGG

GUAG

AACC

AGGU

UCUA

UCCC

CCUU

UGUG

A

CAG

AD-67550.1
A-135283.1
UGCU
1227
871
A-135284.1
UAAG
1317
869
869-891

GGGA

GCAU

GAGA

AUCU

UAUG

CUCC

CCUU

CAGC

A

ACC

AD-67586.1
A-135357.1
UGGG
1228
874
A-135358.1
UUCG
1318
872
872-894

AGAG

AAGG

AUAU

CAUA

GCCU

UCUC

UCGA

UCCC

A

AGC

AD-67576.1
A-135335.1
AGAG
1229
878
A-135336.1
UAUC
1319
876
876-898

AUAU

CUCG

GCCU

AAGG

UCGA

CAUA

GGAU

UCUC

A

UCC

AD-67563.1
A-135309.1
AUAU
1230
882
A-135310.1
UAAA
1320
880
880-902

GCCU

UAUC

UCGA

CUCG

GGAU

AAGG

AUUU

CAUA

A

UCU

AD-67552.1
A-135287.1
UGCC
1231
885
A-135288.1
AUCC
1321
883
883-905

UUCG

AAAU

AGGA

AUCC

UAUU

UCGA

UGGA

AGGC

U

AUA

AD-67608.1
A-135403.1
AUUC
1232
908
A-135404.1
UUCU
1322
906
906-928

AGGU

CUUC

UCUU

CAAG

GGAA

AACC

GAGA

UGAA

A

UGC

AD-67593.1
A-135373.1
CAUC
1233
964
A-135374.1
UAUC
1323
962
962-984

CUCA

CAUC

GAAG

CCUU

GGAU

CUGA

GGAU

GGAU

A

GAC

AD-67609.1
A-135405.1
CCUG
1234
1100
A-135406.1
AUGC
1324
1098
1098-1120

CCCU

UCUC

GGGA

AUCC

UGAG

CAGG

AGCA

GCAG

U

GAU

AD-67597.1
A-135381.1
GACA
1235
1173
A-135382.1
UCUC
1325
1171
1171-1193

AAGG

AUGU

UGGA

AUCC

UACA

ACCU

UGAG

UUGU

A

CUU

AD-67587.1
A-135359.1
AAAG
1236
1176
A-135360.1
UUUG
1326
1174
1174-1196

GUGG

CUCA

AUAC

UGUA

AUGA

UCCA

GCAA

CCUU

A

UGU

AD-67559.1
A-135301.1
GUGG
1237
1180
A-135302.1
AAAU
1327
1178
1178-1200

AUAC

CUUG

AUGA

CUCA

GCAA

UGUA

GAUU

UCCA

U

CCU

AD-67561.1
A-135305.1
UGGA
1238
1181
A-135306.1
UAAA
1328
1179
1179-1201

UACA

UCUU

UGAG

GCUC

CAAG

AUGU

AUUU

AUCC

A

ACC

AD-67551.1
A-135285.1
AUAC
1239
1184
A-135286.1
UUGC
1329
1182
1182-1204

AUGA

AAAU

GCAA

CUUG

GAUU

CUCA

UGCA

UGUA

A

UCC

AD-67591.1
A-135369.1
AGCA
1240
1191
A-135370.1
UAGC
1330
1189
1189-1211

AGAU

AAGU

UUGC

UGCA

AACU

AAUC

UGCU

UUGC

A

UCA

AD-67583.1
A-135351.1
CAAG
1241
1193
A-135352.1
UGUA
1331
1191
1191-1213

AUUU

GCAA

GCAA

GUUG

CUUG

CAAA

CUAC

UCUU

A

GCU

AD-67585.1
A-135355.1
UGCA
1242
1200
A-135356.1
UCUA
1332
1198
1198-1220

ACUU

AUGG

GCUA

GUAG

CCCA

CAAG

UUAG

UUGC

A

AAA

AD-67589.1
A-135363.1
AACU
1243
1203
A-135364.1
UAUC
1333
1201
1201-1223

UGCU

CUAA

ACCC

UGGG

AUUA

UAGC

GGAU

AAGU

A

UGC

AD-67595.1
A-135377.1
GCCA
1244
1266
A-135378.1
UCUC
1334
1264
1264-1286

UUGC

UGGA

GAUU

CAAU

GUCC

CGCA

AGAG

AUGG

A

CAG

AD-67580.1
A-135343.1
GAUU
1245
1274
A-135344.1
UUCA
1335
1272
1272-1294

GUCC

CCAG

AGAG

UCUC

ACUG

UGGA

GUGA

CAAU

A

CGC

AD-67573.1
A-135329.1
UGGU
1246
1288
A-135330.1
UAUC
1336
1286
1286-1308

GACA

UGGA

UGGC

AGCC

UUCC

AUGU

AGAU

CACC

A

AGU

AD-67600.1
A-135387.1
CCAG
1247
1302
A-135388.1
UACA
1337
1300
1300-1322

AUAU

UCGU

GCCC

CGGG

GACG

CAUA

AUGU

UCUG

A

GAA

AD-67603.1
A-135393.1
GUGG
1248
1325
A-135394.1
UAGG
1338
1323
1323-1345

UUGC

UCAC

AGUG

CCAC

GGUG

UGCA

ACCU

ACCA

A

CAG

AD-67598.1
A-135383.1
AGGU
1249
1389
A-135384.1
UCUC
1339
1387
1387-1409

CCCA

ACUG

AAUG

GCAU

CCAG

UUGG

UGAG

GACC

A

UGG

AD-67564.1
A-135311.1
UCAC
1250
1621
A-135312.1
UAGA
1340
1619
1619-1641

UUGA

CUCG

GGAG

CCUC

GCGA

CUCA

GUCU

AGUG

A

ACU

AD-67574.1
A-135331.1
AGUC
1251
1636
A-135332.1
UCUG
1341
1634
1634-1656

UAGC

AAAG

AGAU

AAUC

UCUU

UGCU

UCAG

AGAC

A

UCG

AD-67590.1
A-135365.1
AUUC
1252
1646
A-135366.1
UUUU
1342
1644
1644-1666

UUUC

AGCA

AGAG

CCUC

GUGC

UGAA

UAAA

AGAA

A

UCU

AD-67572.1
A-135327.1
UUCU
1253
1647
A-135328.1
ACUU
1343
1645
1645-1667

UUCA

UAGC

GAGG

ACCU

UGCU

CUGA

AAAG

AAGA

U

AUC

AD-67582.1
A-135349.1
GUGC
1254
1658
A-135350.1
AAAG
1344
1656
1656-1678

UAAA

AUGG

GUUU

GAAA

CCCA

CUUU

UCUU

AGCA

U

CCU

AD-67607.1
A-135401.1
UCCC
1255
1669
A-135402.1
UGUA
1345
1667
1667-1689

AUCU

GCUG

UUGU

CACA

GCAG

AAGA

CUAC

UGGG

A

AAA

AD-67571.1
A-135325.1
CUGC
1256
1713
A-135326.1
UAUC
1346
1711
1711-1733

CUGU

CUCC

GACG

ACGU

UGGA

CACA

GGAU

GGCA

A

GGG

AD-67599.1
A-135385.1
UGUG
1257
1718
A-135386.1
UCUG
1347
1716
1716-1738

ACGU

GGAU

GGAG

CCUC

GAUC

CACG

CCAG

UCAC

A

AGG

AD-67554.1
A-135291.1
UCUG
1258
1740
A-135292.1
AUAA
1348
1738
1738-1760

AGCU

AACC

GAGU

AACU

UGGU

CAGC

UUUA

UCAG

U

AGG

AD-67549.1
A-135281.1
AGUU
1259
1749
A-135282.1
UAGC
1349
1747
1747-1769

GGUU

UUUU

UUAU

CAUA

GAAA

AAAC

AGCU

CAAC

A

UCA

AD-67567.1
A-135317.1
UUGG
1260
1751
A-135318.1
UCUA
1350
1749
1749-1771

UUUU

GCUU

AUGA

UUCA

AAAG

UAAA

CUAG

ACCA

A

ACU

AD-67558.1
A-135299.1
GGUU
1261
1753
A-135300.1
UUCC
1351
1751
1751-1773

UUAU

UAGC

GAAA

UUUU

AGCU

CAUA

AGGA

AAAC

A

CAA

AD-67569.1
A-135321.1
GUUU
1262
1754
A-135322.1
UUUC
1352
1752
1752-1774

UAUG

CUAG

AAAA

CUUU

GCUA

UCAU

GGAA

AAAA

A

CCA

AD-67548.1
A-135279.1
UUUU
1263
1755
A-135280.1
UCUU
1353
1753
1753-1775

AUGA

CCUA

AAAG

GCUU

CUAG

UUCA

GAAG

UAAA

A

ACC

AD-67566.1
A-135315.1
UAUG
1264
1758
A-135316.1
UUUG
1354
1756
1756-1778

AAAA

