Compositions and methods for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3)

Abstract

Compositions and methods are provided that are useful for reducing expression of the patatin-like phospholipase domain-containing 3 (PNPLA3) gene and treating PNPLA3-related diseases and conditions. Provided are PNPLA3 dsRNA agents, PNPLA3 antisense polynucleotide agents, compositions comprising PNPLA3 dsRNA agents, and compositions comprising PNPLA3 antisense polynucleotide agents that can be used to reduce PNPLA3 expression in cells and subjects.

Description

[Technical Field]

[0001] The present invention relates, in part, to compositions and methods that can be used to inhibit patatin-like phospholipase domain-containing 3 (PNPLA3) gene expression. [Background technology]

[0002] Patatin-like phospholipase domain-containing 3 (PNPLA3) is a type II transmembrane protein expressed in various cells, including the liver. In hepatocytes, PNPLA3 is expressed in the endoplasmic reticulum and lipid membranes, and primarily exhibits triacylglycerol hydrolase activity.

[0003] The accumulation of excess triglycerides in the liver, known as hepatic steatosis (or fatty liver), is associated with adverse metabolic consequences such as insulin resistance and dyslipidemia. NAFLD refers to a broad spectrum of liver diseases that can progress from simple fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH) and cirrhosis (irreversible progressive scarring of the liver). Common to all stages of NAFLD is the accumulation of fat (fatty infiltration) in liver cells (hepatocytes).

[0004] Many studies have found a significant association between liver 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 using knock-in mice have demonstrated that expression of a sequence polymorphism in PNPLA3 (rs738409, I148M) results in NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surface of lipid droplets is associated with hepatic triglyceride accumulation (Smagris et al. (2015) Hepatology, 61:108-118). Specifically, the PNPLA3 I148M variant has been 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 production and deposition of extracellular matrix (Chen et al., (2015), World J. Gastroenterol. 21(3):794-802).

[0005] Currently, there is a need for a treatment for subjects suffering from NAFLD. The present invention provides a novel approach for reducing PNPLA3 levels and treating liver diseases such as NAFLD. Summary of the Invention

[0006] According to one aspect of the invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3) is provided, the dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ 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 that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotide positions 2-18 of the antisense strand comprise a region of complementarity to a PNPLA3 RNA transcript, the region of complementarity comprising at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, and optionally a targeting ligand. In some embodiments, the region of complementarity to the PNPLA3 RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than three nucleotides from one of the antisense sequences listed in one of Tables 1-3. In certain embodiments, the antisense strand of the dsRNA is at least substantially complementary to any one of the target regions of SEQ ID NO: 1 and provided in any one of Tables 1-3. In some embodiments, the antisense strand of the dsRNA is fully complementary to any one of the target regions of SEQ ID NO: 1 and provided in any one of Tables 1-3. In some embodiments, the dsRNA agent comprises a sense strand sequence listed in any one of Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In certain embodiments, the dsRNA agent comprises a sense strand sequence listed in any one of Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises an antisense strand sequence listed in any one of Tables 1-3. In some embodiments, the dsRNA agent comprises a sequence set forth as a duplex sequence in any of Tables 1-3.In some embodiments, the dsRNA agent comprises at least one modified nucleotide.In certain embodiments, all or substantially all of the nucleotides of the antisense strand are modified nucleotides.In certain embodiments, all or substantially all of the nucleotides of the sense strand and the antisense strand are modified nucleotides. In some embodiments, at least one of the modified nucleotides comprises a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-seconucleotide mimic, a locked nucleotide, an unlocked nucleic acid nucleotide (UNA), a glycol nucleic acid nucleotide (GNA), a 2'-F-arabinonucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, an inverted 2'-deoxy nucleotide, an isomannide nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a mophorino nucleotide, and a 3'-OMe nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 5'-phosphonate-modified nucleotide, a nucleotide comprising a vinyl phosphonate, or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a phosphoramidite, or a non-natural base containing nucleotide. In some embodiments, the dsRNA agent comprises an E-vinyl phosphonate nucleotide at the 5' end of the guide strand. In certain embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, and fewer than six modified nucleotides are 2'-fluoro nucleotides. In certain embodiments, the antisense strand comprises three or five 2'-fluoro nucleotides, and preferably, the antisense strand comprises five 2'-fluoro nucleotides. In certain embodiments, the antisense strand comprises five 2'-fluoro nucleotides and 5'-phosphonate-modified nucleotides, and preferably, the 5'-phosphonate-modified nucleotides are nucleotides containing vinyl phosphonate. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, and fewer than four modified nucleotides are 2'-fluoro nucleotides. In certain embodiments, the sense strand comprises three 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least 16 modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 2, 5, 7, 12, 14, 16, and / or 18 from the 5' end of the antisense strand are 2'-fluoro nucleotides. In certain embodiments, the nucleotides at positions 2, 7, 12, 14, and 16 from the 5' end of the antisense strand are 2'-fluoro nucleotides. In certain embodiments, the nucleotides at positions 2, 5, 12, 14, and 18 from the 5' end of the antisense strand are 2'-fluoro nucleotides. In certain embodiments, the nucleotides at positions 2, 7, 12, 14, and 16 from the 5' end of the antisense strand are 2'-fluoro nucleotides, and the 5'-terminal nucleotide of the antisense strand is a nucleotide containing a vinyl phosphonate, preferably a VPu* nucleotide as defined herein.In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least 18 modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 9, 11, 13, and / or 14 from the 5' end of the sense strand are 2'-fluoro nucleotides. In certain embodiments, the nucleotides at positions 9, 11, and 13 from the 3' end of the sense strand, counting from the first matching position, are 2'-fluoro nucleotides. In certain embodiments, the nucleotides at positions 8, 11, and 13 from the 3' end of the sense strand, counting from the first matching position, are 2'-fluoro nucleotides.

[0007] In some embodiments, the sense strand sequence can be represented by formula (I):

[0008] [ka] During the ceremony, Each N' F represents a 2'-fluoro modified nucleotide, and each N' N1 , N' N2 , N' N3 , N' N4 , N' N5 , N' N6 , N' N7 , and N8 N' independently represents a modified or unmodified nucleotide, and each N' L independently represent a modified or unmodified nucleotide, but do not represent a 2'-fluoro modified nucleotide; m' and n' are each independently an integer of 0 to 7.

[0009] In some embodiments, the antisense strand sequence can be represented by formula (II):

[0010] [ka] During the ceremony, each NF represents a 2'-fluoro modified nucleotide, and each N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 , and N M8 independently represent modified or unmodified nucleotides, preferably N M1 , N M2 , N M3 , N M6 , and N M7 each independently represents a 2'-fluoro modified nucleotide, and each N L independently represent a modified or unmodified nucleotide, but do not represent a 2'-fluoro modified nucleotide; and n is an integer from 0 to 7.

[0011] In certain embodiments, n' is 1 and m' is 1, or n' is 1 and m' is 2, or n' is 1 and m' is 3, or n' is 1 and m' is 4, or n' is 1 and m' is 5, or n' is 3 and m' is 1, or n' is 3 and m' is 2, or n' is 3 and m' is 3, or n' is 5 and m' is 1.

[0012] In certain embodiments, n is 1, or n is 2, or n is 3.

[0013] In certain embodiments, the modified nucleotide is a modified nucleotide as defined above.

[0014] In certain embodiments, the modified nucleotide is a 2'-OMe modified nucleotide or a 2'-F modified nucleotide.

[0015] In certain embodiments, N M6 , N M3 , and N M2 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M6 , N M3 , and NM2 are all 2'-fluoro modified nucleotides.

[0016] In certain embodiments, N M7 , N M3 , and N M1 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M7 , N M3 , and N M1 are all 2'-fluoro modified nucleotides.

[0017] In some embodiments, the antisense strand sequence can be represented by formula (II').

[0018] [ka] During the ceremony, each N F represents a 2'-fluoro modified nucleotide, and each N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 , and N M8 independently represent modified or unmodified nucleotides, preferably N M1 , N M2 , N M3 , N M6 , and N M7 each independently represents a 2'-fluoro modified nucleotide, and each N L independently represent a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide; N Z represents a nucleotide containing a phosphomimetic, preferably N Z represents a nucleotide containing a vinylphosphonate, and n is an integer of 0 to 7.

[0019] In certain embodiments, n is 1, or n is 2, or n is 3.

[0020] In some embodiments, the modified nucleotide is a modified nucleotide as defined above.

[0021] In some embodiments, the modified nucleotide is a 2'-OMe modified nucleotide or a 2'-F modified nucleotide.

[0022] In certain embodiments, N M6 , N M3 , and N M2 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M6 , N M3 , and N M2 are all 2'-fluoro modified nucleotides.

[0023] In certain embodiments, N M7 , N M3 , and N M1 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M7 , N M3 , and N M1 are all 2'-fluoro modified nucleotides.

[0024] In certain embodiments, N Z is a vinylphosphonate modified nucleotide.

[0025] In certain embodiments, N Z is the structure [ka] VPu* has the following structure.

[0026] In some embodiments, the sense strand and the antisense strand form a dsRNA duplex, wherein the sense strand is complementary to the antisense strand, and the antisense strand comprises a region of complementarity to an mRNA encoding PNPLA3, wherein the region of complementarity comprises at least 15 consecutive nucleotides, and the dsRNA duplex is represented by formula (III).

[0027] [ka] During the ceremony, each N F and N' F independently represents a 2'-fluoro modified nucleotide, and each N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 , N M8 , N' N1 , N' N2 , N' N3 , N' N4 , N' N5 , N' N6 , N' N7 , and N' N8 each independently represents a modified or unmodified nucleotide, and each N L and N' L independently represent a modified or unmodified nucleotide, but do not represent a 2'-fluoro modified nucleotide; m', n', and n are each independently an integer of 0 to 7.

[0028] In certain embodiments, n' is 1 and m' is 1, or n' is 1 and m' is 2, or n' is 1 and m' is 3, or n' is 1 and m' is 4, or n' is 1 and m' is 5, or n' is 3 and m' is 1, or n' is 3 and m' is 2, or n' is 3 and m' is 3, or n' is 5 and m' is 1.

[0029] In certain embodiments, n is 1, or n is 2, or n is 3.

[0030] In certain embodiments, the modified nucleotide is a modified nucleotide as defined above.

[0031] In certain embodiments, the modified nucleotide is a 2'-OMe modified nucleotide or a 2'-F modified nucleotide.

[0032] In certain embodiments, N M6 , N M3 , and N M2 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M6 , N M3 , and N M2 are all 2'-fluoro modified nucleotides.

[0033] In certain embodiments, N M7 , N M3 , and N M1 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M7 , N M3 , and N M1 are all 2'-fluoro modified nucleotides.

[0034] In some embodiments, the sense strand and the antisense strand form a dsRNA duplex, wherein the sense strand is complementary to the antisense strand, and the antisense strand comprises a region of complementarity to an mRNA encoding PNPLA3, wherein the region of complementarity comprises at least 15 consecutive nucleotides, and the dsRNA duplex can be represented by formula (III').

[0035] [ka] During the ceremony, each N F and N' F independently represents a 2'-fluoro modified nucleotide, and each N M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 , N M8 , N' N1 , N' N2 , N'N3 , N' N4 , N' N5 , N' N6 , N' N7 , and N' N8 each independently represents a modified or unmodified nucleotide, and each N L and N' L independently represent a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide; N Z represents a nucleotide containing a phosphomimetic, preferably N Z represents a nucleotide containing a vinylphosphonate, and m', n', and n are each independently an integer of 0 to 7.

[0036] In certain embodiments, n' is 1 and m' is 1, or n' is 1 and m' is 2, or n' is 1 and m' is 3, or n' is 1 and m' is 4, or n' is 1 and m' is 5, or n' is 3 and m' is 1, or n' is 3 and m' is 2, or n' is 3 and m' is 3, or n' is 5 and m' is 1.

[0037] In certain embodiments, n is 1, or n is 2, or n is 3.

[0038] In certain embodiments, the modified nucleotide is a modified nucleotide as defined above.

[0039] In certain embodiments, the modified nucleotide is a 2'-OMe modified nucleotide or a 2'-F modified nucleotide.

[0040] In certain embodiments, N M6 , N M3 , and N M2 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M6 , N M3 , and N M2 are all 2'-fluoro modified nucleotides.

[0041] In certain embodiments, N M7 , N M3 , and N M1 each independently represent a 2'-fluoro modified nucleotide, and optionally, N M7 , N M3 , and N M1 are all 2'-fluoro modified nucleotides.

[0042] In certain embodiments, N Z is a vinylphosphonate modified nucleotide.

[0043] In certain embodiments, N Z is the structure [ka] VPu* has the following structure.

[0044] In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is between 16 and 23 nucleotides in length. In some embodiments, the region of complementarity is 19 to 21 nucleotides in length. In certain embodiments, the region of complementarity is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, each strand is 40 nucleotides or less in length. In some embodiments, each strand is 30 nucleotides or less in length. In some embodiments, each strand is 25 nucleotides or less in length. In some embodiments, each strand is 23 nucleotides or less in length. In some embodiments, each strand is 21 nucleotides or less in length. In certain embodiments, the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups. In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetylgalactosamine (GalNAc).

[0045] In some embodiments, the targeting group has a structure as follows:

[0046] [ka] Each n" is independently selected from 1 or 2.

[0047] In some embodiments, the targeting group has the following structure:

[0048] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0049] In certain embodiments, a dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand. In some embodiments, a dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand. In some embodiments, the antisense strand comprises one inverted abasic residue at the 3'-end. In certain embodiments, the sense strand comprises one or two inverted abasic residues at the 3'-end or / and 5'-end. In certain embodiments, the sense strand comprises one or two iman residues at the 3'-end or / and 5'-end. In certain embodiments, each end of the sense strand comprises one inverted abasic residue. In certain embodiments, each end of the sense strand comprises one iman residue. In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhang of at least one nucleotide. In some embodiments, at least one strand comprises a 3' overhang of at least two nucleotides. In some embodiments, at least one linkage in the sense strand and / or antisense strand is a phosphodiester (PO) linkage. In some embodiments, at least one bond in the sense strand and / or antisense strand is a modified bond. In some embodiments, at least one bond in the sense strand and / or antisense strand is a phosphorothioate (PS) bond. In some embodiments, at least one phosphorothioate (PS) bond is introduced at the 5'-end, 3'-end, or both ends of the sense strand and / or antisense strand. In some embodiments, 1, 2, 3, 4, 5, or 6 phosphorothioate (PS) bonds are introduced at the 5'-end, 3'-end, or both ends of the sense strand and / or antisense strand. In some embodiments, at least two terminal modified or unmodified nucleotides at one or both ends of the antisense strand are linked via phosphorothioate bonds. In some embodiments, three terminal modified or unmodified nucleotides at one or both ends of the antisense strand are linked via phosphorothioate bonds.In some embodiments, at least two terminally modified or unmodified nucleotides at one or both ends of the sense strand are linked via phosphorothioate linkages. In some embodiments, three terminally modified or unmodified nucleotides at one or both ends of the sense strand are linked via phosphorothioate linkages. In some embodiments, three terminally modified or unmodified nucleotides at the 5' end of the sense strand are linked via phosphorothioate linkages, and two terminally modified or unmodified nucleotides at the 3' end of the sense strand are linked via phosphorothioate linkages. In some embodiments, the sense strand contains phosphorothioate linkages between the target group and the inverted abasic or iman residue, and between the inverted abasic or iman residue and the terminally modified or unmodified nucleotide at the 5' end of the sense strand. In some embodiments, the modified sense strand has a modification pattern described in any one of Tables 2-3. In some embodiments, the modified antisense strand has a modification pattern described in any one of Tables 2-3. In some embodiments, the modified sense strand has a modified sense strand sequence described in one of Tables 2-3. In some embodiments, the modified antisense strand is a modified antisense strand sequence set forth in one of Tables 2-3. In certain embodiments, the dsRNA is selected from the group consisting of AD00652, AD00653, AD00654, AD00655, AD00656, AD00657, AD00658, AD00659, AD00660, AD00661, AD00662, AD00663, AD00664, AD00663-1, AD00664-1, AD00815-1, AD00816-1, The duplex comprises a strand selected from the group consisting of AD00819-1, AD00444-1, AD00663-2, AD00664-2, AD00815-2, AD00745, AD00746, AD00747, AD00748, AD00749, AD00750, AD00815, AD00816, AD00817, AD00818, AD00819, and AD00820.

[0050] In some embodiments, any one of the sense strands in Table 1 can be further modified in the pattern shown in formula (I) or (III) above.

[0051] In some embodiments, any one of the antisense strands in Table 1 may be further modified in the pattern shown in formula (II) or (III) above.

[0052] In some embodiments, any one of the duplexes in Table 1 can be further modified in the pattern shown in formula (III) above.

[0053] According to one aspect of the present invention, a composition is provided that comprises any of the above-mentioned dsRNA agent aspects of the present invention.In certain embodiments, the composition also comprises a pharmaceutically acceptable carrier.In some embodiments, the composition also comprises one or more additional therapeutic agents.In certain embodiments, the composition is packaged in a kit, a container, a pack, a dispenser, a pre-filled syringe, or a vial.In some embodiments, the composition is formulated for subcutaneous administration or for intravenous (IV) administration.