CUUC

GCUA

CUAG

GGAA

CUUU

GCAA

UCAU

A

AAA

AD-67613.1
A-135413.1
CGUU
1265
1827
A-135414.1
UCCC
1355
1825
1825-1847

AAUU

AACC

CAGC

AGCU

UGGU

GAAU

UGGG

UAAC

A

GCA

AD-67610.1
A-135407.1
GUUA
1266
1828
A-135408.1
UUCC
1356
1826
1826-1848

AUUC

CAAC

AGCU

CAGC

GGUU

UGAA

GGGA

UUAA

A

CGC

AD-67556.1
A-135295.1
AGCU
1267
1836
A-135296.1
UGUG
1357
1834
1834-1856

GGUU

UCAU

GGGA

UUCC

AAUG

CAAC

ACAC

CAGC

A

UGA

AD-67581.1
A-135345.1
CCUA
1268
1900
A-135346.1
AACA
1358
1898
1898-1920

UUAA

GUCU

UGGU

GACC

CAGA

AUUA

CUGU

AUAG

U

GGC

AD-67560.1
A-135303.1
CUAU
1269
1901
A-135304.1
UAAC
1359
1899
1899-1921

UAAU

AGUC

GGUC

UGAC

AGAC

CAUU

UGUU

AAUA

A

GGG

AD-67596.1
A-135379.1
GCUG
1270
1984
A-135380.1
UAAG
1360
1982
1982-2004

GCCC

AUCA

AUGU

CACA

GUGA

UGGG

UCUU

CCAG

A

CCU

AD-67557.1
A-135297.1
UGGC
1271
1986
A-135298.1
UACA
1361
1984
1984-2006

CCAU

AGAU

GUGU

CACA

GAUC

CAUG

UUGU

GGCC

A

AGC

AD-67584.1
A-135353.1
CCUA
1272
2190
A-135354.1
UUAA
1362
2188
2188-2210

ACUA

ACAU

AAAU

UAUU

AAUG

UUAG

UUUA

UUAG

A

GUG

AD-67575.1
A-135333.1
UUAC
1273
2243
A-135334.1
AAUA
1363
2241
2241-2263

CUGU

CAAA

UGAA

AUUC

UUUU

AACA

GUAU

GGUA

U

ACA

AD-67605.1
A-135397.1
ACCU
1274
2245
A-135398.1
AUAA
1364
2243
2243-2265

GUUG

UACA

AAUU

AAAU

UUGU

UCAA

AUUA

CAGG

U

UAA

AD-67562.1
A-135307.1
UGUA
1275
2258
A-135308.1
UUCA
1365
2256
2256-2278

UUAU

CUGA

GUGA

UUCA

AUCA

CAUA

GUGA

AUAC

A

AAA

AD-67606.1
A-135399.1
UAUG
1276
2263
A-135400.1
AACA
1366
2261
2261-2283

UGAA

UCUC

UCAG

ACUG

UGAG

AUUC

AUGU

ACAU

U

AAU

AD-67604.1
A-135395.1
GAUG
1277
2278
A-135396.1
AAGG
1367
2276
2276-2298

UUAG

CUUA

UAGA

UUCU

AUAA

ACUA

GCCU

ACAU

U

CUC

AD-67565.1
A-135313.1
AUGU
1278
2279
A-135314.1
UAAG
1368
2277
2277-2299

UAGU

GCUU

AGAA

AUUC

UAAG

UACU

CCUU

AACA

A

UCU

AD-67529.1
A-135241.1
UAUA
1279
250
A-135242.1
UCCA
1369
248
248-270

AUGG

UGAG

AGAU

GAUC

CCUC

UCCA

AUGG

UUAU

A

ACG

AD-67533.1
A-135249.1
GUGU
1280
443
A-135250.1
UUUG
1370
441
441-463

CUGA

GAAU

GUUC

GGAA

CAUU

CUCA

CCAA

GACA

A

CCA

AD-67537.1
A-135257.1
AGUC
1281
469
A-135258.1
UACA
1371
467
467-489

GUGG

CCAG

AUGC

GGCA

CCUG

UCCA

GUGU

CGAC

A

UUC

AD-67546.1
A-135275.1
UGCU
1282
770
A-135276.1
UUCC
1372
768
768-790

AUCA

AGGU

AGGG

ACCC

UACC

UUGA

UGGA

UAGC

A

ACA

AD-67547.1
A-135277.1
UCCC
1283
1163
A-135278.1
UAUU
1373
1161
1161-1183

AGGU

CGGG

UUGU

CACA

GCCC

AACC

GAAU

UGGG

A

AUG

AD-67543.1
A-135269.1
CCAG
1284
1165
A-135270.1
UUCA
1374
1163
1163-1185

GUUU

UUCG

GUGC

GGCA

CCGA

CAAA

AUGA

CCUG

A

GGA

AD-67541.1
A-135265.1
UGGA
1285
3032
A-135266.1
UAUC
1375
3030
3030-3052

GCAA

UAGA

CAGU

CACU

GUCU

GUUG

AGAU

CUCC

A

AGA

AD-67535.1
A-135253.1
CUUU
1286
3106
A-135254.1
UUUC
1376
3104
3104-3126

UGGA

CUAG

GGCA

CUGC

GCUA

CUCC

GGAA

AAAA

A

GUA

AD-67530.1
A-135243.1
AAGA
1287
3226
A-135244.1
UAAA
1377
3224
3224-3246

CAAU

CACC

GAUU

AAAU

UGGU

CAUU

GUUU

GUCU

A

UUG

AD-67542.1
A-135267.1
GACA
1288
3228
A-135268.1
UCUA
1378
3226
3226-3248

AUGA

AACA

UUUG

CCAA

GUGU

AUCA

UUAG

UUGU

A

CUU

AD-67528.1
A-135239.1
CAAU
1289
3230
A-135240.1
UUUC
1379
3228
3228-3250

GAUU

UAAA

UGGU

CACC

GUUU

AAAU

AGAA

CAUU

A

GUC

AD-67527.1
A-135237.1
UGCC
1290
3447
A-135238.1
AAAG
1380
3445
3445-3467

AGAU

UAAU

AACU

AAGU

UAUU

UAUC

ACUU

UGGC

U

AGG

AD-67544.1
A-135271.1
ACAC
1291
3473
A-135272.1
AUUA
1381
3471
3471-3493

CUUU

GUAA

GGCU

GAGC

CUUA

CAAA

CUAA

GGUG

U

UCC

AD-67532.1
A-135247.1
CUGG
1292
3629
A-135248.1
UAUA
1382
3627
3627-3649

CUCC

CAAA

AAAU

GAUU

CUUU

UGGA

GUAU

GCCA

A

GUG

AD-67534.1
A-135251.1
UGGC
1293
3630
A-135252.1
UUAU
1383
3628
3628-3650

UCCA

ACAA

AAUC

AGAU

UUUG

UUGG

UAUA

AGCC

A

AGU

AD-67538.1
A-135259.1
CCAA
1294
3635
A-135260.1
UAUG
1384
3633
3633-3655

AUCU

ACUA

UUGU

UACA

AUAG

AAGA

UCAU

UUUG

A

GAG

AD-67545.1
A-135273.1
AGAG
1295
3986
A-135274.1
UAGC
1385
3984
3984-4006

ACAA

CUAG

AGUG

ACAC

UCUA

UUUG

GGCU

UCUC

A

UAG

AD-67539.1
A-135261.1
AAGU
1296
3993
A-135262.1
UUCU
1386
3991
3991-4013

GUCU

GUGU

AGGC

AGCC

UACA

UAGA

CAGA

CACU

A

UUG

AD-67540.1
A-135263.1
AGAA
1297
4283
A-135264.1
UAAA
1387
4281
4281-4303

ACUU

GCAA

CUGC

GGCA

CUUG

GAAG

CUUU

UUUC

A

UAC

AD-67531.1
A-135245.1
GAAG
1298
4540
A-135246.1
UGUG
1388
4538
4538-4560

GAUU

UAUC

GAAU

CAUU

GGAU

CAAU

ACAC

CCUU

A

CUG

AD-67536.1
A-135255.1
GGAU
1299
4543
A-135256.1
UUUG
1389
4541
4541-4563

UGAA

GUGU

UGGA

AUCC

UACA

AUUC

CCAA

AAUC

A

CUU

TABLE 8

PNPLA3 Modified Sequences

Anti-

Sense

sense

Se-

Anti-
Se-

Sense
quence
SEQ
sense
quence
SEQ

Duplex
Oligo
5′ to
ID
Oligo
5′ to
ID

Name
Name
3′
NO
Name
3′
NO

AD-
A-
gsgs
1390
A-
usUf
1480

67524.1
135231.1
cuuc

135232.1
sggu

CfuG

AfgA

fGfG

fAfg

fcuu

cccA

cuac

fgGf

caaL

aagc

96

csgs

c

AD-
A-
usus