[0054] According to another aspect of the present invention, there is provided a cell comprising any of the embodiments of the aforementioned dsRNA agent aspects of the present invention. In some embodiments, the cell is a mammalian cell, optionally a human cell.

[0055] According to another aspect of the present invention, there is provided a method for inhibiting expression of the PNPLA3 gene in a cell, the method comprising: (i) preparing cells comprising an effective amount of any of the aforementioned dsRNA agent embodiments of the present invention or any of the aforementioned composition embodiments of the present invention. In certain embodiments, the method also comprises (ii) inhibiting expression of the PNPLA3 gene in the cell by maintaining the prepared cells for a period of time sufficient to allow degradation of the mRNA transcript of the PNPLA3 gene. In some embodiments, the cells are present in a subject and the dsRNA agent is administered to the subject subcutaneously. In some embodiments, the cells are present in a subject and the dsRNA agent is administered to the subject by IV administration. In certain embodiments, the method also comprises administering a dsRNA agent to the subject and assessing the inhibition of PNPLA3 gene, wherein the means for assessing comprises (i) determining one or more physiological characteristics of PNPLA3-related disease or symptoms in the subject, and (ii) comparing the determined physiological characteristics with the baseline physiological characteristics before treatment of PNPLA3-related disease or symptoms and / or with the control physiological characteristics of PNPLA3-related disease or symptoms, wherein the comparison indicates one or more of the presence or absence of inhibition of the expression of PNPLA3 gene in the subject.In some embodiments, the expression of PNPLA3 gene can be assessed based on the level or level change of any variable related to the expression of PNPLA3 gene, such as PNPLA3 mRNA level, PNPLA3 protein level, liver fat level and / or lipid droplet level, or the number or degree of amyloid deposits.

[0056] According to another aspect of the present invention, there is provided a method for inhibiting the expression of the PNPLA3 gene in a subject, the method comprising administering to the subject an effective amount of the above-mentioned dsRNA agent embodiment of the present invention or the above-mentioned composition embodiment of the present invention. In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered to the subject by IV administration. In some embodiments, the method also comprises assessing the inhibition of the PNPLA3 gene after administration of the dsRNA agent, wherein the means for assessing comprises (i) determining one or more physiological characteristics of the PNPLA3-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics with the baseline physiological characteristics before treatment of the PNPLA3-related disease or condition and / or with the control physiological characteristics of the PNPLA3-related disease or condition, wherein the comparison indicates one or more of the presence or absence of inhibition of the expression of the PNPLA3 gene in the subject. In some embodiments, expression of the PNPLA3 gene can be assessed based on the level or change in level of any variable associated with expression of the PNPLA3 gene, such as PNPLA3 mRNA level, PNPLA3 protein level, liver fat level and / or lipid droplet level, or the number or extent of amyloid deposits.

[0057] According to another aspect of the present invention, there is provided a method for treating a disease or condition associated with the presence of PNPLA3 protein, the method comprising administering to a subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to inhibit the expression of the PNPLA3 gene. In some embodiments, the disease or condition is one or more of liver disease, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, alcoholic liver disease, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, or non-alcoholic fatty liver disease (NAFLD). In some embodiments, the method also comprises administering an additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic regimen includes treatment of a PNPLA3-related disease or condition. In certain embodiments, the additional treatment regimen comprises administering one or more PNPLA3 antisense polynucleotides of the present invention to the subject, administering a non-PNPLA3 dsRNA therapeutic agent to the subject, and behavioral modification in the subject. In some embodiments, the non-PNPLA3 dsRNA therapeutic agent is one or more of an HMG-CoA reductase inhibitor, a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor antagonist, an acyl-CoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol regulator, a bile acid regulator, a peroxisome proliferator-activated receptor (PPAR) agonist, a gene-based therapy, a combined vasoprotectant, a glycoprotein IIb / IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor, a squalene synthase inhibitor, a monocyte chemoattractant protein (MCP)-I inhibitor, or a fish oil. In some embodiments, the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject by IV administration.In some embodiments, the method also includes determining the effectiveness of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject. In some embodiments, the means for determining the effectiveness of treatment in the subject includes (i) determining one or more physiological characteristics of the PNPLA3-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics with baseline pre-treatment physiological characteristics of the PNPLA3-related disease or condition, wherein the comparison indicates one or more of the presence or absence and level of effectiveness of the administration of the double-stranded ribonucleic acid (dsRNA) agent to the subject. In some embodiments, the expression of the PNPLA3 gene can be assessed based on the level or change in level of any variable associated with the expression of the PNPLA3 gene, such as PNPLA3 mRNA level, PNPLA3 protein level, liver fat level and / or lipid droplet level, or the number or extent of amyloid deposits.

[0058] According to another aspect of the present invention, provided is a method for reducing the PNPLA3 protein level of an object compared with the baseline pre-treatment level of the PNPLA3 protein of the object, the method comprises administering to the object an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention, so as to reduce the level of PNPLA3 gene expression.In some embodiments, the dsRNA agent is administered to the object subcutaneously or administered to the object via IV.

[0059] According to another aspect of the present invention, there is provided a method for modifying the physiological characteristics of a subject's PNPLA3-related disease or condition compared with the subject's baseline physiological characteristics before treatment of the PNPLA3-related disease or condition, the method comprising administering to the subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention, to modify the physiological characteristics of the subject's PNPLA3-related disease or condition.In some embodiments, the dsRNA agent is administered to the subject subcutaneously or via IV administration.In certain embodiments, the physiological characteristics are one or more of the levels of PNPLA3 mRNA or PNPLA3 protein in samples (for example, liver or blood) obtained from body fluids or tissues at specific locations within the subject.

[0060] According to another aspect of the present invention, the above-mentioned dsRNA agent is provided for use in the method for treating the disease or condition associated with the presence of PNPLA3 protein.In some embodiments, the disease or condition is one or more of the following liver diseases: fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, inflammation of the liver, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD).Fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), cirrhosis, fat accumulation in the liver, inflammation of the liver, hepatocyte necrosis, hepatocellular carcinoma, hepatic fibrosis, obesity, alcoholic liver disease, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, or non-alcoholic fatty liver disease (NAFLD).

[0061] According to another aspect of the present invention, there is provided an antisense polynucleotide agent for inhibiting expression of PNPLA3 protein, the agent comprising 10 to 30 consecutive nucleotides, at least one of the consecutive nucleotides being a modified nucleotide, and the nucleotide sequence of the agent being about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the equivalent region is any one of the target regions of SEQ ID NO: 1, and the complementary sequence is one of those provided in any one of Tables 1-3. In certain embodiments, the antisense polynucleotide agent comprises one of the antisense sequences provided in any one of Tables 1-3.

[0062] According to another aspect of the present invention, a composition is provided comprising any of the above-described embodiments of an antisense polynucleotide agent. In some embodiments, the composition also comprises a pharmaceutically acceptable carrier. In some embodiments, the composition also comprises one or more additional therapeutic agents for treating PNPLA3-related diseases or conditions. In certain embodiments, the composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial. In certain embodiments, the composition is formulated for subcutaneous or IV administration.

[0063] According to another aspect of the present invention, there is provided a cell comprising any of the foregoing antisense polynucleotide agent embodiments, hi some embodiments, the cell is a mammalian cell, optionally a human cell.

[0064] According to another aspect of the present invention, there is provided a method for inhibiting expression of the PNPLA3 gene in a cell, the method comprising: (i) preparing a cell containing an effective amount of any of the embodiments of the antisense polynucleotide agent described above. In some embodiments, the method also comprises: (ii) maintaining the cells prepared in (i) for a time sufficient to allow degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the cell.

[0065] According to another aspect of the present invention, there is provided a method of inhibiting expression of the PNPLA3 gene in a subject, the method comprising administering to the subject an effective amount of any of the embodiments of the antisense polynucleotide agent described above.

[0066] According to another aspect of the present invention, there is provided a method for treating a disease or condition associated with the presence of the PNPLA3 protein, the method comprising administering to a subject an effective amount of any of the aforementioned antisense polynucleotide agent embodiments of the present invention or any of the aforementioned composition embodiments of the present invention to inhibit expression of the PNPLA3 gene. In certain embodiments, the disease or condition is one or more of liver disease, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, alcoholic liver disease, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, or non-alcoholic fatty liver disease (NAFLD).

[0067] According to another aspect of the present invention, there is provided a method for reducing the level of PNPLA3 protein in a subject compared to the subject's baseline pre-treatment level of PNPLA3 protein, the method comprising administering to the subject an effective amount of any of the above-mentioned antisense polynucleotide agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to reduce the level of PNPLA3 gene expression.In certain embodiments, the antisense polynucleotide agent is administered to the subject by subcutaneous administration or IV administration.

[0068] According to another aspect of the present invention, there is provided an antisense polynucleotide agent for inhibiting expression of the PNPLA3 gene, the agent comprising 10 to 30 consecutive nucleotides, at least one of the consecutive nucleotides being a modified nucleotide, and the nucleotide sequence of the agent being about 80% or about 85% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1.

[0069] According to another aspect of the present invention, there is provided a method for modifying the physiological characteristics of a subject's PNPLA3-related disease or condition compared to the subject's baseline pre-treatment physiological characteristics of the PNPLA3-related disease or condition, the method comprising administering to the subject an effective amount of any of the above-mentioned antisense polynucleotide agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to modify the physiological characteristics of the subject's PNPLA3 disease or condition. In some embodiments, the antisense polynucleotide agent is administered to the subject by subcutaneous administration or IV administration. In some embodiments, the physiological characteristics are one or more of the levels of PNPLA3 mRNA or PNPLA3 protein in samples obtained from body fluids or tissues at specific sites within the subject (e.g., liver or blood). [Brief explanation of the drawings]

[0070] SEQ ID NO:1 and SEQ ID NO:2 (reverse complement) are the Homo sapiens patatin-like phospholipase domain-containing 3 (PNPLA3) mRNA [NCBI Reference Sequence: NM_025225.3].

[0071] SEQ ID NO:3 and SEQ ID NO:4 (reverse complement) are Homo sapiens patatin-like phospholipase domain-containing 3 (PNPLA3) mRNA [Source: HGNC Symbol; Acc: HGNC:18590; Transcript: ENST00000423180.2].

[0072] SEQ ID NO:5 and SEQ ID NO:6 (reverse complement) are the predicted Macaca fascicularis patatin-like phospholipase domain-containing 3 (PNPLA3) mRNA [NCBI Reference Sequence: XM_005567051.2].

[0073] SEQ ID NO:7 and SEQ ID NO:8 (reverse complement) are the predicted Macaca fascicularis patatin-like phospholipase domain-containing 3 (PNPLA3) mRNA [NCBI Reference Sequence: XM_015457081.1].

[0074] SEQ ID NO:9 and SEQ ID NO:10 (reverse complement) are the predicted Macaca fascicularis patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA [Source: HGNC Symbol; Acc: HGNC:18590; Transcript: ENSMFAT00000025830.2].

[0075] SEQ ID NO:11 and SEQ ID NO:12 (reverse complement) are the predicted Macaca mulatta patatin-like phospholipase domain-containing 3 (PNPLA3) mRNA [NCBI Reference Sequence: XM_001109144.4].

[0076] SEQ ID NO:13 and SEQ ID NO:14 (reverse complement) are the predicted Macaca mulatta patatin-like phospholipase domain-containing 3 (PNPLA3) mRNA [NCBI Reference Sequence: XM_015150532.2].

[0077] SEQ ID NO:15 and SEQ ID NO:16 (reverse complement) are the predicted Macaca mulatta patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA [Source: VGNC Symbol; Acc: VGNC:76061; Transcript: ENSMMUT00000023461.4].

[0078] SEQ ID NO: 17 and SEQ ID NO: 18 (reverse complement) are Mus musculus patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA [NCBI Reference Sequence: NM_054088.3].

[0079] SEQ ID NO:19 and SEQ ID NO:20 (reverse complement) are Mus musculus patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA [Source: MGI Symbol, Acc: MGI:2151796, Transcript: ENSMUST00000045289.6].

[0080] SEQ ID NO:21 and SEQ ID NO:22 (reverse complement) are Rattus norvegicus patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA [NCBI Reference Sequence: NM_001282324.1].

[0081] SEQ ID NO:23 and SEQ ID NO:24 (reverse complement) are Rattus norvegicus patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA [Source: RGD Symbol, Acc: 1595843, Transcript: ENSRNOT00000015767.8].

[0082] SEQ ID NOs: 25 to 252 are shown in Table 1 and are sense strand sequences.

[0083] SEQ ID NOs: 253 to 480 are shown in Table 1 and are antisense strand sequences.

[0084] SEQ ID NOs: 481-648 are shown in Table 2 with chemical modifications indicated by uppercase: 2'-fluoro, lowercase: 2'-OMe, and thiophosphate: *, and one skilled in the art will understand that "*" is a symbol indicating a bond relationship, the presence of "*" means that the monomers are bonded to each other via a phosphorothioate diester bond, the absence of "*" between two monomers indicates that the monomers are bonded to each other via a phosphodiester bond, and invab = inverted abasic.

[0085] SEQ ID NOs: 649-742 are shown in Table 3. Delivery molecules are indicated as "GLX-_" at the 3' or 5' end of each sense strand. Chemical modifications are indicated as uppercase: 2'-fluoro, lowercase: 2'-OMe, and thiophosphate: *, where those skilled in the art will recognize that "*" is a symbol indicating a bond relationship; the presence of "*" means that the monomers are linked to each other via a phosphorothioate diester bond; the absence of "*" between two monomers indicates that the monomers are linked to each other via a phosphodiester bond; invab = inverted abasic; imann = at the end of each strand. [ka] or when further conjugated to a delivery molecule [ka] ;VPu*: [ka] It can be understood that this is the case. DETAILED DESCRIPTION OF THE INVENTION

[0086] The present invention includes, in part, RNAi agents, such as, but not limited to, double-stranded (ds) RNAi agents, capable of inhibiting patatin-like phospholipase domain-containing 3 (PNPLA3) gene expression. The present invention also includes, in part, compositions comprising PNPLA3 RNAi agents and methods of using such compositions. The PNPLA3 RNAi agents disclosed herein can be attached to a delivery compound for delivery to cells, including hepatocytes. Pharmaceutical compositions of the present invention can include at least one ds PNPLA3 agent and a delivery compound. In some embodiments of the compositions and methods of the present invention, the delivery compound is a GalNAc-containing delivery compound. The PNPLA3 RNAi agent delivered to a cell can inhibit expression of the PNPLA3 gene, thereby reducing the activity of the gene's PNPLA3 protein product in the cell. The dsRNAi agents of the present invention can be used to treat PNPLA3-related diseases and conditions.

[0087] In some embodiments of the present invention, by reducing PNPLA3 expression in cells or subjects, diseases or conditions associated with PNPLA3 expression in cells or subjects are treated, respectively.Non-limiting examples of diseases and conditions that can be treated by reducing PNPLA3 activity include liver disease, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, alcoholic liver disease, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, or non-alcoholic fatty liver disease (NAFLD), or other diseases that are medically beneficial by reducing the level and activity of PNPLA3 protein.

[0088] As used herein, "G," "C," "A," and "U" typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative replacement moieties, as described in more detail below. Those skilled in the art will understand that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base pairing properties of an oligonucleotide containing a nucleotide having such a replacement moiety. For example, and without limitation, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, in the nucleotide sequences of the present invention, nucleotides containing uracil, guanine, or adenine can be substituted with, for example, a nucleotide containing inosine. Sequences containing such replacement moieties are embodiments of the present invention.

[0089] As used herein, the term "patatin-like phospholipase domain-containing 3" is used interchangeably with the term "PNPLA3" and refers to a naturally occurring gene encoding a triacylglycerol lipase that mediates the hydrolysis of triacylglycerol in adipocytes. The amino acid sequence and complete coding sequence of the reference sequence of the human PNPLA3 gene can be found, for example, in GenBank RefSeq Accession No. NM_025225.3 (SEQ ID NO: 1 and SEQ ID NO: 2), HGNC transcript: ENST00000423180.2 (SEQ ID NO: 3 and SEQ ID NO: 4). Mammalian orthologs of the human PNPLA3 gene are listed, for example, in GenBank RefSeq Accession No. XM_005567051.2, cynomolgus monkey (SEQ ID NO: 5 and SEQ ID NO: 6), RefSeq Accession No. XM_015457081.1, cynomolgus monkey (SEQ ID NO: 7 and SEQ ID NO: 8), HGNC transcript: ENSMFAT00000025830.2, cynomolgus monkey (SEQ ID NO: 9 and SEQ ID NO: 10), GenBank RefSeq Accession No. XM_001109144.4, rhesus monkey (SEQ ID NO: 11 and SEQ ID NO: 12), GenBank RefSeq Accession No. XM_015150532.2, rhesus monkey (SEQ ID NO: 13 and SEQ ID NO: 14), HGNC transcript: ENSMMUT00000023461.4, rhesus monkey (SEQ ID NO: 15 and SEQ ID NO: 16), GenBank RefSeq Accession No. NM_054088.3, ​​mouse (SEQ ID NO: 17 and SEQ ID NO: 18), HGNC transcript: ENSMUST00000045289.6, mouse (SEQ ID NO: 19 and SEQ ID NO: 20), GenBank RefSeq Accession No. NM001282324.1, rat (SEQ ID NO: 21 and SEQ ID NO: 22), HGNC transcript: ENSRNOT00000015767.7, rat (SEQ ID NO: 23 and SEQ ID NO: 24). Additional examples of PNPLA3 mRNA sequences are readily available using public databases such as GenBank, UniProt, Ensembl, and OMIM.

[0090] The following describes the methods for making and using compositions that contain PNPLA3 single-stranded (ssRNA) and dsRNA agents for inhibiting PNPLA3 gene expression, and the compositions and methods for treating diseases and conditions caused by or regulated by PNPLA3 gene expression.The term "RNAi" is known in the art and is sometimes referred to as "siRNA".

[0091] As used herein, the term "RNAi" refers to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region, also defined as a "target region" or "target portion," refers to a contiguous portion of the nucleotide sequence of a messenger RNA (mRNA) molecule formed during transcription of a gene, including the messenger RNA (mRNA) that is the product of RNA processing of the primary transcript. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-induced cleavage at or near that portion. The target sequence may be 8-30 nucleotides in length (inclusive), 10-30 nucleotides in length (inclusive), 12-25 nucleotides in length (inclusive), 15-23 nucleotides in length (inclusive), 16-23 nucleotides in length (inclusive), or 18-23 nucleotides in length (inclusive), including the shorter length within each of the listed ranges. In some embodiments of the invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In particular embodiments, the target sequence is 9 to 26 nucleotides in length (inclusive), including all subranges and integers therebetween. For example, and not intended to be limiting, in certain embodiments of the invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, and the sequence is fully or at least substantially complementary to at least a portion of an RNA transcript of the PNPLA3 gene. Some aspects of the invention include pharmaceutical compositions comprising one or more PNPLA3 dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the present invention, the PNPLA3 RNAi described herein inhibits expression of the PNPLA3 protein.

[0092] As used herein, the term "dsRNA agent" refers to a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of sequence-specifically degrading or inhibiting translation of a messenger RNA (mRNA) transcript of a target mRNA. Without wishing to be limited to a particular theory, the dsRNA agents of the present invention may operate via an RNA interference mechanism (i.e., inducing RNA interference via interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) in mammalian cells) or by any alternative mechanism or pathway. Methods for silencing genes in plant, invertebrate, and vertebrate cells are well known in the art (see, for example, (Sharp et al., Genes Dev. 2001, 15:485; Bernstein et al., (2001) Nature 409:363; Nykanen et al., (2001) Cell 107:309; and Elbashir et al., (2001) Genes Dev. 15:188), the disclosures of each of which are incorporated herein by reference in their entirety).Gene silencing procedures known in the art can be used in combination with the disclosure provided herein to inhibit the expression of PNPLA3.

[0093] The dsRNA agents disclosed herein are composed of a sense strand and an antisense strand, and include, but are not limited to, short interfering RNA (siRNA), RNAi agents, microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the dsRNA agent described herein is at least partially complementary to the target mRNA. It is understood in the art that dsRNA duplex structures of different lengths can be used to inhibit the expression of target genes. For example, dsRNAs with duplex structures of 19, 20, 21, 22, and 23 base pairs are known to be effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). It is also known in the art that shorter or longer RNA duplex structures can also be effective in inducing RNA interference. As used herein, the terms "double-stranded region," "duplex region," and "region of complementarity" can be used interchangeably and refer to the region in which the sense strand is complementary or substantially complementary to the antisense strand, as known in the art. In certain embodiments of the present invention, a PNPLA3 dsRNA can include at least one strand at least 21 nt in length, or can have a shorter duplex based on one of the sequences set forth in any one of Tables 1-3, although dsRNAs with 1, 2, 3, or 4 fewer nucleotides at one or both termini, respectively, compared to dsRNAs set forth in Tables 1-3, can also be effective. In some embodiments of the present invention, a PNPLA3 dsRNA agent can have a subsequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one or more of the sequences in Tables 1-3, and exhibit an ability to inhibit PNPLA3 gene expression that differs by no more than 5%, 10%, 15%, 20%, 25%, or 30% compared to the level of inhibition resulting from a dsRNA containing the entire sequence.The sense sequences, antisense sequences, and duplexes disclosed in Tables 1-3 are sometimes referred to herein as "parent" sequences. The sequences disclosed in Tables 1-3 may be modified, shortened, extended, substituted, etc., as described herein, with the resulting sequences retaining all or at least some of the effectiveness of the parent sequences in the methods and compositions of the present invention. The sense and antisense strands included in the dsRNA of the present invention are independently selected. As used herein, the term "independently selected" means that each of two or more similar elements can be selected independently of the selection of the other elements. For example, and not by way of limitation, two strands of "elements" to be included in a duplex may be selected when preparing a dsRNA of the present invention. One of the selected elements, the sense sequence, may be SEQ ID NO: 650 (shown in Table 3), and the other selected element, the antisense sequence, may be SEQ ID NO: 687 or SEQ ID NO: 687, which may be modified, shortened, extended, and / or contain one, two, or three substitutions compared to the parent sequence SEQ ID NO: 687. It will be understood that a duplex of the invention need not contain both the sense and antisense sequences shown as pairs in the duplexes of Tables 1 to 3. Each sense and antisense strand sequence in the tables is immediately followed by its SEQ ID NO:

[0094] Certain embodiments of the compositions and methods of the present invention comprise single-stranded RNA in the composition and / or administered to a subject. For example, an antisense strand such as those listed in any one of Tables 1-3 can be or be present in the composition administered to a subject to reduce the activity of a PNPLA3 polypeptide and / or the expression of the PNPLA3 gene in the subject. Tables 1-3 show the core stretch base sequences of the antisense and sense strands of certain PNPLA3 dsRNA agents. Single-stranded antisense molecules that can be included in certain compositions of the present invention and / or administered in certain ways are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that can be included in certain compositions of the present invention and / or administered in certain ways are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." The term "base sequence" is used herein to refer to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strand GAGGUCCUCAGAUCUUGUA (SEQ ID NO: 25) shown in Table 1 has the base sequence of SEQ ID NO: 481 in Table 2 and SEQ ID NO: 674 in Table 3, where SEQ ID NO: 481 and SEQ ID NO: 674 are shown together with chemical modifications and / or delivery compounds. The sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified as "sense strand SS#," a single-stranded antisense sequence may be identified as "antisense strand AS#," and a duplex comprising a sense strand and an antisense strand may be identified as "duplex AD# / AV#."

[0095] Table 1 includes sense and antisense strands, and the identification numbers of duplexes formed from the sense and antisense strands are shown in the same row of Table 1. SEQ ID NOs: 177-252 of the sense strand contain random nucleobases (n) at positions 1, 2, 3, and 21 from the 5' end. SEQ ID NOs: 405-480 of the antisense strand contain random nucleobases (n) at positions 1, 19, 20, and 21 from the 5' end. In certain embodiments of the present invention, the antisense sequence contains nucleobase u or nucleobase a at position 1 of the antisense sequence. In certain embodiments of the present invention, the antisense sequence contains nucleobase u at position 1 of the antisense sequence. In the sequences shown in Table 1, "n" can represent a nucleotide containing any one of nucleobases a, u, c, g, and t, and can be selected independently for the sense and antisense strands, and each "n" in the sense and antisense strands can be the same or different. When used in the context of "n" in sense and antisense strands, it will be understood that the nucleobase "n" selected and included at a position in the sense strand is not the same nucleobase as "n" in the antisense strand with which the sense strand is paired, but rather is generally complementary to the nucleobase "n" at the matching position of the opposite strand. As used herein, the term "matching position" in the sense and antisense strands refers to the position of each strand that "pairs" when the two strands are duplex. For example, in a 21-nucleobase sense strand and a 21-nucleobase antisense strand, the nucleobase at position 1 of the sense strand and the nucleobase at position 21 of the antisense strand are "matching positions". In yet another non-limiting example, in a 23-nucleobase sense strand and a 23-nucleobase antisense strand, the nucleobase 2 of the sense strand and the nucleobase 22 of the antisense strand are matching positions. In another non-limiting example, in an 18 nucleobase sense strand and an 18 nucleobase antisense strand, nucleobase at position 1 of the sense strand and nucleobase 18 of the antisense strand are at matching positions, and nucleobase 4 of the sense strand and nucleobase 15 of the antisense strand are at matching positions. A skilled artisan will understand how to identify matching positions within sense and antisense strands that are or become duplex and paired.

[0096] Although (n) can be a, u, c, g, or t, "n" at position 1 of the sense strand is typically complementary to (n) at position 21 of the antisense strand. In two non-limiting examples, (1) if position 1 of the sense strand is "g," then position 21 of the antisense strand is "c," and (2) if position 1 of the sense strand is "a," then position 21 of the antisense strand is "u" or "t." This type of complementary matching pairing applies to (n) at position 2 of the sense strand with (n) at position 20 of the antisense strand, and (n) at position 21 of the sense strand with (n) at position 1 of the antisense strand. At these positions, n can be any nucleotide; however, it will be understood that while nucleotides in the sense and antisense strands are generally complementary (matched), mismatches can occur in certain embodiments. For example, and not by way of limitation, in some embodiments, "n" can be "random," meaning that it may be complementary, but need not be. In certain embodiments, "n" is complementary. As a non-limiting example, "n" at position 1 in the antisense strand is "u" and "n" at position 21 in the sense strand is "a." A skilled artisan will understand how to identify matching positions within the sense and antisense strands that are or become duplexes and pairs.

[0097] The last column of Table 1 indicates the duplex AD# for duplexes containing sense and antisense sequences in the same table row. For example, Table 1 discloses a duplex assigned duplex AD#AD00448.um, containing SEQ ID NO:25 for the sense strand and SEQ ID NO:253 for the antisense strand. Thus, each row of Table 1 identifies a duplex of the invention, each duplex containing the sense and antisense sequences shown in the same row, with the identifier assigned to each duplex shown in the last column of the row.

[0098] In some embodiments of the methods of the invention, an RNAi agent comprising a polynucleotide sequence set forth in Table 1 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex comprising at least one of the base sequences set forth in Table 1, comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. In some embodiments of the methods of the invention, the RNAi agent comprising a polynucleotide sequence set forth in Table 1 is attached to a delivery molecule, non-limiting examples of which include delivery compounds comprising a GalNAc compound or a GLS-15 compound.

[0099] Table 1: Sequences of the antisense and sense strands of unmodified PNPLA3 RNAi agents. All sequences are shown in the 5' to 3' direction. Duplex AD# is the number assigned to the duplex of the two strands in the same row of the table.

[0100] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13

[0101] Table 2 shows the sequences of the antisense and sense strands of certain chemically modified PNPLA3 RNAi agents of the invention. In some embodiments of the methods of the invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a cell and / or subject. In some embodiments of the methods of the invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a subject. In some embodiments of the methods of the invention, the RNAi agent administered to a subject comprises a duplex identified in the first column of a row in Table 2 and includes the sequence modifications shown in the sense and antisense strand sequences, respectively, in columns 3 and 6 of the same row in Table 2. In some embodiments of the methods of the invention, the sequences shown in Table 2 can be attached (also referred to herein as "conjugates") to a compound capable of delivering the RNAi agent to cells and / or tissues of a subject. Non-limiting examples of delivery compounds that can be used in certain embodiments of the invention include GalNAc-containing compounds or GLS-15-containing compounds. Column 1 of Table 2 shows the duplex AV# of the base sequence shown in Table 1. Table 2 discloses the duplex AV# and indicates the chemical modifications contained in the sense and antisense sequences of the duplex. For example, Table 1 lists the single-stranded sequences of SEQ ID NO:25 (sense) and SEQ ID NO:253 (antisense), which together form a double-stranded duplex identified as duplex AD#AD00448.um, and Table 2 lists the duplex AV#AV00448, which indicates that the duplexes of SEQ ID NO:481 and SEQ ID NO:565 contain the sequences of SEQ ID NO:25 and SEQ ID NO:253, respectively, but with the chemical modifications shown in the sense and antisense sequences in columns 3 and 6, respectively. The "Sense Strand SS#" in column 2 of Table 2 is the identifier assigned to the sense sequence (including modifications) shown in column 3 of the same row. The "Antisense Strand AS#" in column 5 of Table 2 is the identifier assigned to the antisense sequence (including modifications) shown in column 6.

[0102] Table 2: Sequences of the antisense and sense strands of chemically modified PNPLA3 RNAi agents are shown. All sequences are shown 5' to 3'. These sequences were used in certain in vitro testing studies described herein. Chemical modifications are shown as uppercase: 2'-fluoro; lowercase: 2'-OMe; thiophosphate: *, where one skilled in the art will understand that "*" is a symbol indicating the bond relationship; the presence of "*" means that the monomers are linked to each other via a phosphorothioate diester bond; the absence of "*" between two monomers indicates that the monomers are linked to each other via a phosphodiester bond; and invab = inverted abasic.

[0103] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

[0104] Table 3 shows the antisense and sense strand sequences of certain chemically modified PNPLA3 RNAi agents of the invention. In some embodiments of the methods of the invention, an RNAi agent shown in Table 3 is administered to a cell and / or a subject. In some embodiments of the methods of the invention, an RNAi agent having a polynucleotide sequence shown in Table 3 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex identified in the first column of a row in Table 3, and includes the sequence modifications and / or delivery compounds shown in the sense and antisense strand sequences, respectively, in columns 3 and 6 of the same row in Table 3. These sequences were used in certain in vivo testing studies described elsewhere herein. In some embodiments of the methods of the invention, the sequences shown in Table 3 may be attached to a compound for delivery (also referred to herein as a "conjugate"), a non-limiting example of which is a GalNAc-containing compound, and the delivery compound is identified as "GLX-n" on the sense strand in column 3 of Table 3. As used herein, "GLX-n" is used to represent either a "GLS-n" or a "GLO-n" delivery compound (where "X" can be either an "S" or an "O"), and GLX-0 can be either a "GLS-n" or a "GLO-n" delivery compound that can be attached to the 3' or 5' end of an oligonucleotide during synthesis. As used herein and as shown in Table 3, "GLX-n" is used to indicate that the attached GalNAc-containing compound is any of the compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are provided elsewhere herein.Those skilled in the art can prepare and use dsRNA compounds of the invention in which the attached delivery compound is one of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16. Column 1 of Table 3 lists the duplex AD# assigned to the sense and antisense sequence duplexes in that row of the table. For example, duplex AD# AD00451 is a duplex of SEQ ID NO: 650 as the sense strand and SEQ ID NO: 687 as the antisense strand. Each row in Table 3 lists a sense strand and an antisense strand, and discloses the indicated sense and antisense strand duplex. The "Sense Strand SS#" in column 2 of Table 3 is the identifier assigned to the sense sequence (including modifications) shown in column 3 of the same row. The "Antisense Strand AS#" in column 5 of Table 3 is the identifier assigned to the antisense sequence (including modifications) shown in column 6. Identifiers for particular attached GalNAc-containing "GLO-n" or "GLS-n" compounds are designated as GLS-5, GLS-15, or GLX-0; it will be understood that instead of the compound designated as GLO-0, other "GLO-n" or "GLS-n" compounds may be used, and the resulting compounds are included in embodiments of the methods and / or compositions of the present invention. GLO-0 refers to the GalNAc3 compound of Jayaprakash et al. (2014) J. Am. Chem. Soc., 136, 16958-16961.

[0105] Table 3 shows the sequences of the antisense and sense strands of chemically modified PNPLA3 RNAi agents. All sequences are shown 5' to 3'. These sequences were used in specific in vivo testing studies described elsewhere herein. The delivery molecules are designated as "GLO-n" or "GLS-n" at the 3' or 5' end of each sense strand for in vivo studies. The chemical modifications are designated as uppercase: 2'-fluoro; lowercase: 2'-OMe; thiophosphate: *, where those skilled in the art will recognize that "*" denotes the bond relationship; the presence of "*" means that the monomers are linked to each other via a phosphorothioate diester bond; the absence of "*" between two monomers indicates that the monomers are linked to each other via a phosphodiester bond; invab = inverted abasic; iman = at the end of each strand. [ka] or when further conjugated to a delivery molecule [ka] ;VPu*: [ka] and NAG37 can be understood to have the structure shown in US20190256849 as "NAG37", and one of skill in the art would recognize the binding mode in the presence of a phosphorothioate bond (represented by "*").