1391
A-
usAf
1481

67611.1
135409.1
gugc

135410.1
scuc

GfgA

CfuG

fAfG

fGfc

fgcc

cuuC

agga

fcGf

guaL

caca

96

asgs

a

AD-
A-
asas
1392
A-
asAf
1482

67601.1
135389.1
ggcc

135390.1
sugu

AfgG

UfcC

fAfG

fGfa

fucg

cucC

gaac

fuGf

auuL

gccu

96

uscs

c

AD-
A-
asgs
1393
A-
usAf
1483

67579.1
135341.1
gcca

135342.1
saug

GfgA

UfuC

fGfU

fCfg

fcgg

acuC

aaca

fcUf

uuaL

ggcc

96

usus

c

AD-
A-
asas
1394
A-
asAf
1484

67588.1
135361.1
cguu

135362.1
sagu

CfuG

CfaG

fGfU

fAfc

fguc

accA

ugac

fgAf

uuuL

acgu

96

usus

u

AD-
A-
csus
1395
A-
usGf
1485

67602.1
135391.1
gacu

135392.1
sucu

UfuC

UfuG

fGfG

fGfa

fucc

ccgA

aaag

faAf

acaL

guca

96

gsas

c

AD-
A-
uscs
1396
A-
asCf
1486

67570.1
135323.1
gguc

135324.1
sgac

CfaA

UfuC

fAfG

fGfu

facg

cuuU

aagu

fgGf

cguL

accg

96

asas

a

AD-
A-
csgs
1397
A-
usAf
1487

67553.1
135289.1
gucc

135290.1
scga

AfaA

CfuU

fGfA

fCfg

fcga

ucuU

aguc

fuGf

guaL

gacc

96

gsas

a

AD-
A-
gsas
1398
A-
usAf
1488

67612.1
135411.1
cgaa

135412.1
sagg

GfuC

CfaU

fGfU

fCfc

fgga

acgA

ugcc

fcUf

uuaL

ucgu

96

csus

u

AD-
A-
csus
1399
A-
asUf
1489

67525.1
135233.1
uggu

135234.1
sgaa

AfuG

GfcA

fUfU

fGfg

fccu

aacA

gcuu

fuAf

cauL

ccaa

96

gsgs

c

AD-
A-
gsgs
1400
A-
usAf
1490

67526.1
135235.1
ccuu

135236.1
sagg

AfuC

AfaG

fCfC

fGfa

fucc

gggA

uucc

fuAf

uuaL

aggc

96

csas

c

AD-
A-
asgs
1401
A-
usGf
1491

67592.1
135371.1
gagu

135372.1
suac

GfaG

GfuU

fUfG

fGfu

faca

cacU

acgu

fcAf

acaL

cucc

96

uscs

c

AD-
A-
gsus
1402
A-
usAf
1492

67578.1
135339.1
gagu

135340.1
sagg

GfaC

GfuA

fAfA

fCfg

fcgu

uugU

accc

fcAf

uuaL

cuca

96

csus

c

AD-
A-
usgs
1403
A-
usUf
1493

67555.1
135293.1
augc

135294.1
sgau

CfaA

GfgU

fAfA

fUfg

fcaa

uuuU

ccau

fgGf

caaL

cauc

96

asas

u

AD-
A-
csgs
1404
A-
usUf
1494

67577.1
135337.1
acau

135338.1
sgac

CfuG

UfuU

fCfC

fAfg

fcua

ggcA

aagu

fgAf

caaL

uguc

96

gsus

a

AD-
A-
ascs
1405
A-
usUf
1495

67594.1
135375.1
gaac

135376.1
scca

UfuU

CfaU

fCfU

fGfa

fuca

agaA

ugug

faGf

gaaL

uucg

96

usgs

g

AD-
A-
gscs
1406
A-
usAf
1496

67568.1
135319.1
acag

135320.1
sagg

GfgA

UfaG

fAfC

fAfg

fcuc

guuC

uacc

fcCf

uuaL

ugug

96

csas

g

AD-
A-
usgs
1407
A-
usAf
1497

67550.1
135283.1
cugg

135284.1
sagg

GfaG

CfaU

fAfG

fAfu

faua

cucU

ugcc

fcCf

uuaL

cagc

96

ascs

c

AD-
A-
usgs
1408
A-
usUf
1498

67586.1
135357.1
ggag

135358.1
scga

AfgA

AfgG

fUfA

fCfa

fugc

uauC

cuuc

fuCf

gapL

uccc

96

asgs

c

AD-
A-
asgs
1409
A-
usAf
1499

67576.1
135335.1
agau

135336.1
succ

AfuG

UfcG

fCfC

fAfa

fuuc

ggcA

gagg

fuAf

auaL

ucuc

96

uscs

c

AD-
A-
asus
1410
A-
usAf
1500

67563.1
135309.1
augc

135310.1
saau

CfuU

AfuC

fCfG

fCfu

fagg

cgaA

auau

fgGf

uuaL

caua

96

uscs

u

AD-
A-
usgs
1411
A-
asUf
1501

67552.1
135287.1
ccuu

135288.1
scca

CfgA

AfaU

fGfG

fAfu

faua

ccuC

uuug

fgAf

gauL

aggc

96

asus

a

AD-
A-
asus
1412
A-
usUf
1502

67608.1
135403.1
ucag

135404.1
scuc

GfuU

UfuC

fCfU

fCfa

fugg

agaA

aaga

fcCf

gapL

ugaa

96

usgs

c

AD-
A-
csas
1413
A-
usAf
1503

67593.1
135373.1
uccu

135374.1
succ

CfaG

AfuC

fAfA

fCfc

fggg

uucU

augg

fgAf

auaL

ggau

96

gsas

c

AD-
A-
cscs
1414
A-
asUf
1504

67609.1
135405.1
ugcc

135406.1
sgcu

CfuG

CfuC

fGfG

fAfu

faug

cccA

agag

fgGf

cauL

gcag

96

gsas

u

AD-
A-
gsas
1415
A-
usCf
1505

67597.1
135381.1
caaa

135382.1
suca

GfgU

UfgU

fGfG

fAfu

faua

ccaC

caug

fcUf

agaL

uugu

96

csus

u

AD-
A-
asas
1416
A-
usUf
1506

67587.1
135359.1
aggu

135360.1
sugc

GfgA

UfcA

fUfA

fUfg

fcau

uauC

gagc

fcAf

aaaL

ccuu

96

usgs

u

AD-
A-
gsus
1417
A-
asAf
1507

67559.1
135301.1
ggau

135302.1
sauc

AfcA

UfuG

fUfG

fCfu

fagc

cauG

aaga

fuAf

uuuL

ucca

96

cscs

u

AD-
A-
usgs
1418
A-
usAf
1508

67561.1
135305.1
gaua

135306.1
saau

CfaU

CfuU

fGfA

fGfc

fgca

ucaU

agau

fgUf

uuaL

aucc

96

ascs

c

AD-
A-
asus
1419
A-
usUf
1509

67551.1
135285.1
acau

135286.1
sgca

GfaG

AfaU

fCfA

fCfu

faga

ugcU

uuug

fcAf

caaL

ugua

96

uscs

c

AD-
A-
asgs
1420
A-
usAf
1510

67591.1
135369.1
caag

135370.1
sgca

AfuU

AfgU

fUfG

fUfg

fcaa

caaA

cuug

fuCf

cuaL

uugc

96

uscs

a

AD-
A-
csas
1421
A-
usGf
1511

67583.1
135351.1
agau

135352.1
suag

UfuG

CfaA

fCfA

fGfu

facu

ugcA

ugcu

faAf

acaL

ucuu

96

gscs

u

AD-
A-
usgs
1422
A-
usCf
1512

67585.1
135355.1
caac

135356.1
suaa

UfuG

UfgG

fCfU

fGfu

facc

agcA

cauu

faGf

agaL

uugc

96

asas

a

AD-
A-
asas
1423
A-
usAf
1513

67589.1
135363.1
cuug

135364.1
succ

CfuA

UfaA

fCfC

fUfg

fcau

gguA

uagg

fgCf

auaL

aagu

96

usgs

c

AD-
A-
gscs
1424
A-
usCf
1514

67595.1
135377.1
cauu

135378.1
sucu

GfcG

GfgA

fAfU

fCfa

fugu

aucG

ccag

fcAf

agaL

augg

96

csas

g

AD-
A-
gsas
1425
A-
usUf
1515

67580.1
135343.1
uugu

135344.1
scac

CfcA

CfaG

fGfA

fUfc