[0106] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0107] In certain embodiments of the invention, a dsRNA (also referred to herein as a "duplex") is disclosed in one of Tables 1-3. Each row in Tables 1-3 discloses a duplex comprising the sense strand sequence and the antisense strand sequence of that table row. In addition to the duplexes disclosed in Tables 1-3, it will be understood that in some embodiments, a duplex of the invention can include sense and antisense sequences set forth in Tables 1-3 that differ by 0, 1, 2, or 3 nucleotides from the sequences set forth in Tables 1-3. Thus, by way of non-limiting example, in some embodiments, the antisense strand in a duplex of the invention is SEQ ID NO: 253, 565, 609, 635, 648, 702, 709, 710, or 717, which differs from the nucleotides in SEQ ID NO: 253, 565, 609, 635, 648, 702, 709, 710, or 717 by 0, 1, 2, or 3 nucleotides, respectively.

[0108] It will be understood that the sequences of the sense and antisense strands in a duplex of the present invention can be selected independently. Thus, a dsRNA of the present invention can comprise the sense and antisense strands of a duplex disclosed in a row in Tables 1-3. Alternatively, in a dsRNA of the present invention, one or both of the selected sense and antisense strands in the dsRNA comprise a sequence shown in Tables 1-3, but one or both of the sense and antisense sequences contain one, two, three, or more nucleobase substitutions from the parent sequence. In some embodiments, the selected sequence may be longer or shorter than its parent sequence. Thus, a dsRNA agent included in the present invention can, but need not, comprise the exact sequence of the sense and antisense pair disclosed as a duplex in Tables 1-3.

[0109] In some embodiments, a dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotide positions 2-18 of the antisense strand comprise a region of complementarity to a PNPLA3 RNA transcript, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, and optionally comprises a target ligand. Optionally, the region of complementarity to a PNPLA3 RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3. In some embodiments of a dsRNA agent of the invention, the antisense strand of the dsRNA is at least substantially complementary to any one of the target regions of SEQ ID NO: 1, provided in any one of Tables 1-3. In some embodiments, the antisense strand of a dsRNA agent of the invention is fully complementary to any one of the target regions of SEQ ID NO: 1, provided in any one of Tables 1-3. In some embodiments, a dsRNA agent comprises a sense strand sequence set forth in any one of Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In other embodiments, a dsRNA agent of the invention comprises a sense strand sequence set forth in any one of Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent. In some cases, a dsRNA agent of the invention comprises an antisense strand sequence set forth in any one of Tables 1-3. Some embodiments of a dsRNA agent of the invention comprise a sense sequence and an antisense sequence that are disclosed as a duplex in any of Tables 1-3. It will be understood that, as described herein, the sense and antisense strands in a duplex of the invention can be independently selected.

[0110] mismatch Those skilled in the art know that mismatches are tolerated for the efficacy of dsRNA, particularly mismatches within the terminal regions of the dsRNA. Certain mismatches are more tolerated for efficacy, for example, mismatches of wobble base pairs G:U and A:C (Du et al., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33(5): 1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005; 33(11): 3698). In some embodiments of the methods and compounds of the present invention, the PNPLA3 dsRNA agent may contain one or more mismatches relative to the PNPLA3 target sequence. In some embodiments, the PNPLA3 dsRNA agent of the present invention contains no mismatches. In certain embodiments, the PNPLA3 dsRNA agent of the present invention contains one or less mismatches. In some embodiments, a PNPLA3 dsRNA agent of the invention contains two or fewer mismatches. In certain embodiments, a PNPLA3 dsRNA agent of the invention contains three or fewer mismatches. In some embodiments of the invention, the antisense strand of a PNPLA3 dsRNA agent contains a mismatch to a PNPLA3 target sequence that is not located in the center of the complementary region. In some embodiments, the antisense strand of a PNPLA3 dsRNA agent contains one, two, three, four, or more mismatches located within the last 5, 4, 3, 2, or 1 nucleotide from either or both of the 5' end or 3' end of the complementary region. Methods described herein and / or known in the art can be used to determine whether a PNPLA3 dsRNA agent containing mismatches to a PNPLA3 target sequence is effective in inhibiting expression of the PNPLA3 gene.

[0111] Complementarity As used herein, unless otherwise specified, the term "complementary," when describing a first nucleotide sequence (e.g., the sense strand of a PNPLA3 dsRNA agent or a target PNPLA3 mRNA) in relation to a second nucleotide sequence (e.g., the antisense strand of a PNPLA3 dsRNA agent or a single-stranded antisense polynucleotide), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (forming base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) and, under specified conditions, form a duplex or double-helix structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence. Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, are also applicable. A skilled artisan can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, so long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modifications.

[0112] For example, a complementary sequence within a PNPLA3 dsRNA described herein includes base pairing across the entire length of one or both nucleotide sequences of an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. In embodiments, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, it will be understood that such overhangs are not considered mismatches for purposes of determining complementarity herein. For example, a PNPLA3 dsRNA agent comprising one 19-nucleotide long oligonucleotide and another 20-nucleotide long oligonucleotide, although the longer oligonucleotide contains a 19-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for purposes described herein. Thus, as used herein, "fully complementary" means that all (100%) of the bases in a contiguous sequence of a first polynucleotide hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence may include all or part of the first or second nucleotide sequence.

[0113] As used herein, the term "substantially complementary" means that in a hybridized pair of nucleobase sequences, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not all, of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. The term "substantially complementary" can be used in reference to a first sequence relative to a second sequence if the two sequences contain one or more, e.g., at least 1, 2, 3, 4, or 5 mismatched base pairs upon hybridization, forming a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp), while retaining the ability to hybridize under conditions most relevant to the end use, e.g., inhibition of PNPLA3 gene expression via the RISC pathway.

[0114] As used herein, the term "partially complementary" can be used to refer to a pair of hybridized nucleobase sequences in which at least 75%, but not all, of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide.

[0115] As used herein, the terms "complementary," "fully complementary," "substantially complementary," and "partially complementary" are used in reference to base matches between the sense and antisense strands of a PNPLA3 dsRNA agent, between the antisense strand of a PNPLA3 dsRNA agent and the sequence of a target PNPLA3 mRNA, or between a single-stranded antisense oligonucleotide and the sequence of a target PNPLA3 mRNA. It will be understood that the term "antisense strand of a PNPLA3 dsRNA agent" can refer to the same sequence of a "PNPLA3 antisense polynucleotide agent."

[0116] As used herein, the term "substantially identical" or "substantial identity" in reference to a nucleic acid sequence refers to a nucleic acid sequence that contains a sequence having at least about 85% sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, relative to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions where identical nucleic acid bases occur in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. The inventions disclosed herein encompass nucleotide sequences that are substantially identical to those disclosed herein, for example, in Tables 1-3. In some embodiments, the sequences disclosed herein are completely identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, those disclosed herein, e.g., in Tables 1-3.

[0117] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referenced using standard nucleotide nomenclature. The term "double-stranded RNA" or "dsRNA" as used herein refers to an RNAi comprising an RNA molecule or molecular complex having a hybridized duplex region comprising two antiparallel and substantially or completely complementary nucleic acid strands, said to have "sense" and "antisense" orientations relative to the target PNPLA3 RNA. The duplex region can be any length that allows for specific degradation of the desired target PNPLA3 RNA via the RISC pathway, but is typically 9-30 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 30 base pairs, the duplex can be any length within this range, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-23 base pairs, 18-22 base pairs, 18-24 base pairs, 18-26 base pairs, 18-24 base pairs, 18-28 base pairs, 18-30 base pairs, 18-32 base pairs, 18-34 base pairs, 18-36 base pairs, 18-38 base pairs, 18-39 base pairs, 18-40 base pairs, 18-41 base pairs, 18-42 base pairs, 18-43 base pairs, 18-44 base pairs, 18-45 base pairs, 18-46 base pairs, 18-47 base pairs, 18-48 base pairs, 18-49 base pairs, 18-50 base pairs, 18-51 base pairs, 18-52 base pairs, 18-53 base pairs, 18-54 base pairs, 18-55 base pairs, 18-56 base pairs, 18-57 base pairs, 18- The length of the PNPLA3 dsRNA agent can be any subrange therebetween, including, but not limited to, 8-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. PNPLA3 dsRNA agents produced in cells by processing with Dicer and similar enzymes are typically in the 19-22 base pair range. One strand of the duplex region of a PNPLA3 dsDNA agent contains a sequence that is substantially complementary to a region of the target PNPLA3 RNA. The two strands that form the duplex structure can be formed from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules.When the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single strand of nucleotides (referred to herein as a "hairpin loop") between the 3' end of one strand and the 5' end of the other strand that forms the duplex structure. In some embodiments of the present invention, the hairpin loop contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more unpaired nucleotides. When the two substantially complementary strands of a PNPLA3 dsRNA agent are composed of separate RNA molecules, the molecules do not need to be covalently connected, but can be. When the two strands are covalently connected by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" is also used herein to refer to the dsRNA agents described herein.

[0118] In some embodiments of the present invention, a PNPLA3 dsRNA agent may comprise a sense sequence and an antisense sequence that do not have unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. An end without unpaired nucleotides is referred to as a "blunt end" and is considered to have no nucleotide overhangs. If both ends of a dsRNA agent are blunt, the dsRNA is referred to as a "blunt end." In some embodiments of the present invention, the first end of the dsRNA agent is blunt, and in some embodiments, the second end of the dsRNA agent is blunt, and in certain embodiments of the present invention, both ends of a PNPLA3 dsRNA agent are blunt.

[0119] In some embodiments of the dsRNA agent of the present invention, the dsRNA does not have one or two blunt ends. In such cases, at least one unpaired nucleotide is present at the end of the strand of the dsRNA agent. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA can include an overhang of at least 1, 2, 3, 4, 5, 6, or more nucleotides. The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. In some embodiments, the nucleotide overhang is located on the sense strand of the dsRNA agent, on the antisense strand of the dsRNA agent, or at both ends of the dsRNA agent, and it will be understood that the nucleotides of the overhang can be located at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA. In certain embodiments of the present invention, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0120] As used herein, the term "antisense strand" or "guide strand" refers to the strand of a PNPLA3 dsRNA agent that contains a region that is substantially complementary to a PNPLA3 target sequence.As used herein, the term "sense strand" or "passenger strand" refers to the strand of a PNPLA3 dsRNA agent that contains a region that is substantially complementary to a region of the antisense strand of a PNPLA3 dsRNA agent.

[0121] qualification In some embodiments of the present invention, the RNA of the PNPLA3 RNAi agent is chemically modified to enhance stability and / or one or more other beneficial properties. In certain embodiments of the present invention, the nucleic acids 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, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that can be present in certain embodiments of the PNPLA3 dsRNA agent of the present invention include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotide, reverse linkage, etc.), (b) base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanded repertoire of partners, removal of a base (abasic nucleotide), or conjugated base, (c) sugar modifications (e.g., 2'-position or 4'-position) or sugar substitution, and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of RNA compounds useful in certain embodiments of the PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and PNPLA3 sense polynucleotide of the present invention include, but are not limited to, RNAs containing modified backbones or non-natural internucleoside linkages. As a non-limiting example, RNAs with modified backbones may not have a phosphorus atom in the backbone. RNAs that do not have a phosphorus atom in their internucleoside backbone may be referred to as oligonucleosides. In certain embodiments of the invention, modified RNAs have a phosphorus atom in their internucleoside backbone.

[0122] It will be understood that the term "RNA molecule" or "RNA" or "ribonucleic acid molecule" includes not only RNA molecules expressed or found in nature, but also RNA analogs and derivatives that contain one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. As used herein, the terms "ribonucleoside" and "ribonucleotide" may be used interchangeably. RNA molecules can be modified, for example, within the nucleobase structure or the ribose-phosphate backbone structure, as described herein below, and molecules that contain ribonucleoside analogs or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecules can also include at least one modified ribonucleoside, including, but not limited to, a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate, or a non-natural base containing nucleoside, or any combination thereof. In some embodiments of the invention, an RNA molecule includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to the full length of ribonucleosides in a PNPLA3 dsRNA agent molecule, modified ribonucleosides. The modifications need not be identical for each of the multiple modified ribonucleosides in such an RNA molecule.

[0123] In some embodiments, the dsRNA agents, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides of the present invention may contain one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester linkages. As used herein, the terms "internucleotide linkage," "internucleoside linkage," "linkage," and "linker" are used interchangeably and refer to linking groups between unmodified or modified nucleosides and / or between unmodified or modified nucleosides and one or more targeting groups. In some embodiments, linkages may be independently selected from phosphodiester (PO) linkages, phosphorothioate (PS) linkages, and / or phosphorodithioate (PS2) linkages of dinucleotides at any position in a single-stranded or double-stranded oligonucleotide. As used herein, the term "independently selected" in reference to selected elements such as modified nucleotides and non-phosphodiester linkages means that two or more selected elements can be identical to each other, but are not necessarily identical.

[0124] As used herein, a "nucleotide base," "nucleotide," or "nucleobase" refers to a heterocyclic pyrimidine or purine compound that is a standard component of all nucleic acids, including the bases that form the nucleotides adenine, guanine, cytosine, thymine, and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases. As used herein, the term "ribonucleotide" or "nucleotide" can refer to an unmodified nucleotide, a modified nucleotide, or an alternative replacement moiety. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base pairing properties of an oligonucleotide containing a nucleotide having such a replacement moiety.

[0125] As used herein, "optionally" or "optionally" means that the event or circumstance described below may occur, but need not occur, and includes cases where the event or circumstance occurs or does not occur. For example, "C alkyl optionally substituted with halogen or cyano" means that halogen or cyano may be present, but is not necessarily present, and includes cases where the alkyl is substituted with halogen or cyano and cases where the alkyl is not substituted with halogen and cyano.

[0126] In the chemical structures of the compounds of the present disclosure, the bonds used herein are [ka] represents a non-specific configuration, i.e., if chiral isomers exist in a chemical structure, the bond [ka] teeth, [ka] or [ka] Some of the structural formulas above are depicted as isomers for simplicity, but the present disclosure includes all isomers, including tautomers, rotamers, and mixtures thereof. Suitable chiral compounds include geometric isomers, diastereomers, racemates, and enantiomers.

[0127] As used herein, the compounds used in the chemical formulas of the present disclosure [ka] may be attached to any one or more groups in accordance with the scope of the invention described herein.

[0128] In one embodiment, the modified RNA considered for use in the methods and compositions described herein is peptide nucleic acid (PNA) that has the ability to form the required double-stranded structure, and allows or mediates the specific degradation of target RNA via the RISC pathway.In certain embodiments of the present invention, PNPLA3 RNA interference agents include single-stranded RNA that interacts with target PNPLA3 RNA sequence and induces the cleavage of target PNPLA3 RNA.

[0129] Modified RNA backbones can 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'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, in which 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. Means for preparing phosphorus-containing linkages are routinely practiced in the art, and such methods can be used to prepare the specific modified PNPLA3 dsRNA agents, specific modified PNPLA3 antisense polynucleotides, and / or specific modified PNPLA3 sense polynucleotides of the invention.

[0130] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages, including morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH moieties. Means for preparing modified RNA backbones that do not contain a phosphorus atom are routinely practiced in the art, and such methods can be used to prepare the specific modified PNPLA3 dsRNA agents, specific modified PNPLA3 antisense polynucleotides, and / or specific modified PNPLA3 sense polynucleotides of the invention.

[0131] In certain embodiments of the present invention, PNPLA3 dsRNA, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide contain RNA mimics, including, but not limited to, replacement of the sugar and internucleoside linkage, i.e., backbone, of nucleotide units with novel groups. In such embodiments, the base units are maintained for hybridization with appropriate PNPLA3 nucleic acid target compounds. One such oligomeric compound is an RNA mimic, known as a peptide nucleic acid (PNA), which has been shown to have excellent hybridization properties. In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Means for preparing RNA mimics are routinely practiced in the art, and such methods can be used to prepare certain modified PNPLA3 dsRNA agents of the present invention.

[0132] Some embodiments of the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, specifically -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene(methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2- [the natural phosphodiester backbone is represented as -OPO-CH2-]. Means for preparing RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare specific modified PNPLA3 dsRNA agents, specific PNPLA3 antisense polynucleotides, and / or specific PNPLA3 sense polynucleotides of the invention.

[0133] Modified RNAs can contain one or more substituted sugar moieties. The PNPLA3 dsRNAs, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides of the present invention can contain any 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It may be alkenyl or alkynyl. Examples of suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, and n and m are from 1 to about 10. In other embodiments, the dsRNA comprises any of the following at the 2' position: C1 to C 10lower 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, RNA cleaving group, reporter group, intercalator, group that improves the pharmacokinetic properties of a PNPLA3 dsRNA agent, or group for improving the pharmacodynamic properties of a PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide, and other substituents with similar properties. In some embodiments, modifications include 2'-methoxyethoxy (2'-O-CH2CHOCH3, 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., O(CH2)2ON(CH3)2, known as 2'-DMAOE and described herein in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2). Means for preparing modified RNAs as described are routinely practiced in the art, and such methods can be used to prepare certain modified PNPLA3 dsRNA agents of the invention.

[0134] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of a PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide of the invention, particularly on the 3'-terminal nucleotide or the 3' position of the sugar of a 2'-5'-linked PNPLA3 dsRNA, PNPLA3 antisense polynucleotide, or PNPLA3 sense polynucleotide, and the 5' position of the 5'-terminal nucleotide. PNPLA3 dsRNA agents, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Means for preparing modified RNAs as described are routinely practiced in the art, and such methods can be used to prepare specific modified PNPLA3 dsRNA agents, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides of the invention.

[0135] PNPLA3 dsRNA agents, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides may, in some embodiments, include modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine, and uracil. Modified nucleobases include 5-methylcytosine (5-Me-C), 5-hydroxymethylcytosine, 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), and cytosine (cytosine). Other synthetic and natural nucleobases include uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine, and 3-deazaguanine and 3-deazaadenine.Additional nucleobases that can be included in certain embodiments of the PNPLA3 dsRNA agents of the invention are known in the art, see, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JL, Ed. John Wiley & Sons, 1990; English et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B. Ed., CRC Press, 1993. Means for preparing dsRNA, PNPLA3 antisense strand polynucleotides, and / or PNPLA3 sense strand polynucleotides containing nucleobase modifications and / or substitutions as described herein are routinely practiced in the art, and such methods can be used to prepare specific modified PNPLA3 dsRNA agents, PNPLA3 sense polynucleotides, and / or PNPLA3 antisense polynucleotides of the invention.Teachings regarding the synthesis of specific modified oligonucleotides can be found in the following publications: U.S. Pat. No. 5,218,105 for polyamine-conjugated oligonucleotides; U.S. Pat. No. 5,541,307 for oligonucleotides with modified backbones; U.S. Pat. No. 5,521,302 for processes for preparing oligonucleotides with chiral phosphorus linkages; U.S. Pat. No. 5,539,082 for peptide nucleic acids; U.S. Pat. No. 5,554,746 for oligonucleotides with 3-lactam backbones; U.S. Pat. No. 5,571,902 for methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,571,902 for nucleosides having alkylthio groups, which may be used as linkers to other moieties attached at any of various positions on the nucleoside; No. 8,718, U.S. Pat. No. 5,587,361 relating to oligonucleotides having phosphorothioate linkages of high chiral purity, U.S. Pat. No. 5,506,351 relating to a process for preparing 2'-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds, U.S. Pat. No. 5,587,469 relating to oligonucleotides having N-2 substituted purines, U.S. Pat. No. 5,587,470 relating to oligonucleotides having 3-deazapurines, U.S. Pat. No. 5,608,046 both relating to conjugated 4'-desmethyl nucleoside analogs, U.S. Pat. No. 5,610,289 relating to backbone-modified oligonucleotide analogs, and U.S. Pat. No. 6,262,241 relating to a method for synthesizing 2'-fluoro oligonucleotides, among others.

[0136] Certain embodiments of the PNPLA3 dsRNA agents, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides of the present invention include RNAs modified to contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural conformation. The addition of a locked nucleic acid to a PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide of the invention can improve serum stability and 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). Means for preparing dsRNA agents, PNPLA3 antisense polynucleotides, and / or PNPLA3 sense polynucleotides containing locked nucleic acids are routinely practiced in the art, and such methods can be used to prepare certain modified PNPLA3 dsRNA agents of the invention.

[0137] Certain embodiments of the PNPLA3 dsRNA compounds, sense polynucleotides, and / or antisense polynucleotides of the present invention include at least one modified nucleotide, and the at least one modified nucleotide is selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seconucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, mopholino nucleotides, and 3'-OMe nucleotides, nucleotides containing 5'-phosphorothioate groups, nucleosides containing vinyl phosphonates, and the like. The nucleotides include nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S-isomers, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, nucleotides containing 2'-deoxyadenosine-3'-phosphate, nucleotides containing 2'-deoxycytidine-3'-phosphate, nucleotides containing 2'-deoxyuridine-3'-phosphate, or terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2'-amino-modified nucleotides, phosphoramidates, or unnatural bases including nucleotides. In some embodiments, the PNPLA3 dsRNA compound includes an E-vinylphosphonate nucleotide at the 5' end of the antisense strand, also referred to herein as the guide strand.

[0138] Certain embodiments of the PNPLA3 dsRNA compounds of the present invention, the 3' and 5' ends of the sense polynucleotide, and / or the 3' end of the antisense polynucleotide contain at least one modified nucleotide, including an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, or an inverted 2'-deoxynucleotide. Those skilled in the art know that including an abasic nucleotide or an inverted abasic nucleotide at the end of an oligonucleotide improves stability (Czauderna et al., Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003;31(11):2705-2716, doi:10.1093 / nar / gkg393). In some embodiments, the PNPLA3 dsRNA compounds contain one or more inverted abasic residues (invab) at either the 3' or 5' end, or at both the 3' and 5' ends. Examples of inverted abasic residues (invabs) include, but are not limited to:

[0139] [ka] Certain embodiments of the PNPLA3 dsRNA compounds of the present invention, the 3' and 5' ends of the sense polynucleotides, and / or the 3' end of the antisense polynucleotides comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises an isomannide nucleotide. Specific examples of isomannide nucleotides include, but are not limited to, the following:

[0140] [ka] wherein the term "Olig" independently represents a polynucleotide moiety. Exemplary isomannide residues (imanes) include, but are not limited to:

[0141] [ka]

[0142] Certain embodiments of the PNPLA3 dsRNA compounds and antisense polynucleotides of the present invention include at least one modified nucleotide, including an unlocked nucleic acid nucleotide (UNA) and / or a glycol nucleic acid nucleotide (GNA). Those skilled in the art are aware that UNA and GNA are thermally destabilizing chemical modifications that can significantly improve the off-target profile of siRNA compounds (Janas et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018;9(1):723. doi:10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010;6:862-70).

[0143] Certain embodiments of the PNPLA3 dsRNA compound of the present invention, antisense polynucleotide, further comprise a phosphate moiety.As used herein, phosphate moiety refers to the phosphate group that comprises phosphate or phosphate mimic attached to the sugar moiety (for example, ribose or deoxyribose or their analogues) of nucleotide.The nucleotide that comprises phosphate mimic can also be defined as phosphonate-modified nucleotide.

[0144] In some embodiments, the phosphate mimetic is a 5'-vinyl phosphonate (VP). In an exemplary embodiment, a vinyl phosphonate of the disclosure has the following structure:

[0145] [ka] The vinyl phosphonates of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain preferred embodiments, the vinyl phosphonates of the present disclosure are attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0146] In certain embodiments, the vinylphosphonate-modified nucleotides of the present disclosure have the structure of formula (IV):

[0147] [ka] X is O or S; R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy); R5' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z orientation (e.g., the E orientation); and B is a nucleobase or a modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0148] In certain embodiments, R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E orientation. In certain embodiments, R is methoxy, R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E orientation. In certain embodiments, X is S, R is methoxy, R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E orientation.

[0149] Vinyl phosphonate modifications are also contemplated in the dsRNAs, compositions, and methods of this disclosure. Exemplary vinyl phosphonate structures are as follows:

[0150] [ka] In certain embodiments, the vinylphosphonate modified nucleotide is VPu*, which has the structure: [ka]

[0151] Protecting groups are often used during the preparation of the compounds of the present invention. As used herein, the term "protected" means that a protecting group is attached to the indicated moiety. In some embodiments of the present invention, the compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the present invention. Generally, protecting groups render chemical functional groups inert to specific reaction conditions and can be added to or removed from such functional groups within a molecule without causing substantial damage to the remainder of the molecule. Protecting groups in general, and hydroxyl protecting groups in particular, are well known in the art (Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2nd Edition, John Wiley & Sons, New York, 1991).

[0152] Examples of protecting groups (e.g., hydroxyl protecting groups) used herein include methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, tert-butoxymethyl, methoxymethyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, allyl, cyclohexyl, 9-fluorenylmethoxycarbonyl (Fmoc), methanesulfonate, toluenesulfonate, triflate, benzoyl, benzoylformate, p-phenylbenzoyl, 4-methoxybenzyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 4-chlorobenzyl, 4-nitrobenzyl, 2,4-dinitrophenyl, 4-acyloxybenzyl, 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 2,6-dichlorobenzyl, diphenylmethyl, triphenylmethyl, 4-methylthio-1-butyl, S-acetylthioacetate (SATA), 2- Cyanoethyl, 2-cyanol, 1-dimethylethyl (CDM), 4-cyano-2-butenyl, 2-(trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl, 2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2,2-trifluoroethyl, phenylthio, 2-chloro-4-tritylphenyl, 2-bromophenyl, 2-[N-isopropyl-N-(4- 4-(N-trifluoroacetylamino)butyl, 4-oxopentyl, 4-tritylaminophenyl, 4-benzylaminophenyl, tetrahydropyranyl, morpholino, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, pivaloyloxymethyl (POM), and 9-phenylxanthin-9-yl.

[0153] As used herein, examples of amino-protecting groups include, but are not limited to, carbamate protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butyloxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, pivaloyl, trihaloacetyl, benzoyl, and 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups such as phthalimide and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the present invention.

[0154] Another modification that can be included in the RNA of certain embodiments of the PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide of the present invention includes chemically linking one or more ligands, moieties, or conjugates to the RNA, which each enhance one or more properties of the PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide. Non-limiting examples of properties that can be enhanced include the activity of the PNPLA3 dsRNA agent, PNPLA3 antisense polynucleotide, and / or PNPLA3 sense polynucleotide, cellular distribution, delivery of the PNPLA3 dsRNA agent, the pharmacokinetic properties of the PNPLA3 dsRNA agent, and the cellular uptake of the PNPLA3 dsRNA agent. In some embodiments of the present invention, the PNPLA3 dsRNA agent includes one or more targeting groups or linking groups, which are conjugated to the sense strand in certain embodiments of the PNPLA3 dsRNA agent of the present invention. Non-limiting examples of targeting groups include compounds containing N-acetylgalactosamine (GalNAc). The terms "targeting group," "targeting agent," "linking agent," "targeting compound," and "targeting ligand" may be used interchangeably herein. In certain embodiments of the invention, a PNPLA3 dsRNA agent includes a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the invention, a PNPLA3 dsRNA agent includes a targeting compound conjugated to the 3'-end of the sense strand. In some embodiments of the invention, a PNPLA3 dsRNA agent includes a targeting group containing GalNAc. In certain embodiments of the invention, a PNPLA3 dsRNA agent does not include a targeting compound conjugated to either or both of the 3'-end and 5'-end of the sense strand. In certain embodiments of the invention, a PNPLA3 dsRNA agent does not include a GalNAc-containing targeting compound conjugated to either or both of the 5'-end and 3'-end of the sense strand.

[0155] Additional targeting agents and linking agents are well known in the art; for example, targeting agents and linking agents that may be used in certain embodiments of the invention include cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, 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), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains such as dodecanediol 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), phospholipids such as 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), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or lipid moieties such as octadecylamine or hexylaminocarbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0156] Certain embodiments of compositions comprising a PNPLA3 dsRNA agent, a PNPLA3 antisense polynucleotide, and / or a PNPLA3 sense polynucleotide may include a ligand that alters the distribution, targeting, etc. of the PNPLA3 dsRNA agent. In some embodiments of compositions comprising a PNPLA3 dsRNA agent of the invention, the ligand enhances affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, compared to a species in which such ligand is absent. Ligands useful in the compositions and / or methods of the invention can be naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids or polyamines. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0157] The ligand included in the compositions and / or methods of the invention may comprise a targeting group, non-limiting examples of which include a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a kidney cell or liver cell. The targeting group can be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0158] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, etc.), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric 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)cholenoic acid, dimethoxytrityl, phenoxazine, etc.), 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 enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0159] The ligand included in the compositions and / or methods of the invention may be a protein, e.g., a glycoprotein or peptide, e.g., a molecule with a specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specific cell type, such as cancer cells, endothelial cells, cardiac cells, or bone cells. Ligands useful in embodiments of the compositions and / or methods of the invention may be hormones or hormone receptors. Ligands useful in embodiments of the compositions and / or methods of the invention may be lipids, lectins, carbohydrates, vitamins, coenzymes, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine, multivalent mannose, or multivalent fucose. Ligands useful in embodiments of the compositions and / or methods of the invention may be substances that can increase cellular uptake of a PNPLA3 dsRNA agent, for example, by disrupting the cytoskeleton of a cell, e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments. Non-limiting examples of such agents include taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, and myoservin.

[0160] In some embodiments, the ligand attached to the PNPLA3 dsRNA agent of the present invention functions as a pharmacokinetic (PK) regulator. Examples of PK regulators that can be used in the compositions and methods of the present invention include, but are not limited to, lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and aptamers that bind to serum proteins. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins, and therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5, 10, 15, or 20 bases, can also be used as ligands in the compositions and / or methods of the present invention.

[0161] PNPLA3 dsRNA agent compositions In some embodiments of the present invention, the PNPLA3 dsRNA agent is present in a composition. The compositions of the present invention may include one or more PNPLA3 dsRNA agents and, optionally, one or more of a pharmaceutically acceptable carrier, a delivery agent, a targeting agent, a detectable label, etc. Non-limiting examples of targeting agents that may be useful in some embodiments of the methods of the present invention include agents that direct the PNPLA3 dsRNA agent of the present invention to and / or within cells to be treated. The targeting agent selected will depend on factors such as the nature of the PNPLA3-related disease or condition and the type of cell to be targeted. As a non-limiting example, in some embodiments of the present invention, it may be desirable to target the PNPLA3 dsRNA agent to and / or within liver cells. In some embodiments of the methods of the present invention, the therapeutic agent will be understood to include a PNPLA3 dsRNA agent that includes only a delivery agent, such as a delivery agent containing N-acetylgalactosamine (GalNAc), without any additional attachment elements. For example, in some embodiments of the invention, a PNPLA3 dsRNA agent may be attached to a delivery compound that includes GalNAc, included in a composition that includes a pharmaceutically acceptable carrier, and administered to a cell or subject without a detectable label or targeting agent attached to the PNPLA3 dsRNA agent.

[0162] When the PNPLA3 dsRNA agent of the present invention is administered with and / or attached to one or more delivery agents, targeting agents, labeling agents, etc., skilled artisans can recognize and select suitable agents for use in the methods of the present invention. Labeling agents may be used to determine the location of the PNPLA3 dsRNA agent in cells and tissues in certain methods of the present invention, and may also be used to determine the location of a therapeutic composition containing a PNPLA3 dsRNA agent administered in a method of the present invention in cells, tissues, or organs. Procedures for attaching and utilizing labeling agents, such as enzyme labels, dyes, and radioactive labels, are well known in the art. It will be understood that in some embodiments of the compositions and methods of the present invention, a labeling agent is attached to one or both of the sense polynucleotide and the antisense polynucleotide contained in the PNPLA3 dsRNA agent.

[0163] Delivery of PNPLA3 dsRNA and PNPLA3 antisense polynucleotide agents Certain embodiments of the method of the present invention include delivering PNPLA3 dsRNA agents into cells. As used herein, the term "delivery" refers to promoting or achieving cellular uptake or absorption. Absorption or uptake of PNPLA3 dsRNA agents can occur through unassisted diffusion processes or active cellular processes, or by using delivery agents, targeting agents, etc., which can be associated with the PNPLA3 dsRNA agents of the present invention. Delivery means suitable for use in the method of the present invention include, but are not limited to, in vivo delivery, in which the PNPLA3 dsRNA agent is injected into a tissue site or administered systemically. In some embodiments of the present invention, the PNPLA3 dsRNA agent is attached to a delivery agent.

[0164] Non-limiting examples of methods that can be used to deliver PNPLA3 dsRNA agents to cells, tissues, and / or subjects include PNPLA3 dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents for the treatment of various diseases and conditions, including, but not limited to, liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. Details of various delivery means can be found in publications such as Nikam, R.R. & K.R. Gore (2018) Nucleic Acid Ther, 28(4), 209-224, Aug 2018; Springer A.D. & S.F. Dowdy (2018) Nucleic Acid Ther. Jun 1;28(3):109-118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; and Nair, J.K. et al., (2014) J.Am.Chem.Soc. 136:16958-16961, the contents of each of which are incorporated herein by reference.

[0165] Some embodiments of the present invention include using lipid nanoparticles (LNPs) to deliver PNPLA3 dsRNA agents of the present invention to cells, tissues, and / or subjects. LNPs are routinely used for in vivo delivery of PNPLA3 dsRNA agents, including therapeutic PNPLA3 dsRNA agents. One advantage of using LNPs or other delivery agents is that the stability of the PNPLA3 RNA agent is improved when delivered to a subject using LNPs or other delivery agents. In some embodiments of the present invention, the LNPs comprise cationic LNPs loaded with one or more PNPLA3 RNAi molecules of the present invention. When the LNPs containing the PNPLA3 RNAi molecules are administered to a subject, the LNPs and the attached PNPLA3 RNAi molecules are taken up into the cells by endocytosis, resulting in the release of the RNAi trigger molecule due to their presence, thereby mediating RNAi.