fgac

ucuG

uggu

fgAf

gauL

caau

96

csgs

c

AD-
A-
usgs
1426
A-
usAf
1516

67573.1
135329.1
guga

135330.1
sucu

CfaU

GfgA

fGfG

fAfg

fcuu

ccaU

ccag

fgUf

auaL

cacc

96

asgs

u

AD-
A-
cscs
1427
A-
usAf
1517

67600.1
135387.1
agau

135388.1
scau

AfuG

CfgU

fCfC

fCfg

fcga

ggcA

cgau

fuAf

guaL

ucug

96

gsas

a

AD-
A-
gsus
1428
A-
usAf
1518

67603.1
135393.1
gguu

135394.1
sggu

GfcA

CfaC

fGfU

fCfc

fggg

acuG

ugac

fcAf

cuaL

acca

96

csas

g

AD-
A-
asgs
1429
A-
usCf
1519

67598.1
135383.1
gucc

135384.1
suca

CfaA

CfuG

fAfU

fGfc

fgcc

auuU

agug

fgGf

agaL

gacc

96

usgs

g

AD-
A-
uscs
1430
A-
usAf
1520

67564.1
135311.1
acuu

135312.1
sgac

GfaG

UfcG

fGfA

fCfc

fggc

uccU

gagu

fcAf

cuaL

agug

96

ascs

u

AD-
A-
asgs
1431
A-
usCf
1521

67574.1
135331.1
ucua

135332.1
suga

GfcA

AfaG

fGfA

fAfa

fuuc

ucuG

uuuc

fcUf

agaL

agac

96

uscs

g

AD-
A-
asus
1432
A-
usUf
1522

67590.1
135365.1
ucuu

135366.1
suua

ticA

GfcA

fGfA

fCfc

fggu

ucuG

gcua

faAf

aaaL

agaa

96

uscs

u

AD-
A-
usus
1433
A-
asCf
1523

67572.1
135327.1
cuuu

135328.1
suuu

CfaG

AfgC

fAfG

fAfc

fgug

cucU

cuaa

fgAf

aguL

aaga

96

asus

c

AD-
A-
gsus
1434
A-
asAf
1524

67582.1
135349.1
gcua

135350.1
saga

AfaG

UfgG

fUfU

fGfa

fucc

aacU

cauc

fuUf

uuuL

agca

96

cscs

u

AD-
A-
uscs
1435
A-
usGf
1525

67607.1
135401.1
ccau

135402.1
suag

CfuU

CfuG

fUfG

fCfa

fugc

caaA

agcu

fgAf

acaL

uggg

96

asas

a

AD-
A-
csus
1436
A-
usAf
1526

67571.1
135325.1
gccu

135326.1
succ

GfuG

UfcC

fAfC

fAfc

fgug

gucA

gagg

fcAf

auaL

ggca

96

gsgs

g

AD-
A-
usgs
1437
A-
usCf
1527

67599.1
135385.1
ugac

135386.1
sugg

GfuG

GfaU

fGfA

fCfc

fgga

uccA

uccc

fcGf

agaL

ucac

96

asgs

g

AD-
A-
uscs
1438
A-
asUf
1528

67554.1
135291.1
ugag

135292.1
saaa

CfuG

AfcC

fAfG

fAfa

fuug

cucA

guuu

fgCf

uauL

ucag

96

asgs

g

AD-
A-
asgs
1439
A-
usAf
1529

67549.1
135281.1
uugg

135282.1
sgcu

UfuU

UfuU

fUfA

fCfa

fuga

uaaA

aaag

faCf

cuaL

caac

96

uscs

a

AD-
A-
usus
1440
A-
usCf
1530

67567.1
135317.1
gguu

135318.1
suag

UfuA

CfuU

fUfG

fUfu

faaa

cauA

agcu

faAf

agaL

acca

96

ascs

u

AD-
A-
gsgs
1441
A-
usUf
1531

67558.1
135299.1
uuuu

135300.1
sccu

AfuG

AfgC

fAfA

fUfu

faag

uucA

cuag

fuAf

gauL

aaac

96

csas

a

AD-
A-
gsus
1442
A-
usUf
1532

67569.1
135321.1
uuua

135322.1
succ

UfgA

UfaG

fAfA

fCfu

fagc

uuuC

uagg

faUf

aaaL

aaaa

96

cscs

a

AD-
A-
usus
1443
A-
usCf
1533

67548.1
135279.1
uuau

135280.1
suuc

GfaA

CfuA

fAfA

fGfc

fgcu

uuuU

agga

fcAf

agaL

uaaa

96

ascs

c

AD-
A-
usas
1444
A-
usUf
1534

67566.1
135315.1
ugaa

135316.1
sugc

AfaG

UfuC

fCfU

fCfu

fagg

agcU

aagc

fuUf

aauL

ucau

96

asas

a

AD-
A-
csgs
1445
A-
usCf
1535

67613.1
135413.1
uuaa

135414.1
scca

UfuC

AfcC

fAfG

fAfg

fcug

cugA

guug

faUf

ggaL

uaac

96

gscs

a

AD-
A-
gsus
1446
A-
usUf
1536

67610.1
135407.1
uaau

135408.1
sccc

ticA

AfaC

fGfC

fCfa

fugg

gcuG

uugg

faAf

gauL

uuaa

96

csgs

c

AD-
A-
asgs
1447
A-
usGf
1537

67556.1
135295.1
cugg

135296.1
sugu

UfuG

CfaU

fGfG

fUfu

faaa

cccA

ugac

faCf

acaL

cagc

96

usgs

a

AD-
A-
cscs
1448
A-
asAf
1538

67581.1
135345.1
uauu

135346.1
scag

AfaU

UfcU

fGfG

fGfa

fuca

ccaU

gacu

fuAf

guuL

auag

96

gsgs

c

AD-
A-
csus
1449
A-
usAf
1539

67560.1
135303.1
auua

135304.1
saca

AfuG

GfuC

fGfU

fUfg

fcag

accA

acug

fuUf

uuaL

aaua

96

gsgs

g

AD-
A-
gscs
1450
A-
usAf
1540

67596.1
135379.1
uggc

135380.1
saga

CfcA

UfcA

fUfG

fCfa

fugu

cauG

gauc

fgGf

uuaL

ccag

96

cscs

u

AD-
A-
usgs
1451
A-
usAf
1541

67557.1
135297.1
gccc

135298.1
scaa

AfuG

GfaU

fUfG

fCfa

fuga

cacA

ucuu

fuGf

guaL

ggcc

96

asgs

c

AD-
A-
cscs
1452
A-
usUf
1542

67584.1
135353.1
uaac

135354.1
saaa

UfaA

CfaU

fAfA

fUfa

fuaa

uuuU

uguu

faGf

uaaL

uuag

96

gsus

g

AD-
A-
usus
1453
A-
asAf
1543

67575.1
135333.1
accu

135334.1
suac

GfuU

AfaA

fGfA

fAfu

fauu

ucaA

uugu

fcAf

auuL

ggua

96

ascs

a

AD-
A-
ascs
1454
A-
asUf
1544

67605.1
135397.1
cugu

135398.1
saau

UfgA

AfcA

fAfU

fAfa

fuuu

auuC

guau

faAf

uauL

cagg

96

usas

a

AD-
A-
usgs
1455
A-
usUf
1545

67562.1
135307.1
uauu

135308.1
scac

AfuG

UfgA

fUfG

fUfu

faau

cacA

cagu

fuAf

gaaL

auac

96

asas

a

AD-
A-
usas
1456
A-
asAf
1546

67606.1
135399.1
ugug

135400.1
scau

AfaU

CfuC

fCfA

fAfc

fgug

ugaU

agau

fuCf

guuL

acau

96

asas

u

AD-
A-
gsas
1457
A-
asAf
1547

67604.1
135395.1
uguu

135396.1
sggc

AfgU

UfuA

fAfG

fUfu

faau

cuaC

aagc

fuAf

cuuL

acau

96

csus

c

AD-
A-
asus
1458
A-
usAf
1548

67565.1
135313.1
guua

135314.1
sagg

GfuA

CfuU

fGfA

fAfu

faua

ucuA

agcc

fcUf

uuaL

aaca

96

uscs

u

AD-
A-
usas
1459
A-
usCf
1549

67529.1
135241.1
uaau

135242.1
scau

GfgA

GfaG

fGfA

fGfa

fucc

ucuC

ucau

fcAf

ggaL

uuau

96

ascs

g

AD-
A-
gsus
1460
A-
usUf
1550

67533.1
135249.1
gucu

135250.1
sugg

GfaG

AfaU

fUfU

fGfg

fcca

aacU

uucc

fcAf

aaaL

gaca

96

cscs

a

AD-
A-
asgs
1461
A-
usAf
1551

67537.1
135257.1
ucgu

135258.1
scac

GfgA

CfaG

fUfG

fGfg

fccc

cauC

uggu

fcAf

guaL

cgac

96

usus

c

AD-
A-
usgs
1462
A-
usUf
1552

67546.1
135275.1
cuau

135276.1
scca

CfaA

GfgU

fGfG

fAfc

fgua

ccuU

ccug

fgAf

gauL

uagc

96

ascs

a

AD-
A-
uscs
1463
A-
usAf
1553

67547.1
135277.1
ccag

135278.1
suuc

GfuU

GfgG

fUfG

fCfa

fugc

caaA

ccga

fcCf

auaL

uggg

96

asus

g

AD-
A-
cscs
1464
A-
usUf
1554

67543.1
135269.1
aggu

135270.1
scau

UfuG

UfcG

fUfG

fGfg

fccc

cacA

gaau

faAf

gaaL

ccug

96

gsgs

a

AD-
A-
usgs
1465
A-
usAf
1555

67541.1
135265.1
gagc

135266.1
sucu

AfaC

AfgA

fAfG

fCfa

fugu

cugU

cuag

fuGf

auaL

cucc

96

asgs

a

AD-
A-
csus
1466
A-
usUf
1556

67535.1
135253.1
uuug

135254.1
succ

GfaG

UfaG

fGfC

fCfu

fagc

gccU

uagg

fcCf

aauL

aaaa

96

gsus

a

AD-
A-
asas
1467
A-
usAf
1557

67530.1
135243.1
gaca

135244.1
saac

AfuG

AfcC

fAfU

fAfa

fuug

aucA

gugu

fuUf

uuaL

gucu

96

usus

g

AD-
A-
gsas
1468
A-
usCf
1558

67542.1
135267.1
caau

135268.1
suaa

GfaU

AfcA

fUfU

fCfc

fggu

aaaU

guuu

fcAf

agaL

uugu

96

csus

u

AD-
A-
csas
1469
A-
usUf
1559

67528.1
135239.1
auga

135240.1
sucu

UfuU

AfaA

fGfG

fCfa

fugu

ccaA

uuag

faUf

aaaL

cauu

96

gsus

c

AD-
A-
usgs
1470
A-
asAf
1560

67527.1
135237.1
ccag

135238.1
sagu

AfuA

AfaU

fAfC

fAfa

fuua

guuA

uuac

fuCf

uuuL

uggc

96

asgs

g

AD-
A-
ascs
1471
A-
asUf
1561

67544.1
135271.1
accu

135272.1
suag

UfuG

UfaA

fGfC

fGfa

fucu

gccA

uacu

faAf

aauL

ggug

96

uscs

c

AD-
A-
csus
1472
A-
usAf
1562

67532.1
135247.1
ggcu

135248.1
suac

CfcA

AfaA

fAfA

fGfa

fucu

uuuG

uugu

fgAf

auaL

gcca

96

gsus

g

AD-
A-
usgs
1473
A-
usUf
1563

67534.1
135251.1
gcuc

135252.1
saua

CfaA

CfaA

fAfU

fAfg

fcuu

auuU

ugua

fgGf

uaaL

agcc

96

asgs

u

AD-
A-
cscs
1474
A-
usAf
1564

67538.1
135259.1
aaau

135260.1
suga

CfuU

CfuA

fUfG

fUfa

fuau

caaA

aguc

fgAf

auaL

uuug

96

gsas

g

AD-
A-
asgs
1475
A-
usAf
1565

67545.1
135273.1
agac

135274.1
sgcc

AfaA

UfaG

fGfU

fAfc

fguc

acuU

uagg

fuGf

cuaL

ucuc

96

usas

g

AD-
A-
asas
1476
A-
usUf
1566

67539.1
135261.1
gugu

135262.1
scug

CfuA

UfgU

fGfG

fAfg

fcua

ccuA

caca

fgAf

gaaL

cacu

96

usus

g

AD-
A-
asgs
1477
A-
usAf
1567

67540.1
135263.1
aaac

135264.1
saag

UfuC

CfaA

fUfG

fGfg

fccu

cagA

ugcu

faGf

uuaL

uuuc

96

usas

c

AD-
A-
gsas
1478
A-
usGf
1568

67531.1
135245.1
agga

135246.1
sugu

UfuG

AfuC

fAfA

fCfa

fugg

uucA

auac

faUf

acaL

ccuu

96

csus

g

AD-
A-
gsgs
1479
A-
usUf
1569

67536.1
135255.1
auug

135256.1
sugg

AfaU

UfgU

fGfG

fAfu

faua

ccaU

cacc

fuCf

aaaL

aauc

96

csus

u

TABLE 9

PNPLA3 mRNA Target Sequences of Modifed

PNPLA3 Agents in Table 8

Anti-
mRNA

Sense-
ssense
target
SEQ

Duplex
Oligo
Oligo
se-
ID

Name
Name
Name
quence
NO

AD-
A-
A-
GCGGC
1570

67524.1
135231.1
135232.1
UUCCU

GGGCU

UCUAC

CAC

AD-
A-
A-
UCUUG
1571

67611.1
135409.1
135410.1
UGCGG

AAGGC

CAGGA

GUC

AD-
A-
A-
GGAAG
1572

67601.1
135389.1
135390.1
GCCAG

GAGUC

GGAAC

AUU

AD-
A-
A-
GAAGG
1573

67579.1
135341.1
135342.1
CCAGG

AGUCG

GAACA

UUG

AD-
A-
A-
AAAAC
1574

67588.1
135361.1
135362.1
GUUCU

GGUGU

CUGAC

UUU

AD-
A-
A-
GUCUG
1575

67602.1
135391.1
135392.1
ACUUU

CGGUC

CAAAG

ACG

AD-
A-
A-
UUUCG
1576

67570.1
135323.1
135324.1
GUCCA

AAGAC

GAAGU

CGU

AD-
A-
A-
UUCGG
1577

67553.1
135289.1
135290.1
UCCAA

AGACG

AAGUC

GUG

AD-
A-
A-
AAGAC
1578

67612.1
135411.1
135412.1
GAAGU

CGUGG

AUGCC

UUG

AD-
A-
A-
GCCUU
1579

67525.1
135233.1
135234.1
GGUAU

GUUCC

UGCUU

CAU

AD-
A-
A-
GUGGC
1580

67526.1
135235.1
135236.1
CUUAU

CCCUC

CUUCC

UUC

AD-
A-
A-
GGAGG
1581

67592.1
135371.1
135372.1
AGUGA

GUGAC

AACGU

ACC

AD-
A-
A-
GAGUG
1582

67578.1
135339.1
135340.1
AGUGA

CAACG

UACCC

UUC

AD-
A-
A-
AUUGA
1583

67555.1
135293.1
135294.1
UGCCA

AAACA

ACCAU

CAC

AD-
A-
A-
UACGA
1584

67577.1
135337.1
135338.1
CAUCU

GCCCU

AAAGU

CAA

AD-
A-
A-
CCACG
1585

67594.1
135375.1
135376.1
AACUU

UCUUC

AUGUG

GAC

AD-
A-
A-
CUGCA
1586

67568.1
135319.1
135320.1
CAGGG

AACCU

CUACC

UUC

AD-
A-
A-
GGUGC
1587

67550.1
135283.1
135284.1
UGGGA

GAGAU

AUGCC

UUC

AD-
A-
A-
GCUGG
1588

67586.1
135357.1
135358.1
GAGAG

AUAUG

CCUUC

GAG

AD-
A-
A-
GGAGA
1589

67576.1
135335.1
135336.1
GAUAU

GCCUU

CGAGG

AUA

AD-
A-
A-
AGAUA
1590

67563.1
135309.1
135310.1
UGCCU

UCGAG

GAUAU

UUG

AD-
A-
A-
UAUGC
1591

67552.1
135287.1
135288.1
CUUCG

AGGAU

AUUUG

GAU

AD-
A-
A-
GCAUU
1592

67608.1
135403.1
135404.1
CAGGU

UCUUG

GAAGA

GAA

AD-
A-
A-
GUCAU
1593

67593.1
135373.1
135374.1
CCUCA

GAAGG

GAUGG

AUC

AD-
A-
A-
AUCCU
1594

67609.1
135405.1
135406.1
GCCCU

GGGAU

GAGAG

CAU

AD-
A-
A-
AAGAC
1595

67597.1
135381.1
135382.1
AAAGG

UGGAU

ACAUG

AGC

AD-
A-
A-
ACAAA
1596