[0166] Another non-limiting example of a delivery agent that can be used in embodiments of the present invention to deliver a PNPLA3 dsRNA agent of the present invention to a cell, tissue, and / or subject is an agent comprising at least one GalNAc targeting ligand attached to the PNPLA3 dsRNA agent of the present invention and delivering the PNPLA3 dsRNA agent to a cell, tissue, and / or subject. Additional examples of specific delivery agents containing GalNAc that can be used in certain embodiments of the methods and compositions of the present invention are disclosed in PCT Application WO2020191183A1 (incorporated herein in its entirety). Non-limiting examples of GalNAc targeting ligands that can be used in the compositions and methods of the present invention to deliver a PNPLA3 dsRNA agent to a cell include targeting ligand clusters. Examples of targeting ligand clusters provided herein are referred to as GalNAc ligands with phosphodiester linkages (GLO) and GalNAc ligands with phosphorothioate linkages (GLS). The term "GLX-n" may be used herein to indicate that the attached GalNAc-containing compound is any one of compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are shown below, with the right-hand end of each structure indicating the position of attachment of a GalNAc targeting ligand to an RNAi agent of the invention ( (View in JPEG2025542119000060.jpg66). It will be understood that any RNAi and dsRNA molecule of the present invention can be attached to GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16. The structures of GLO-1 through GLO-16 and GLS-1 through GLS-16 are shown below.

[0167] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0168] In certain embodiments, the aforementioned isomannide nucleotides may be further conjugated to one or more GalNAc targeting ligands. Specific examples of isomannide nucleotides conjugated to GalNAc targeting ligands include: [ka] and each term "olig" independently represents a polynucleotide portion.

[0169] In some embodiments of the present invention, in vivo delivery can be achieved by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005 / 0281781, which are incorporated herein by reference in their entireties. In vitro introduction of PNPLA3 RNAi agents into cells can be achieved using known methods, such as electroporation and lipofection. In certain embodiments of the methods of the present invention, PNPLA3 dsRNAs are delivered without a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, the PNPLA3 dsRNAs of the present invention can be administered to a subject in the form of a pharmaceutical composition containing an RNAi agent but not a targeting agent, such as a GalNAc targeting compound, to treat a PNPLA3-related disease or condition, such as liver disease, in the subject.

[0170] In addition to the specific delivery means described herein, it will be understood that RNAi delivery means, such as, but not limited to, those described herein and those used in the art, can be used with the PNPLA3 RNAi agent and method of treatment embodiments described herein.

[0171] The PNPLA3 dsRNA agent of the present invention can be administered to a subject in an amount and manner effective to reduce the level and activity of PNPLA3 polypeptide in cells and / or subjects.In some embodiments of the method of the present invention, one or more PNPLA3 dsRNA agents are administered to cells and / or subjects to treat diseases or conditions related to PNPLA3 expression and activity.In some embodiments, the method of the present invention comprises administering one or more PNPLA3 dsRNA agents to a subject in need of such treatment to alleviate diseases or conditions related to PNPLA3 expression in the subject.The PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention can be administered to reduce PNPLA3 expression and / or activity in one or more of in vitro, ex vivo, and in vivo cells.

[0172] In some embodiments of the present invention, delivering (e.g., introducing) a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent into a cell reduces the level and, therefore, activity of PNPLA3 polypeptide within the cell. Targeting agents and methods can be used to assist in the delivery of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent to specific cell types, cell subtypes, organs, or spatial regions within a subject and / or a subcellular region within a cell. In certain methods of the present invention, a PNPLA3 dsRNA agent can be administered alone or in combination with one or more additional PNPLA3 dsRNA agents. In some embodiments, two, three, four, or more independently selected PNPLA3 dsRNA agents are administered to a subject.

[0173] In certain embodiments of the present invention, a PNPLA3 dsRNA agent is administered to a subject in combination with one or more additional therapeutic regimens for treating PNPLA3-related diseases or symptoms, thereby treating PNPLA3-related diseases or symptoms. Non-limiting examples of additional therapeutic regimens include administering one or more PNPLA3 antisense polynucleotides of the present invention, administering a non-PNPLA3 dsRNA therapeutic agent, and behavioral modification. The additional therapeutic regimen can be administered at one or more time points before, simultaneously with, and after the administration of the PNPLA3 dsRNA agent of the present invention. As used herein, "simultaneous" refers to within 5 minutes from time zero, within 10 minutes from time zero, within 30 minutes from time zero, within 45 minutes from time zero, and within 60 minutes from time zero, and "time zero" is understood to refer to the time when the PNPLA3 dsRNA agent of the present invention is administered to the subject. Non-limiting examples of non-PNPLA3 dsRNA therapeutic agents include HMG-CoA reductase inhibitors, fibrates, bile acid sequestrants, niacin, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol regulators, bile acid regulators, peroxisome proliferator-activated receptor (PPAR) agonists, gene-based therapy, combined vascular protective agents, glycoprotein IIb / IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors, monocyte chemoattractant protein (MCP)-I inhibitors, or fish oil, and therapeutic agents that can reduce PNPLA3 levels and / or accumulation in subjects. Non-limiting examples of behavioral modification include dietary therapy, counseling, and exercise therapy. These and other therapeutic agents and behavioral modifications are known in the art and are used to treat a PNPLA3 disease or condition in a subject, and may be administered to a subject in combination with administration of one or more PNPLA3 dsRNA agents of the invention to treat a PNPLA3 disease or condition.The PNPLA3 dsRNA agents of the present invention administered to a cell or subject to treat a PNPLA3-associated disease or condition may act synergistically with one or more other therapeutic agents or therapeutic activities to enhance the effectiveness of the one or more therapeutic agents or therapeutic activities and / or enhance the effectiveness of the PNPLA3 dsRNA agent in treating a PNPLA3-associated disease or condition.

[0174] Therapeutic methods of the invention involving administration of a PNPLA3 dsRNA agent can be used before the onset of a PNPLA3-related disease or condition and / or when a PNPLA3-related disease or condition is present, including the early, middle, and late stages of the disease or condition, as well as any time point before or after these stages. The methods of the invention can also treat subjects who have previously been treated for a PNPLA3-related disease or condition with one or more other therapeutic agents and / or therapeutic activities, but which have been ineffective, have had little effect, and / or are no longer effective in treating the PNPLA3-related disease or condition in the subject.

[0175] Vector-encoded dsRNA In certain embodiments of the present invention, PNPLA3 dsRNA agents can be delivered into cells using vectors. The PNPLA3 dsRNA agent transcription unit can be contained in a DNA or RNA vector. The preparation and use of such vectors encoding transgenes for delivering sequences to cells and / or subjects is well known in the art. In the methods of the present invention, vectors can be used to transiently express PNPLA3 dsRNA, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. The length of transient expression can be determined using routine methods based on factors such as, but not limited to, the specific vector construct selected and the target cell and / or tissue. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. Transgenes can also be constructed to be inherited as extrachromosomal plasmids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0176] One or more individual strands of PNPLA3 dsRNA agent can be transcribed from a promoter on an expression vector.For example, when expressing two separate strands to produce dsRNA, two separate expression vectors can be co-introduced into cells using means such as transfection or infection.In certain embodiments, each individual strand of the PNPLA3 dsRNA agent of the present invention can be transcribed by a promoter contained in the same expression vector.In certain embodiments of the present invention, the PNPLA3 dsRNA agent is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence, so that the PNPLA3 dsRNA agent has a stem and loop structure.

[0177] Non-limiting examples of RNA expression vectors include DNA plasmids or viral vectors.The expression vectors useful in the embodiments of the present invention are compatible with eukaryotic cells.Eukaryotic cell expression vectors are routinely used in the art and are available from many commercial sources.The PNPLA3 dsRNA expression vector can be delivered systemically, such as by intravenous or intramuscular administration, by administration to target cells removed from a subject and then reintroduced into the subject, or by any other means that allows introduction into desired target cells.

[0178] Viral vector systems that can be included in method embodiments include, but are not limited to, (a) adenoviral vectors, (b) retroviral vectors, including but not limited to lentiviral vectors and Moloney murine leukemia virus, (c) adeno-associated virus vectors, (d) herpes simplex virus vectors, (e) SV40 vectors, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox, and (j) helper-dependent or helpless adenoviruses. Constructs for recombinant expression of PNPLA3 dsRNA agents may include regulatory elements, such as promoters and enhancers, which can be selected to provide constitutive or regulated / inducible expression. The use of viral vector systems, promoters, and enhancers is routine in the art and can be used in conjunction with the methods and compositions described herein.

[0179] Certain embodiments of the present invention include the use of viral vectors to deliver PNPLA3 dsRNA agents to cells.In the art, a number of adenovirus-based delivery systems are routinely used, for example, to deliver to lung, liver, central nervous system, endothelial cells, muscle, etc.The non-limiting examples of viral vectors that can be used in the method of the present invention include AAV vectors, smallpox viruses such as vaccinia virus, modified virus Ankara (MVA), NYVAC, avian pox viruses such as fowl pox or canary pox.

[0180] Certain embodiments of the present invention include the method of using vector to deliver PNPLA3 dsRNA agent to cell, and such vector can be present in a pharmaceutically acceptable carrier, which can comprise, but does not necessarily comprise, the slow-release matrix in which gene delivery vehicle is embedded.In some embodiments, the vector for delivering PNPLA3 dsRNA can be produced from recombinant cells, and the pharmaceutical composition of the present invention can comprise one or more cells that produce PNPLA3 dsRNA delivery system.

[0181] Pharmaceutical compositions containing PNPLA3 dsRNA or ssRNA agents Certain embodiments of the present invention include the use of pharmaceutical compositions containing a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent can be used in the methods of the present invention to reduce intracellular PNPLA3 gene expression and PNPLA3 activity, and are useful for treating PNPLA3-related diseases or conditions. Such pharmaceutical compositions can be formulated based on the delivery mode. Non-limiting examples of delivery mode formulations include compositions formulated for subcutaneous delivery, compositions formulated for systemic administration via parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, and compositions formulated for direct delivery into the brain. The administration of the pharmaceutical composition of the present invention for delivering PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent to cells can be carried out by one or more means, such as topical (for example, by transdermal patch), pulmonary, for example, by inhalation or insufflation of powder or aerosol by nebulizer, intratracheal, intranasal, epidermal and transdermal, oral or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, for example, subcutaneous administration via implantable device, or intracranial administration, for example, by intraparenchymal, intrathecal or intraventricular administration.PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent can also be delivered directly to target tissue, for example, directly to liver or directly to kidney. It will be understood that "delivering a PNPLA3 dsRNA agent" or "delivering a PNPLA3 antisense polynucleotide agent" to a cell includes direct delivery of the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent, respectively, as well as expression of the PNPLA3 dsRNA agent within the cell from an encoding vector delivered to the cell or by any suitable means by which the PNPLA3 dsRNA or PNPLA3 antisense polynucleotide agent becomes present within the cell. The preparation and use of formulations and means for delivering inhibitory RNA are well known and routinely used in the art.

[0182] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered agents, pharmaceutically acceptable carriers include pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives, but are not limited to these. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while cornstarch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are typically magnesium stearate, stearic acid, or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in the formulation are described further below.

[0183] As used herein, the terms "pharmacologically effective amount," "therapeutically effective amount," and "effective amount" refer to the amount of the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention to produce the intended pharmacological, therapeutic, or preventive results.For example, if a given clinical treatment is considered effective when the measurable parameter related to a disease or disorder is reduced by at least 10%, the therapeutically effective amount of the agent for treating that disease or disorder is the amount required to reduce that parameter by at least 10%.For example, a therapeutically effective amount of a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent can reduce the level of PNPLA3 polypeptide by at least 10%.

[0184] Effective doseIn some embodiments, the methods of the present invention include contacting cells with a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent in an amount effective to reduce PNPLA3 gene expression in the contacted cells. Certain embodiments of the methods of the present invention include administering a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent to a subject in an amount effective to reduce PNPLA3 gene expression and treat a PNPLA3-related disease or condition in the subject. An "effective amount" used to reduce PNPLA3 expression and / or treat a PNPLA3-related disease or condition is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent for treating a PNPLA3-related disease or condition can be the amount necessary (i) to slow or stop the progression of the disease or condition, or (ii) to reverse, alleviate, or eliminate one or more symptoms of the disease or condition. In some embodiments of the present invention, an effective amount is an amount of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent that, when administered to a subject in need of treatment for a PNPLA3-related disease or condition, results in a therapeutic response that prevents and / or treats the disease or condition. According to some embodiments of the present invention, an effective amount is an amount that, when administered in combination or simultaneously with another therapeutic treatment for a PNPLA3-related disease or condition, results in a therapeutic response that prevents and / or treats the disease or condition. In some embodiments of the present invention, the biological effect of treating a subject with a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent of the present invention can be an improvement and / or complete elimination of symptoms caused by a PNPLA3-related disease or condition. In some embodiments of the present invention, the biological effect is, for example, the complete disappearance of a PNPLA3-related disease or condition, as evidenced by a diagnostic test showing that the subject is free of a PNPLA3-related disease or condition. Non-limiting examples of physiological symptoms that can be detected include a decrease in PNPLA3 levels in the subject's liver after administration of an agent of the present invention.Additional means known in the art for assessing the status of a PNPLA3-associated disease or condition can be used to determine the effect of the agents and / or methods of the invention on a PNPLA3-associated disease or condition.

[0185] Typically, the effective amount of a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent that reduces PNPLA3 polypeptide activity to a level that can treat a PNPLA3-related disease or condition is determined in a clinical trial, and effective dosages are established for test and control populations in blinded studies. In some embodiments, an effective amount is an amount that produces a desired response, for example, an amount that alleviates a PNPLA3-related disease or condition in cells, tissues, and / or subjects with the disease or condition. Thus, an effective amount of a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent for treating a PNPLA3-related disease or condition that can be treated by reducing PNPLA3 polypeptide activity can be an amount that, when administered, can reduce PNPLA3 polypeptide activity in a subject to an amount lower than the amount that would be present in the cells, tissues, and / or subject if the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent were not administered. In certain aspects of the present invention, the level of PNPLA3 polypeptide activity and / or PNPLA3 gene expression present in cells, tissues, and / or subjects that have not been contacted with or administered a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention is referred to as a "control" amount. In some embodiments of the methods of the present invention, the control amount for a subject is the pre-treatment amount for the subject; in other words, the level in the subject before administration of the PNPLA3 agent serves as the control level for that subject, and can be compared with the PNPLA3 polypeptide activity and / or PNPLA3 gene expression level in the subject after administration of the siRNA to the subject. When treating a PNPLA3-related disease or condition, the desired response can be the reduction or elimination of one or more symptoms of the disease or condition in cells, tissues, and / or subjects. The reduction or elimination can be temporary or permanent. It is understood that the status of a PNPLA3-related disease or condition can be monitored using methods to determine PNPLA3 polypeptide activity, PNPLA3 gene expression, symptom assessment, clinical trials, etc. In some embodiments of the present invention, the desired response to treatment of a PNPLA3-related disease or condition is to delay or even prevent the onset of the disease or condition.

[0186] The effective amount of a compound that reduces PNPLA3 polypeptide activity can also be determined by assessing the physiological effects of administering a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent to a cell or subject, such as the reduction of PNPLA3-related disease or symptoms after administration. Subject assays and / or symptom monitoring can be used to determine the effectiveness of the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention that can be administered in the medical composition of the present invention, and to determine whether or not there is a response to treatment. Non-limiting examples include one or more art-known tests for alanine aminotransferase (ALT) or aspartate aminotransferase (AST) profile. As another non-limiting example, one or more known tests for liver function can be used to determine the status of a PNPLA3-related liver disease or condition in a subject before and after treatment with a PNPLA3 dsRNA agent of the present invention.

[0187] Some embodiments of the present invention include methods for determining the effectiveness of a dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention administered to a subject in treating a PNPLA3-related disease or condition by assessing and / or monitoring one or more "physiological characteristics" of the PNPLA3-related disease or condition in the subject. Non-limiting examples of physiological characteristics of a PNPLA3-related disease or condition include PNPLA3 mRNA levels, PNPLA3 protein levels, or the number or extent of amyloid deposits. Standard means for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging tests, physical examinations, etc.

[0188] It will be understood that the amount of PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent administered to a subject can be varied, at least in part, based on the determination of the state and / or physiological characteristics of the disease and / or condition determined for the subject. The therapeutic amount can be varied, for example, by increasing or decreasing the amount of PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent, changing the composition in which the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent, respectively, is administered, changing the route of administration, changing the timing of administration, etc. The effective amount of PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent will vary depending on the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concomitant therapy (if applicable), the particular route of administration, and other factors within the knowledge and expertise of the medical professional. For example, the effective amount can depend on the desired level of PNPLA3 polypeptide activity and / or PNPLA3 gene expression effective to treat a PNPLA3-related disease or condition. A skilled artisan can empirically determine the effective amount of a specific PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention used in the method of the present invention without undue experimentation.In combination with the teachings provided herein, by selecting from the various PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents of the present invention and considering factors such as efficacy, relative bioavailability, patient weight, the severity of adverse side effects, and preferred administration mode, an effective preventive or therapeutic treatment regimen can be designed for treating a specific subject.When used in the embodiments of the present invention, the effective amount of a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention can be the amount that produces the desired biological effect in cells when contacted with cells.