67587.1
135359.1
135360.1
GGUGG

AUACA

UGAGC

AAG

AD-
A-
A-
AGGUG
1597

67559.1
135301.1
135302.1
GAUAC

AUGAG

CAAGA

UUU

AD-
A-
A-
GGUGG
1598

67561.1
135305.1
135306.1
AUACA

UGAGC

AAGAU

UUG

AD-
A-
A-
GGAUA
1599

67551.1
135285.1
135286.1
CAUGA

GCAAG

AUUUG

CAA

AD-
A-
A-
UGAGC
1600

67591.1
135369.1
135370.1
AAGAU

UUGCA

ACUUG

CUA

AD-
A-
A-
AGCAA
1601

67583.1
135351.1
135352.1
GAUUU

GCAAC

UUGCU

ACC

AD-
A-
A-
UUUGC
1602

67585.1
135355.1
135356.1
AACUU

GCUAC

CCAUU

AGG

AD-
A-
A-
GCAAC
1603

67589.1
135363.1
135364.1
UUGCU

ACCCA

UUAGG

AUA

AD-
A-
A-
CUGCC
1604

67595.1
135377.1
135378.1
AUUGC

GAUUG

UCCAG

AGA

AD-
A-
A-
GCGAU
1605

67580.1
135343.1
135344.1
UGUCC

AGAGA

CUGGU

GAC

AD-
A-
A-
ACUGG
1606

67573.1
135329.1
135330.1
UGACA

UGGCU

UCCAG

AUA

AD-
A-
A-
UUCCA
1607

67600.1
135387.1
135388.1
GAUAU

GCCCG

ACGAU

GUC

AD-
A-
A-
CUGUG
1608

67603.1
135393.1
135394.1
GUUGC

AGUGG

GUGAC

CUC

AD-
A-
A-
CCAGG
1609

67598.1
135383.1
135384.1
UCCCA

AAUGC

CAGUG

AGC

AD-
A-
A-
AGUCA
1610

67564.1
135311.1
135312.1
CUUGA

GGAGG

CGAGU

CUA

AD-
A-
A-
CGAGU
1611

67574.1
135331.1
135332.1
CUAGC

AGAUU

CUUUC

AGA

AD-
A-
A-
AGAUU
1612

67590.1
135365.1
135366.1
CUUUC

AGAGG

UGCUA

AAG

AD-
A-
A-
GAUUC
1613

67572.1
135327.1
135328.1
UUUCA

GAGGU

GCUAA

AGU

AD-
A-
A-
AGGUG
1614

67582.1
135349.1
135350.1
CUAAA

GUUUC

CCAUC

UUU

AD-
A-
A-
UUUCC
1615

67607.1
135401.1
135402.1
CAUCU

UUGUG

CAGCU

ACC

AD-
A-
A-
CCCUG
1616

67571.1
135325.1
135326.1
CCUGU

GACGU

GGAGG

AUC

AD-
A-
A-
CCUGU
1617

67599.1
135385.1
135386.1
GACGU

GGAGG

AUCCC

AGC

AD-
A-
A-
CCUCU
1618

67554.1
135291.1
135292.1
GAGCU

GAGUU

GGUUU

UAU

AD-
A-
A-
UGAGU
1619

67549.1
135281.1
135282.1
UGGUU

UUAUG

AAAAG

CUA

AD-
A-
A-
AGUUG
1620

67567.1
135317.1
135318.1
GUUUU

AUGAA

AAGCU

AGG

AD-
A-
A-
UUGGU
1621

67558.1
135299.1
135300.1
UUUAU

GAAAA

GCUAG

GAA

AD-
A-
A-
UGGUU
1622

67569.1
135321.1
135322.1
UUAUG

AAAAG

CUAGG

AAG

AD-
A-
A-
GGUUU
1623

67548.1
135279.1
135280.1
UAUGA

AAAGC

UAGGA

AGC

AD-
A-
A-
UUUAU
1624

67566.1
135315.1
135316.1
GAAAA

GCUAG

GAAGC

AAC

AD-
A-
A-
UGCGU
1625

67613.1
135413.1
135414.1
UAAUU

CAGCU

GGUUG

GGA

AD-
A-
A-
GCGUU
1626

67610.1
135407.1
135408.1
AAUUC

AGCUG

GUUGG

GAA

AD-
A-
A-
UCAGC
1627

67556.1
135295.1
135296.1
UGGUU

GGGAA

AUGAC

ACC

AD-
A-
A-
GCCCU
1628

67581.1
135345.1
135346.1
AUUAA

UGGUC

AGACU

GUU

AD-
A-
A-
CCCUA
1629

67560.1
135303.1
135304.1
UUAAU

GGUCA

GACUG

UUC

AD-
A-
A-
AGGCU
1630

67596.1
135379.1
135380.1
GGCCC

AUGUG

UGAUC

UUG

AD-
A-
A-
GCUGG
1631

67557.1
135297.1
135298.1
CCCAU

GUGUG

AUCUU

GUG

AD-
A-
A-
CACCU
1632

67584.1
135353.1
135354.1
AACUA

AAAUA

AUGUU

UAA

AD-
A-
A-
UGUUA
1633

67575.1
135333.1
135334.1
CCUGU

UGAAU

UUUGU

AUU

AD-
A-
A-
UUACC
1634

67605.1
135397.1
135398.1
UGUUG

AAUUU

UGUAU

UAU

AD-
A-
A-
UUUGU
1635

67562.1
135307.1
135308.1
AUUAU

GUGAA

UCAGU

GAG

AD-
A-
A-
AUUAU
1636

67606.1
135399.1
135400.1
GUGAA

UCAGU

GAGAU

GUU

AD-
A-
A-
GAGAU
1637

67604.1
135395.1
135396.1
GUUAG

UAGAA

UAAGC

CUU

AD-
A-
A-
AGAUG
1638

67565.1
135313.1
135314.1
UUAGU

AGAAU

AAGCC

UUA

AD-
A-
A-
CGUAU
1639

67529.1
135241.1
135242.1
AAUGG

AGAUC

CUCAU

GGA

AD-
A-
A-
UGGUG
1640

67533.1
135249.1
135250.1
UCUGA

GUUCC

AUUCC

AAA

AD-
A-
A-
GAAGU
1641

67537.1
135257.1
135258.1
CGUGG

AUGCC

CUGGU

GUG

AD-
A-
A-
UGUGC
1642

67546.1
135275.1
135276.1
UAUCA

AGGGU

ACCUG

GAC

AD-
A-
A-
CAUCC
1643

67547.1
135277.1
135278.1
CAGGU

UUGUG

CCCGA

AUG

AD-
A-
A-
UCCCA
1644

67543.1
135269.1
135270.1
GGUUU

GUGCC

CGAAU

GAC

AD-
A-
A-
UCUGG
1645

67541.1
135265.1
135266.1
AGCAA

CAGUG

UCUAG

AUG

AD-
A-
A-
UACUU
1646

67535.1
135253.1
135254.1
UUGGA

GGCAG

CUAGG

AAG

AD-
A-
A-
CAAAG
1647

67530.1
135243.1
135244.1
ACAAU

GAUUU

GGUGU

UUA

AD-
A-
A-
AAGAC
1648

67542.1
135267.1
135268.1
AAUGA

UUUGG

UGUUU

AGA

AD-
A-
A-
GACAA
1649

67528.1
135239.1
135240.1
UGAUU

UGGUG

UUUAG

AAA

AD-
A-
A-
CCUGC
1650

67527.1
135237.1
135238.1
CAGAU

AACUU

AUUAC

UUU

AD-
A-
A-
GGACA
1651

67544.1
135271.1
135272.1
CCUUU

GGCUC

UUACU

AAU

AD-
A-
A-
CACUG
1652

67532.1
135247.1
135248.1
GCUCC

AAAUC

UUUGU

AUA

AD-
A-
A-
ACUGG
1653

67534.1
135251.1
135252.1
CUCCA

AAUCU

UUGUA

UAG

AD-
A-
A-
CUCCA
1654

67538.1
135259.1
135260.1
AAUCU

UUGUA

UAGUC

AUC

AD-
A-
A-
CUAGA
1655

67545.1
135273.1
135274.1
GACAA

AGUGU

CUAGG

CUA

AD-
A-
A-
CAAAG
1656

67539.1
135261.1
135262.1
UGUCU

AGGCU

ACACA

GAA

AD-
A-
A-
GUAGA
1657

67540.1
135263.1
135264.1
AACUU

CUGCC

UUGCU

UUG

AD-
A-
A-
CAGAA
1658

67531.1
135245.1
135246.1
GGAUU

GAAUG

GAUAC

ACC

AD-
A-
A-
AAGGA
1659

67536.1
135255.1
135256.1
UUGAA

UGGAU

ACACC

AAA

TABLE 10

Hep3B PNPLA3 endogenous in vitro 10 nM and

0.1 nM single dose screen

Duplex
10
10
0.1
0.1

Name
nM_AVG
nM_STDEV
nM_AVG
nM_STDEV

AD-67524.1
85.34
6.47
90.33
14.17

AD-67611.1
111.68
23.20