[0189] It will be appreciated that PNPLA3 gene silencing can be determined in any cell that expresses PNPLA3, either constitutively or by genome engineering, and by any suitable assay. In some embodiments of the present invention, administration of a PNPLA3 dsRNA agent of the present invention reduces PNPLA3 gene expression by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the present invention, administration of a PNPLA3 dsRNA agent of the present invention reduces PNPLA3 gene expression by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%.

[0190] dosage The PNPLA3 dsRNA agent and PNPLA3 antisense polynucleotide agent are delivered in a pharmaceutical composition at a dose sufficient to inhibit expression of the PNPLA3 gene. In certain embodiments of the invention, the dose of the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent is in the range of 0.01 to 200.0 milligrams per kilogram of recipient body weight per day, generally in the range of 1 to 50 mg / kilogram of body weight, 5 to 40 mg / kg of body weight, 10 to 30 mg / kg of body weight, 1 to 20 mg / kg of body weight, 1 to 10 mg / kg of body weight, or 4 to 15 mg / kg of body weight per day, inclusive. For example, The dsRNA agent or PNPLA3 antisense polynucleotide agent may be administered at a dose of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 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 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 5.9 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3m g / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg , 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg , 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 1 It can be administered in the following amounts: 5mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg, and 50mg / kg.

[0191] The dosage and delivery timing of the PNPLA3 dsRNA agent of the present invention can be determined by various factors.The absolute amount of the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent to be delivered will depend on various factors, including simultaneous treatment, administration frequency, and individual subject parameters such as age, physical condition, size and weight.These are factors well known to those skilled in the art and can be addressed by simple experimentation.In some embodiments, the maximum dosage, i.e., the maximum safe dosage according to sound medical judgment, can be used.

[0192] In some embodiments, the methods of the present invention may include administering to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent. In some cases, a pharmaceutical compound (e.g., comprising a PNPLA3 dsRNA agent or comprising a PNPLA3 antisense polynucleotide agent) can be administered to a subject at least daily, every other day, weekly, biweekly, monthly, etc. Doses can be administered once a day or more than once a day, for example, two, three, four, five, or more times within a 24-hour period. The pharmaceutical compositions of the present invention may be administered once a day, or the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent may be administered in divided doses two, three, or more times at appropriate intervals throughout the day, or may be administered using continuous infusion or delivery by controlled-release formulation. In some embodiments of the methods of the invention, a pharmaceutical composition of the invention is administered to a subject one or more times daily, one or more times weekly, one or more times monthly, or one or more times yearly.

[0193] In some embodiments, the methods of the present invention involve administering a pharmaceutical compound alone or in combination with one or more other PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents, and / or in combination with other medications or therapeutic activities or regimens administered to a subject with a PNPLA3-related disease or condition. The pharmaceutical compound may be administered as a pharmaceutical composition. The pharmaceutical compositions used in the methods of the present invention are sterile and may contain an amount of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent in a weight or volume unit suitable for administration to a subject that reduces PNPLA3 polypeptide activity to a level sufficient to produce the desired response. The dose at which a pharmaceutical composition containing a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent is administered to a subject to reduce PNPLA3 protein activity can be selected depending on various parameters, particularly the mode of administration used and the subject's condition. Other factors include the desired duration of treatment. If the subject does not respond adequately to the initial dose, a higher dose (or a higher dose that becomes effective using a different, more localized route of administration) may be used, as tolerated by the patient.

[0194] treatment PNPLA3-related diseases and conditions in which reducing the level and / or activity of PNPLA3 polypeptide is effective in treating the disease or condition can be treated by inhibiting PNPLA3 expression using the methods and PNPLA3 dsRNA agents of the present invention. Examples of diseases and conditions that can be treated using the PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents of the present invention and the methods of the present invention include, but are not limited to, liver disease, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, alcoholic liver disease, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, or non-alcoholic fatty liver disease (NAFLD). Herein, such diseases and conditions may be referred to as "PNPLA3-related diseases and conditions" and "diseases and conditions caused and / or regulated by PNPLA3."

[0195] In certain embodiments of the present invention, a subject may be administered a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent of the present invention at one or more times before or after the diagnosis of a PNPLA3-related disease or condition. In some embodiments of the present invention, a subject has or is at risk of developing a PNPLA3-related disease or condition. A subject at risk of developing a PNPLA3-related disease or condition is a subject who has a higher probability of developing a PNPLA3-related disease or condition compared to the control risk of developing a PNPLA3-related disease or condition. In some embodiments of the present invention, the level of risk may be statistically significant compared to the control level of risk. At-risk subjects may include, for example, subjects who have an existing disease and / or genetic abnormality, and are therefore more susceptible to or will be more susceptible to a PNPLA3-related disease or condition than control subjects without an existing disease or genetic abnormality, subjects with a family history and / or personal history of a PNPLA3-related disease or condition, and subjects who have previously received treatment for a PNPLA3-related disease or condition. It will be understood that the pre-existing disease and / or genetic abnormality that predisposes a subject to a PNPLA3-associated disease or condition may be a disease or genetic abnormality that, when present, has previously been identified as being correlated with an increased likelihood of developing a PNPLA3-associated disease or condition.

[0196] It will be understood that PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents can be administered to a subject based on the individual subject's medical condition. For example, the healthcare provided to a subject may assess the PNPLA3 level measured in a sample obtained from the subject and determine that it is desirable to administer a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention to reduce the subject's PNPLA3 level or liver lipid droplet level. In this example, even if the subject has not been diagnosed with a PNPLA3-related disease as disclosed herein, the PNPLA3 level may be considered a physiological characteristic of a PNPLA3-related condition. A healthcare provider may monitor changes in the subject's PNPLA3 level as a measure of the effectiveness of the administered PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention. As a non-limiting example, a biological sample, such as a liver or serum sample, may be obtained from the subject, and the subject's PNPLA3 level in the sample may be determined. PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent is administered to subject, and after administration, liver or serum sample is obtained from subject, and this sample is used to determine PNPLA3 level, and the result is compared with the result determined in subject's pre-administration (pre-) sample.Compared with pre-administration level, the PNPLA3 level of subject in post-administration sample is reduced, indicating that the administered PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent has the effect of reducing subject's lipid level, liver fat or liver lipid droplets.

[0197] Certain embodiments of the methods of the present invention include adjusting treatment, including administering a dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention to a subject, based at least in part on assessing one or more changes in the physiological characteristics of the subject with a PNPLA3-related disease or condition that occur as a result of treatment.For example, in some embodiments of the present invention, the effect of the administered dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention is determined for the subject, and can then be used to assist in adjusting the amount of the dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention administered to the subject.As a non-limiting example, a dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention is administered to a subject, and the subject's PNPLA3 level is determined after administration, and based at least in part on the determined level, it is determined that a larger amount of the dsRNA agent or PNPLA3 antisense polynucleotide agent is desirable to enhance the physiological effect of the administered agent, for example, to reduce or further reduce the subject's PNPLA3 level. In another non-limiting example, it is desirable to administer to a subject a dsRNA agent or PNPLA3 antisense polynucleotide agent of the invention, determine the subject's PNPLA3 levels after administration, and decrease the amount of dsRNA agent or PNPLA3 antisense polynucleotide agent administered to the subject based at least in part on the determined level.

[0198] Thus, some embodiments of the invention include assessing changes in one or more physiological characteristics resulting from a subject's previous treatment and then adjusting the amount of a dsRNA agent or PNPLA3 antisense polynucleotide agent of the invention administered to the subject. Some embodiments of the methods of the invention include determining the physiological characteristics of a PNPLA3-related disease or condition one, two, three, four, five, six, or more times to assess and / or monitor the effectiveness of an administered PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the invention, and optionally using the determinations to adjust one or more of the dose, administration regimen, and / or administration frequency of the dsRNA agent or PNPLA3 antisense polynucleotide agent of the invention to treat the subject's PNPLA3-related disease or condition. In some embodiments of the methods of the invention, the desired result of administering to a subject an effective amount of a dsRNA agent or PNPLA3 antisense polynucleotide agent of the invention is a reduction in the subject's PNPLA3 mRNA level, PNPLA3 protein level, liver fat level and / or lipid droplet level, or the number or extent of amyloid deposits, etc., compared to a previous level or control level determined for the subject.

[0199] As used herein, the terms "treat," "treated," or "treating" when used in reference to a PNPLA3-related disease or condition can refer to prophylactic treatment that reduces the likelihood that a subject will develop a PNPLA3-related disease or condition, or can refer to treatment administered after a subject has developed a PNPLA3-related disease or condition to eliminate or reduce the level of the PNPLA3-related disease or condition, prevent the PNPLA3-related disease or condition from progressing further (e.g., becoming more severe), and / or slow the progression of the PNPLA3-related disease or condition in a subject compared to a subject not receiving treatment that reduces the activity of a PNPLA3 polypeptide in the subject.

[0200] Certain embodiments of the agent, composition and method of the present invention can be used to inhibit the expression of PNPLA3 gene.In this specification, the terms " inhibit ", " silence ", " reduce ", " down-regulation " and " knockdown " used in relation to the expression of PNPLA3 gene refer to the expression of PNPLA3 gene, when the cell, cell group, tissue, organ or subject in which PNPLA3 gene is transcribed is contacted (for example, treated) with the PNPLA3 dsRNA agent of the present invention or the PNPLA3 antisense polynucleotide agent, the expression of PNPLA3 gene is measured by one or more of the RNA level transcribed from PNPLA3 gene, the activity level of PNPLA3 expressed and the PNPLA3 polypeptide, protein or protein subunit level translated from mRNA in cells, and are reduced respectively compared with the control level of the RNA transcribed from PNPLA3 gene, the activity level of PNPLA3 expressed and the PNPLA3 level translated from mRNA. In some embodiments, the control level is the level in a cell, tissue, organ, or subject not contacted (eg, untreated) with a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent.

[0201] Administration method The methods of the present invention can use various routes of administration for PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents. The specific administration method selected will depend, at least in part, on the specific condition being treated and the dosage required for therapeutic effect. Generally speaking, the methods of the present invention can be carried out using any medically acceptable administration mode, i.e., any mode that produces an effective level for treating PNPLA3-related diseases or conditions without causing clinically unacceptable side effects. In some embodiments of the present invention, PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents can be administered orally, enterally, mucosally, subcutaneously, and / or parenterally. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other administration routes include, but are not limited to, nasal (e.g., via a gastronasogastric tube), skin, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention can include intrathecal, intraventricular, or intracranial. In some embodiments of the present invention, PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents can be placed in a sustained-release matrix and administered to a subject by placing the matrix in the subject.In some aspects of the present invention, PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents can be delivered to a subject's cells using nanoparticles coated with a delivery agent that targets specific cells or organelles.Various delivery means, methods, and agents are known in the art.Non-limiting examples of delivery methods and delivery agents are further described elsewhere herein.In some embodiments of the invention, the term "delivery" with respect to a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent can refer to administering one or more "naked" PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent sequences to a cell or subject, and in particular embodiments of the invention, "delivery" refers to administering to a cell or subject via transfection means, delivering to a subject a cell containing a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent, delivering to a cell and / or subject a vector encoding a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent, etc. Delivery of a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent using transfection means can include administering the vector to a cell and / or subject.

[0202] In some methods of the present invention, one or more PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents are administered in a formulation, which is administered in a pharmaceutically acceptable solution, which may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In some embodiments of the present invention, a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent may be formulated with another therapeutic agent for simultaneous administration. According to the methods of the present invention, a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent may be administered as a pharmaceutical composition. Generally, the pharmaceutical composition comprises a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent and, optionally, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. As used herein, a pharmaceutically acceptable carrier refers to a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient, for example, the ability of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent to inhibit PNPLA3 gene expression in a cell or subject. Numerous methods for administering and delivering dsRNA agents or PNPLA3 antisense polynucleotide agents for therapeutic purposes are known in the art and can be used in the methods of the present invention.

[0203] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, and other carriers are known to those skilled in the art. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and, optionally, other therapeutic agents. While salts for pharmaceutical use must be pharmaceutically acceptable, non-pharmaceutically acceptable salts may also be conveniently used to prepare pharmaceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacological and pharmaceutically acceptable salts include, but are not limited to, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, and succinic acid. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, and calcium salts.

[0204] Some embodiments of the methods of the present invention include directly administering one or more PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which a PNPLA3-related disease or condition exists or may occur, including, but not limited to, the liver or kidney. Direct tissue administration can be achieved by direct injection or other means. Many orally administered compounds naturally migrate to and pass through the liver and kidney. Some embodiments of the therapeutic methods of the present invention include orally administering one or more PNPLA3 dsRNA agents to a subject. The PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents may be administered alone or in combination with other therapeutic agents, either once or, alternatively, in multiple doses. When administered multiple times, the PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents may be administered by different routes. For example, and not by way of limitation, the first (or first few) doses may be administered subcutaneously, and one or more additional doses may be administered orally and / or systemically.

[0205] In embodiments of the present invention in which systemic administration of a PNPLA3 dsRNA agent or a PNPLA3 antisense polynucleotide agent is desired, the PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent can be formulated for parenteral administration, for example, by injection via bolus injection or continuous infusion. Injectable formulations can be provided in unit dosage forms, such as ampoules or multi-dose containers, with or without added preservatives. Formulations of PNPLA3 dsRNA agents (also referred to as pharmaceutical compositions) can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents.

[0206] Formulations for parenteral administration include aqueous or non-aqueous sterile solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Vehicles for intravenous administration include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases. Other forms of administration, such as intravenous administration, require lower dosages. If the subject does not respond adequately to the initial dose, a higher dose (or a higher dose efficacious using a different, more localized route of administration) may be used, as tolerated by the patient. Multiple doses per day may be administered, if necessary, to achieve adequate systemic or local levels of one or more PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents to adequately reduce PNPLA3 protein activity.

[0207] In yet other embodiments, the methods of the present invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant, suitable for implantation into a recipient, e.g., a subject. Exemplary biodegradable implants that may be useful according to this method are described in PCT Publication No. WO 95 / 24929 (incorporated herein by reference), which describes biocompatible, biodegradable polymeric matrices for housing biopolymers.

[0208] In the methods of the present invention, both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the desired period of release, typically ranging from a few hours to a year or more. Typically, release periods ranging from a few hours to 3-12 months can be used. The polymer is optionally in the form of a hydrogel capable of absorbing up to about 90% of its weight in water and, further, optionally crosslinked with multivalent ions or other polymers.

[0209] Generally, in some embodiments of the present invention, PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents can be delivered by diffusion or by degradation of a polymer matrix using biodegradable implants. Exemplary synthetic polymers for such use are well known in the art. Biodegradable and non-biodegradable polymers can be used to deliver PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents using methods known in the art. Bioadhesive polymers, such as biodegradable hydrogels (see H.S.Sawhney, C.P.Pathak, and J.A.Hubell, Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference), can also be used to deliver PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents for the treatment of PNPLA3-related diseases or conditions. Additional suitable delivery systems can include time-release, delayed-release, or sustained-release delivery systems. Such a system can avoid repeated administration of PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents, improving convenience for patients and medical professionals. Many types of release delivery systems are available and known to those skilled in the art. (See, for example, U.S. Patent Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686 (the teachings of each patent are incorporated herein by reference).) In addition, pump-based hardware delivery systems can also be used, some of which are adapted for implantation.

[0210] The use of long-term sustained release implants can be suitable for the prophylactic treatment of subjects or for subjects at risk of recurrence of PNPLA3-related diseases or symptoms.As used herein, long-term release means that the implant is constructed and arranged to deliver therapeutic levels of PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent for at least 10 days, 20 days, 30 days, 60 days, 90 days, 6 months, 1 year or more.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems mentioned above.

[0211] Therapeutic formulations of PNPLA3 dsRNA agents or PNPLA3 antisense polynucleotide agents can be prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing the molecule or compound having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer [Remington's Pharmaceutical Sciences 21 stedition, (2006)]. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acid salts, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, etc. or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0212] Cells, subjects, and controls The methods of the present invention can be used in conjunction with cells, tissues, organs, and / or subjects. In some embodiments of the present invention, the subject is a vertebrate, including, but not limited to, a human or a primate, such as a dog, cat, horse, cow, goat, mouse, rat, or monkey. Thus, the present invention can be used to treat PNPLA3-related diseases or conditions in both human and non-human subjects. In some embodiments of the present invention, the subject can be a farm animal, zoo animal, livestock, or non-livestock animal, and the methods of the present invention can be used in veterinary prophylactic and therapeutic regimens. In some embodiments of the present invention, the subject is a human, and the methods of the present invention can be used in human prophylactic and therapeutic regimens.