95.28
9.27

AD-67601.1
86.74
11.59
86.84
15.67

AD-67579.1
89.60
12.00
58.82
15.51

AD-67588.1
57.73
16.50
64.15
8.44

AD-67602.1
66.35
12.49
86.76
8.21

AD-67570.1
72.72
8.21
85.53
5.27

AD-67553.1
67.90
6.88
84.25
11.40

AD-67612.1
62.03
12.34
51.52
5.24

AD-67525.1
42.50
9.07
65.77
14.98

AD-67526.1
45.14
9.69
58.35
4.26

AD-67592.1
55.32
9.06
58.01
6.50

AD-67578.1
51.16
8.74
53.17
15.71

AD-67555.1
92.88
23.69
66.11
10.08

AD-67577.1
53.93
9.32
55.41
5.82

AD-67594.1
79.39
12.41
78.57
7.33

AD-67568.1
43.12
7.69
65.24
11.56

AD-67550.1
62.65
16.23
87.64
22.99

AD-67586.1
57.51
11.23
66.30
21.67

AD-67576.1
62.33
9.41
66.43
17.91

AD-67563.1
56.23
17.97
69.60
6.43

AD-67552.1
55.69
5.10
103.09
5.25

AD-67608.1
51.30
15.89
53.54
16.44

AD-67593.1
52.04
9.82
69.34
7.89

AD-67609.1
90.41
32.12
73.63
16.54

AD-67597.1
78.98
19.93
90.94
16.10

AD-67587.1
81.37
16.51
70.07
28.64

AD-67559.1
71.11
9.40
96.14
12.25

AD-67561.1
50.85
14.84
56.18
15.19

AD-67551.1
37.30
6.63
53.00
4.52

AD-67591.1
70.98
19.00
93.65
11.21

AD-67583.1
65.57
7.72
80.60
14.05

AD-67585.1
53.90
14.18
52.77
10.67

AD-67589.1
43.29
5.45
54.29
4.43

AD-67595.1
83.09
44.03
88.45
13.90

AD-67580.1
88.42
14.74
74.18
8.01

AD-67573.1
60.57
4.91
71.22
17.26

AD-67600.1
70.88
0.97
65.57
10.49

AD-67603.1
100.97
25.43
86.68
16.12

AD-67598.1
55.25
6.91
79.47
10.06

AD-67564.1
65.67
14.01
60.23
4.86

AD-67574.1
63.24
16.91
68.91
19.35

AD-67590.1
70.11
7.76
68.94
18.75

AD-67572.1
86.54
6.37
95.11
36.91

AD-67582.1
57.31
14.76
52.76
8.24

AD-67607.1
59.03
14.94
59.28
10.58

AD-67571.1
99.63
15.80
89.53
6.64

AD-67599.1
94.78
19.21
87.91
7.53

AD-67554.1
36.53
8.09
56.06
5.32

AD-67549.1
56.20
20.65
56.90
10.27

AD-67567.1
57.81
4.61
67.97
17.13

AD-67558.1
57.17
10.26
60.10
11.12

AD-67569.1
66.43
25.81
58.49
14.52

AD-67548.1
52.14
8.72
75.41
15.44

AD-67566.1
54.88
11.91
51.93
11.84

AD-67613.1
83.78
26.96
79.37
8.59

AD-67610.1
78.50
18.94
80.88
11.97

AD-67556.1
87.08
5.39
87.94
8.28

AD-67581.1
52.21
11.55
84.89
7.12

AD-67560.1
51.65
4.09
67.85
6.59

AD-67596.1
82.71
20.80
76.57
11.58

AD-67557.1
56.15
8.28
90.70
5.11

AD-67584.1
42.16
6.42
38.63
13.85

AD-67575.1
42.62
11.19
54.35
9.20

AD-67605.1
43.75
11.62
59.95
7.68

AD-67562.1
73.26
11.12
72.58
11.11

AD-67606.1
86.42
38.80
75.45
12.67

AD-67604.1
64.47
6.80
72.33
10.76

AD-67565.1
49.43
3.37
54.34
12.25

AD-67529.1
96.11
23.73
104.54
5.56

AD-67533.1
91.29
27.25
102.72
10.83

AD-67537.1
96.12
30.20
90.92
17.55

AD-67546.1
117.18
35.85
90.75
10.80

AD-67547.1
109.66
23.27
110.07
17.90

AD-67543.1
106.67
27.98
103.10
22.41

AD-67541.1
112.89
34.51
105.50
18.29

AD-67535.1
95.95
17.30
111.96
8.37

AD-67530.1
86.64
13.15
89.64
10.56

AD-67542.1
108.30
12.22
111.03
18.93

AD-67528.1
86.06
15.40
100.52
11.52

AD-67527.1
94.22
9.43
103.95
8.31

AD-67544.1
95.63
16.01
94.25
5.66

AD-67532.1
96.24
10.13
114.20
14.38

AD-67534.1
104.27
20.55
101.24
14.18

AD-67538.1
108.29
29.79
99.37
10.01

AD-67545.1
110.68
11.06
143.56
45.88

AD-67539.1
106.92
43.45
107.56
15.77

AD-67540.1
104.01
18.83
105.58
12.67

AD-67531.1
117.06
37.65
102.32
27.15

AD-67536.1
104.51
7.42
110.11
14.23

TABLE 11

Cynomolgus monkey PNPLA3 endogenous in vitro 10 nM and

0.1 nM single dose screen

Duplex
10
10
0.1
0.1

Name
nM_AVG
nM_STDEV
nM_AVG
nM_STDEV

AD-67524.1
64.22
12.50
98.57
57.56

AD-67611.1
201.95
55.02
147.71
34.65

AD-67601.1
106.76
23.66
104.01
20.80

AD-67579.1
69.15
24.02
39.69
7.49

AD-67588.1
34.18
13.04
58.34
19.48

AD-67602.1
64.07
21.95
114.16
40.22

AD-67570.1
45.66
21.83
92.73
22.46

AD-67553.1
61.54
20.51
78.87
33.03

AD-67612.1
49.05
10.63
68.98
21.48

AD-67525.1
58.61
6.56
83.50
29.86

AD-67526.1
48.75
19.00
81.70
44.79

AD-67592.1
54.34
23.45
107.45
52.70

AD-67578.1
54.22
18.19
62.05
18.44

AD-67555.1
83.45
13.63
96.21
32.86

AD-67577.1
41.40
13.97
50.80
20.40

AD-67594.1
71.17
26.23
90.30
12.23

AD-67568.1
28.74
8.05
56.57
12.90

AD-67550.1
67.27
14.09
102.11
22.04

AD-67586.1
44.83
10.13
52.06
1.96

AD-67576.1
61.04
36.58
78.16
7.18

AD-67563.1
85.83
27.55
88.34
7.26

AD-67552.1
70.65
36.42
112.67
14.77

AD-67608.1
65.16
37.26
90.87
21.05

AD-67593.1
72.95
19.92
108.58
27.09

AD-67609.1
83.80
52.06
113.25
23.43

AD-67597.1
57.86
7.16
101.52
29.68

AD-67587.1
71.36
33.38
83.46
28.71

AD-67559.1
38.13
5.57
85.54
20.52

AD-67561.1
49.61
17.03
75.51
35.59

AD-67551.1
24.74
13.01
57.84
19.55

AD-67591.1
65.58
11.64
70.61
18.06

AD-67583.1
35.16
12.01
56.71
13.29

AD-67585.1
51.64
38.68
91.09
23.58

AD-67589.1
30.43
8.50
55.59
15.49

AD-67595.1
64.53
12.69
108.07
61.59

AD-67580.1
52.22
14.63
59.80
19.21

AD-67573.1
47.55
19.02
69.12
8.02

AD-67600.1
55.58
11.69
92.41
26.52

AD-67603.1
119.04
50.54
152.95
37.00

AD-67598.1
51.72
17.51
84.34
25.38

AD-67564.1
58.62
27.17
77.33
37.58

AD-67574.1
33.51
14.78
45.90
17.45

AD-67590.1
40.45
9.84
56.63
12.25

AD-67572.1
47.06
14.49
77.89
27.67

AD-67582.1
27.10
5.89
49.41
18.11

AD-67607.1
43.61
8.27
72.35
13.09

AD-67571.1
109.27
56.41
69.47
23.50

AD-67599.1
83.03
58.74
83.94
15.01

AD-67554.1
19.86
10.03
85.24
13.88

AD-67549.1
38.63
14.53
94.17
23.93

AD-67567.1
31.60
11.57
71.66
11.57

AD-67558.1
39.31
19.51
67.91
23.15

AD-67569.1
35.42
13.52
37.45
9.62

AD-67548.1
84.14
21.27
83.38
26.65

AD-67566.1
26.85
5.90
47.24
9.79

AD-67613.1
90.32
43.98
110.21
22.37

AD-67610.1
76.90
29.15
116.95
25.59

AD-67556.1
99.65
38.94
78.32
27.42

AD-67581.1
31.34
9.79
69.04
11.53

AD-67560.1
25.86
10.00
49.96
14.82

AD-67596.1
70.39
22.12
83.45
32.21

AD-67557.1
30.36
3.67
77.23
33.61

AD-67584.1
30.34
10.44
35.30
9.75

AD-67575.1
29.04
9.17
48.86
8.65

AD-67605.1
62.92
35.30
97.67
46.22

AD-67562.1
149.14
76.05
137.22
31.54

AD-67606.1
53.08
12.65
76.76
17.13

AD-67604.1
45.22
6.49
90.48
27.49

AD-67565.1
58.35
24.21
60.94
28.29

AD-67529.1
158.59
45.47
150.25
53.50

AD-67533.1
142.31
43.60
146.81
39.93

AD-67537.1
141.43
43.53
173.26
50.75

AD-67546.1
176.38
88.77
147.25
35.28

AD-67547.1
160.76
104.70
125.22
35.52

AD-67543.1
117.94
26.94
178.90
44.99

AD-67541.1
171.26
52.40
148.66
41.86

AD-67535.1
117.80
12.65
154.87
34.75

AD-67530.1
130.28
46.60
124.85
37.32

AD-67542.1
130.98
44.83
158.70
46.06

AD-67528.1
131.06
56.44
149.25
40.56

AD-67527.1
128.94
29.24
154.29
24.13

AD-67544.1
122.80
57.17
155.85
21.25

AD-67532.1
73.68
20.38
130.31
58.83

AD-67534.1
173.61
86.25
174.54
61.47

AD-67538.1
153.55
53.00
170.55
45.06

AD-67545.1
139.49
20.95
128.18
37.75

AD-67539.1
258.50
123.06
144.40
39.80

AD-67540.1
139.83
54.43
134.33
34.88

AD-67531.1
131.80
41.72
155.34
63.49

AD-67536.1
143.28
42.58
150.88
41.76

TABLE 12

Cynomolgus monkey PNPLA3 endogenous in

vitro dose response screen

Duplex Name
IC₅₀(nM)

AD-67525.1
0.003

AD-67526.1
0.005

AD-67551.1
0.298

AD-67554.1
0.003

AD-67560.1
0.034

AD-67568.1
0.049

AD-67575.1
0.317

AD-67577.1
0.001

AD-67578.1
0.001

AD-67581.1
0.081

AD-67582.1
0.058

AD-67584.1
0.001

AD-67585.1
0.038

AD-67592.1
0.216

AD-67605.1
0.123

AD-67612.1
1.381

TABLE 13

Mouse PNPLA3 endogenous in vitro 10 nM and

0.1 nM single dose screen

Duplex
10
10
0.1
0.1

Name
nM_AVG
nM_STDEV
nM_AVG
nM_STDEV

AD-67524.1
59.20
19.07
172.14
102.64

AD-67611.1
101.53
39.04
166.84
16.96

AD-67601.1
106.69
30.70
133.40
10.57

AD-67579.1
93.33
23.59
111.59
17.65

AD-67588.1
87.03
12.30
114.60
9.93

AD-67602.1
99.22
9.70
127.47
8.95

AD-67570.1
19.23
14.62
74.87
29.73

AD-67553.1
15.43
7.94
60.49
46.00

AD-67612.1
28.43
11.87
91.40
64.47

AD-67525.1
39.75
26.63
140.88
39.79

AD-67526.1
16.16
5.74
97.66
43.58

AD-67592.1
25.04
16.02
117.35
19.59

AD-67578.1
27.07
18.71
138.94
57.91

AD-67555.1
43.71
36.83
148.89
53.90

AD-67577.1
35.95
24.04
106.43
62.19

AD-67594.1
118.99
24.43
118.98
22.56

AD-67568.1
22.01
15.09
104.04
28.73

AD-67550.1
128.32
33.32
153.22
21.77

AD-67586.1
74.59
3.44
106.75
20.87

AD-67576.1
79.48
5.82
129.09
31.89

AD-67563.1
141.90
59.30
132.24
34.38

AD-67552.1
143.18
49.65
124.20
18.26

AD-67608.1
154.76
66.58
190.93
42.10

AD-67593.1
112.21
46.12
116.10
14.11

AD-67609.1
164.89
46.54
171.23
27.85

AD-67597.1
145.67
37.55
143.83
34.86

AD-67587.1
102.09
20.42
106.63
18.56

AD-67559.1
126.57
13.72
137.00
24.64

AD-67561.1
121.82
26.66
151.62
35.30

AD-67551.1
152.46
60.75
133.95
21.61

AD-67591.1
166.93
65.07
145.36
28.46

AD-67583.1
127.60
32.09
142.44
42.81

AD-67585.1
99.84
23.69
148.35
48.23

AD-67589.1
106.32
18.73
156.26
53.14

AD-67595.1
105.40
18.78
123.24
28.02

AD-67580.1
105.49
27.33
127.82
10.97

AD-67573.1
17.45
7.76
126.15
41.24

AD-67600.1
86.36
21.17
126.08
20.47

AD-67603.1
104.95
35.50
142.30
15.52

AD-67598.1
95.85
23.74
172.20
35.33

AD-67564.1
109.00
17.65
121.28
23.44

AD-67574.1
86.31
11.33
131.22
27.38

AD-67590.1
136.21
58.52
123.09
14.38

AD-67572.1
139.23
24.55
115.97
17.05

AD-67582.1
126.01
33.04
165.25
40.55

AD-67607.1
94.42
35.45
121.14
19.57

AD-67571.1
112.27
26.92
120.03
21.17

AD-67599.1
171.97
24.42
113.09
20.69

AD-67554.1
125.76
26.14
118.60
35.12

AD-67549.1
119.56
65.06
150.20
16.69

AD-67567.1
133.44
93.04
144.51
44.20

AD-67558.1
158.66
58.69
115.42
22.09

AD-67569.1
123.35
42.23
150.79
30.96

AD-67548.1
130.24
31.18
126.72
29.14

AD-67566.1
97.88
15.18
161.34
45.64

AD-67613.1
133.15
53.50
164.06
35.86

AD-67610.1
125.68
41.94
123.89
17.82

AD-67556.1
129.25
45.87
156.50
34.41

AD-67581.1
81.75
13.75
127.10
26.78

AD-67560.1
119.69
56.51
127.65
12.06

AD-67596.1
104.08
30.46
128.33
24.04

AD-67557.1
78.91
9.50
127.50
9.39

AD-67584.1
131.87
19.40
128.29
20.96

AD-67575.1
124.30
43.53
151.26
44.05

AD-67605.1
122.92
37.28
120.37
16.17

AD-67562.1
124.35
35.73
109.59
17.85

AD-67606.1
160.77
45.92
152.73
33.64

AD-67604.1
111.98
13.56
167.29
31.28

AD-67565.1
135.81
13.80
120.59
16.82

AD-67529.1
38.14
15.75
121.25
64.05

AD-67533.1
12.73
5.26
16.84
7.89

AD-67537.1
86.70
19.46
92.22
10.90

AD-67546.1
51.08
19.80