[0213] Non-limiting examples of subjects to which the present invention can be applied include subjects who have been diagnosed with, are suspected of having, or are at risk for a disease or condition associated with higher than desired levels of PNPLA3 expression and / or activity, also referred to as "elevated PNPLA3 expression levels." Non-limiting examples of diseases and conditions associated with higher than desired levels of PNPLA3 expression and / or activity are described elsewhere herein. The methods of the present invention can be applied to subjects who, upon treatment, have been diagnosed with a disease or condition associated with higher than desired levels of PNPLA3 expression and / or activity, or subjects who are believed to have or be at risk for developing a disease or condition associated with higher than desired levels of PNPLA3 expression and / or activity. In some embodiments of the present invention, the disease or condition associated with higher than desired levels of PNPLA3 expression and / or activity is an acute disease or condition, while in certain embodiments of the present invention, the disease or condition associated with higher than desired levels of PNPLA3 expression and / or activity is a chronic disease or condition.

[0214] As a non-limiting example, the PNPLA3 dsRNA agent of the present invention is administered to a subject who has been diagnosed with, is suspected of, or is at risk of having non-alcoholic steatohepatitis (NASH), a disease for which it is desirable to reduce the expression of PNPLA3.The method of the present invention can be applied to a subject who has been diagnosed with a disease or condition, or a subject who is thought to have or be at risk of developing a disease or condition during treatment.

[0215] As another non-limiting example, the PNPLA3 dsRNA agent of the present invention is administered to a subject who has been diagnosed with, is suspected of having, or is at risk for non-alcoholic fatty liver disease (NAFLD), a disease for which it is desirable to reduce the expression of PNPLA3.The method of the present invention can be applied to a subject who has been diagnosed with a disease or condition, or a subject who is thought to have or be at risk for developing a disease or condition at the time of treatment.

[0216] Cells to which the methods of the present invention can be applied include in vitro, in vivo, and ex vivo cells. Cells may be in a subject, in culture, and / or in suspension, or under other suitable conditions. Cells to which the methods of the present invention can be applied may be liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In certain aspects of the present invention, cells to which the methods of the present invention can be applied are healthy, normal cells that are not known to be diseased cells. In certain embodiments of the present invention, the cells to which the methods and compositions of the present invention can be applied are liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, and / or kidney cells. While in certain aspects of the present invention, the control cells are normal cells, it will be understood that cells with a disease or condition can also serve as control cells in certain situations, such as when comparing the results of treated cells with a disease or condition to untreated cells with a disease or condition.

[0217] According to the methods of the present invention, the level of PNPLA3 polypeptide activity can be determined and compared to a control level of PNPLA3 polypeptide activity. The control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as a median or mean value. It can be established based on comparison groups, such as a group with normal levels of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity and a group with increased levels of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity. Other non-limiting examples of comparison groups include a group with one or more symptoms of a PNPLA3-related disease or condition or a diagnosis thereof, a group without one or more symptoms of a disease or condition or a diagnosis thereof, a group of subjects administered an siRNA treatment of the present invention, and a group of subjects not administered an siRNA treatment of the present invention. Typically, the control can be based on apparently healthy normal individuals of an appropriate age group or apparently healthy cells. It will be understood that the control according to the present invention can be a predetermined value as well as a sample of material tested in parallel with the experimental material. Examples include samples from a control population and control samples generated through manufacturing to be tested in parallel with the experimental sample. In some embodiments of the invention, the control includes cells or subjects that have not been contacted or treated with a PNPLA3 dsRNA agent of the invention, and in such cases, the level of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity in the control can be compared to the level of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity in cells or subjects that have been contacted with a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the invention.

[0218] In some embodiments of the present invention, the PNPLA3 polypeptide level determined for a subject can be a control level compared to the PNPLA3 polypeptide level determined for the same subject at a different time point.As a non-limiting example, the PNPLA3 level is determined in a biological sample obtained from a subject who has not been administered the PNPLA3 treatment of the present invention.In some embodiments, the biological sample is a serum sample.The PNPLA3 polypeptide level determined in a sample obtained from a subject can serve as the subject's baseline or control value.In the treatment methods of the present invention, after administering a PNPLA3 dsRNA agent to the subject one or more times, one or more additional serum samples can be collected from the subject, and the PNPLA3 polypeptide level in the one or more subsequent samples can be compared with the subject's control / baseline level.Such comparisons can be used to assess the onset, progression, or regression of PNPLA3-related diseases or symptoms in a subject. For example, if the level of PNPLA3 polypeptide in a baseline sample obtained from a subject is higher than the level obtained from the same subject after administering to the subject a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention, this indicates regression of the PNPLA3-related disease or condition and indicates the effectiveness of the administered PNPLA3 dsRNA agent of the present invention in treating the PNPLA3-related disease or condition.

[0219] In some embodiments of the present invention, one or more values ​​of the level of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity determined for a subject serve as a control value for later comparison of the level of PNPLA3 polypeptide and / or PNPLA3 activity in the same subject, thereby assessing changes from "baseline" PNPLA3 polypeptide activity in the subject. Thus, an initial PNPLA3 polypeptide level and / or initial PNPLA3 polypeptide activity level may be present and / or measured in a subject, and the methods and compounds of the present invention may be used to reduce the level of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity in a subject, with the initial level serving as the control level for the subject.

[0220] Using the method of the present invention, the PNPLA3 dsRNA agent and / or PNPLA3 antisense polynucleotide agent of the present invention can be administered to subject.The effectiveness of the administration and treatment of the present invention can be assessed when the level of PNPLA3 polypeptide in the serum sample obtained from subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, compared with the pre-administration level of PNPLA3 polypeptide in the serum sample obtained from subject at a previous time point, or compared with the level of PNPLA3 polypeptide in the serum sample of non-contact control, for example, the level of PNPLA3 polypeptide in control serum sample.It will be understood that the level of PNPLA3 polypeptide and the level of PNPLA3 polypeptide activity are both correlated with the level of PNPLA3 gene expression. Certain embodiments of the methods of the present invention include administering to a subject a PNPLA3 dsRNA and / or PNPLA3 antisense agent of the present invention in an amount effective to inhibit PNPLA3 gene expression, thereby reducing the level of PNPLA3 polypeptide in the subject and reducing the level of PNPLA3 polypeptide activity.

[0221] Some embodiments of the present invention include determining the presence, absence, and / or amount (also referred to herein as level) of PNPLA3 polypeptide in one or more biological samples obtained from one or more subjects. This determination can be used to assess the effectiveness of the treatment of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of PNPLA3 polypeptide in biological samples obtained from subjects previously treated with the administration of a PNPLA3 dsRNA agent and / or a PNPLA3 antisense agent of the present invention. If the level of PNPLA3 polypeptide determined in a serum sample obtained from a treated subject is at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more lower than the pre-treatment level of PNPLA3 polypeptide determined for the subject or compared to the level in an untreated control biological sample, this indicates the level of effectiveness of the treatment administered to the subject.

[0222] In some embodiments of the present invention, the physiological characteristics of a PNPLA3-related disease or condition determined for a subject can be compared with a control determination of the physiological characteristics of the same subject at a different time point. Non-limiting examples include physiological characteristics such as PNPLA3 mRNA levels, PNPLA3 protein levels, or the number and extent of amyloid deposits, determined in a liver or serum sample from a subject not receiving the PNPLA3 treatment of the present invention. The PNPLA3 mRNA level (and / or other physiological characteristics of a PNPLA3 disease or condition) determined in a sample from the subject can serve as a baseline or control value for the subject. In the treatment methods of the present invention, after one or more administrations of a PNPLA3 dsRNA agent to the subject, one or more additional liver or serum samples can be obtained from the subject, and the PNPLA3 mRNA level and / or PNPLA3 protein level in the subsequent one or more samples can be compared with the subject's control / baseline level and / or ratio, respectively. Such comparisons can be used to assess the onset, progression, or regression of a PNPLA3-related disease or condition in a subject. For example, if the PNPLA3 mRNA level in a baseline sample obtained from a subject is higher than the PNPLA3 mRNA level determined in a sample obtained from the same subject after administering a PNPLA3 dsRNA agent or PNPLA3 antisense polynucleotide agent of the present invention to the subject, this indicates regression of the PNPLA3-related disease or condition and indicates the effectiveness of administering a PNPLA3 dsRNA agent of the present invention for treating a PNPLA3-related disease or condition.

[0223] In some embodiments of the present invention, the value of one or more physiological characteristics of a PNPLA3-related disease or condition determined for a subject can serve as a control value for later comparison of physiological characteristics of the same subject, allowing changes from the subject's "baseline" physiological characteristics to be assessed. Thus, an initial physiological characteristic may be present and / or determined in a subject, and the methods and compounds of the present invention may be used to reduce the level of PNPLA3 polypeptide and / or PNPLA3 polypeptide activity in the subject, with the initial physiological characteristic determination serving as a control for the subject.

[0224] Using the methods of the present invention, PNPLA3 dsRNA agents and / or PNPLA3 antisense polynucleotide agents of the present invention can be administered to a subject in an amount effective to treat PNPLA3 diseases or conditions. The effectiveness of the administration and treatment of the present invention can be assessed by determining changes in one or more physiological characteristics of PNPLA3 diseases or conditions. In a non-limiting example, the PNPLA3 mRNA level in a serum sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to the pre-administration lipids in a serum sample obtained from the subject at a previous time point, or compared to the PNPLA3 mRNA level in an untreated control, for example, a control serum sample. It will be understood that the PNPLA3 mRNA level, PNPLA3 protein level, or the number and extent of amyloid deposits in a subject are each correlated with the level of PNPLA3 gene expression. Certain embodiments of the methods of the present invention include administering to a subject an effective amount of a PNPLA3 dsRNA and / or PNPLA3 antisense agent of the present invention to inhibit PNPLA3 gene expression, thereby reducing PNPLA3 mRNA levels, PNPLA3 protein levels, or the number or extent of amyloid deposits in the subject, or positively affecting the physiological characteristics of a PNPLA3-related disease or condition in the subject.

[0225] Some embodiments of the present invention include, but are not limited to, determining the presence, absence, and / or alteration of physiological characteristics of a PNPLA3-associated disease or condition using methods such as: (1) evaluating one or more biological samples obtained from one or more subjects for physiological characteristics, (2) imaging the subjects (e.g., but not limited to, obtaining liver images), and (3) physical examination of the subjects. This determination can be used to assess the effectiveness of the treatment methods of the present invention.

[0226] kit The present invention also encompasses kits containing one or more PNPLA3 dsRNA agents and / or PNPLA3 antisense polynucleotide agents and instructions for their use in the methods of the invention. The kits of the invention may include one or more PNPLA3 dsRNA agents, PNPLA3 sense polynucleotides, and PNPLA3 antisense polynucleotide agents that can be used to treat PNPLA3-related diseases or conditions. Kits containing one or more PNPLA3 dsRNA agents, PNPLA3 sense polynucleotides, and PNPLA3 antisense polynucleotide agents can be prepared for use in the therapeutic methods of the invention. The components of the kits of the invention may be packaged in either aqueous media or lyophilized form. The kits of the invention may comprise a compartmentalized carrier for hermetically containing one or more container means or a series of container means, such as test tubes, vials, flasks, bottles, syringes, etc. A first container means or a series of container means may contain one or more compounds, such as a PNPLA3 dsRNA agent and / or a PNPLA3 sense or antisense polynucleotide agent. A second container means or series of container means may contain a targeting agent, labeling agent, delivery agent, etc. that may be included as part of the PNPLA3 dsRNA agent and / or PNPLA3 antisense polynucleotide administered in embodiments of the treatment methods of the present invention.

[0227] Kits of the invention may also include instructions, typically in written form, that provide guidance for carrying out the treatment provided by the kit and for making decisions based on that treatment.

[0228] The following examples are provided to illustrate specific embodiments of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to those skilled in the art, the present invention has application in a variety of compositions and methods. [Example]

[0229] Example 1 Preparation of Intermediate A and Intermediate B.

[0230] As shown in Scheme 1 below, intermediate A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). Subsequent glycosylation with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol afforded compound II. The Cbz-protecting group was removed by hydrogenation to afford intermediate A as the trifluoroacetate (TFA) salt. Intermediate B was synthesized according to the same scheme, except that Cbz-protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as the starting material.

[0231] [ka] Scheme 1 To a solution of compound I (20.0 g, 51.4 mmol) in 100 mL of 1,2-dichloroethane (DCE), TMSOTf (17.1 g, 77.2 mmol) was added. The resulting reaction solution was stirred at 60 °C for 2 h and then at 25 °C for 1 h. Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) in DCE (100 mL) dried over 4 Å powdered molecular sieves (10 g) was added dropwise to the above reaction solution at 0 °C under a N atmosphere. The resulting reaction mixture was stirred at 25 °C for 16 h under a N atmosphere. The reaction mixture was filtered and washed with saturated NaHCO (200 mL), water (200 mL), and saturated brine (200 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude product, which was triturated with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80 L) for 2 h. The resulting mixture was filtered and dried to give compound II (15.0 g, 50.3% yield) as a white solid.

[0232] To a dry, argon-purged hydrogenation bottle, 10% Pd / C (1.50 g) was carefully added, followed by 10 mL of tetrahydrofuran (THF), followed by a solution of compound II (15.0 g, 26.4 mmol) in THF (300 mL) and TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol). The resulting mixture was degassed, purged with H2 three times, and stirred under an atmosphere of H2 (45 psi) at 25 °C for 3 h. Thin-layer chromatography (TLC, solvent: DCM:MeOH = 10:1) indicated that compound II was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. This process was repeated three times to give intermediate A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400 MHz DMSO-d6): δppm7.90(d,J=9.29Hz, 1H), 7.78(br s, 3H), 5.23(d, J=3.26Hz, 1H), 4.98(dd, J=11.29, 3.26Hz, 1H), 4.56(d, J=8. 53Hz, 1H), 3.98~4.07(m, 3H), 3.79~3.93(m, 2H), 3.55~3.66(m, 5H), 2.98(br d, J=4.77Hz, 2H), 2.11(s, 3H), 2.00(s, 3H), 1.90(s, 3H), 1.76(s, 3H).

[0233] Intermediate B was synthesized using a procedure similar to that of Intermediate A. 1 H NMR(400MHz DMSO-d6):δppm7.90(br d, J=9.03Hz, 4H), 5.21(d, J=3.51Hz, 1H), 4.97(dd, J=11.1Hz, 1H), 4.54(d, J=8.53Hz, 1H), 3.98~4.06(m, 3H), 3.88(dt, J=10.9Hz, 1H), 3.76~3.83(m, 1H), 3.49~3.61(m, 9H), 2.97(br s, 2H), 2.10(s, 3H), 1.99(s, 3H), 1.88(s, 3H), 1.78(s, 3H). C 20 H 34 N2O 11 Calculated mass: 478.22; Found: 479.3 (M+H + ).

[0234] Example 2 Synthesis of GalNAc Ligand Cluster Phosphoramidites GLPA1, GLPA2, and GLPA15.

[0235] To prepare GLPA1 and GLPA2, the following scheme 2 was followed. Starting from benzyl-protected propane-1,3-diamine, alkylation with tert-butyl 2-bromoacetate gave triester compound I. The benzyl protecting group was removed by hydrogenation to give secondary amine compound II. Amide coupling with 6-hydroxyhexanoic acid gave compound III. Subsequent treatment with HCl in dioxane removed the tert-butyl protecting group to generate triacid compound IV. Amide coupling of triacid compound IV with intermediate A or intermediate B gave compound Va or Vb. Phosphoramidite GLPA1 or GLPA2 was synthesized by phosphitylation of compound Va or Vb with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

[0236] [ka] Scheme 2 To a solution of N-benzyl-1,3-propanediamine (5.00 g, 30.4 mmol) in dimethylformamide (DMF, 100 mL) was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol), followed by the dropwise addition of diisopropylethylamine (DIEA, 23.61 g, 182 mmol). The resulting reaction mixture was stirred at 25–30 °C for 16 h. LCMS showed that N-benzyl-1,3-propanediamine was completely consumed. The reaction mixture was diluted with HO (500 mL) and extracted with EtOAc (500 mL × 2). The combined organics were washed with saturated brine (1 L), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 20:1 to 5:1). Compound I (12.1 g, 78.4% yield) was obtained as a colorless oil. 1H NMR (400MHz, CDCl3): δppm7.26~7.40(m,5H),3.79(s,2H),3.43(s,4H),3.21(s,2) H), 2.72 (dt, J=16.9, 7.34Hz, 4H), 1.70 (quin, J=7.2Hz, 2H), 1.44~1.50 (m, 27H).

[0237] A dry hydrogenation bottle was purged with argon three times. Pd / C (200 mg, 10%) was added, followed by MeOH (5 mL), followed by a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with H. This process was repeated three times. The mixture was stirred under an H (15 psi) atmosphere at 25 °C for 12 h. LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under a N atmosphere. The filtrate was concentrated under reduced pressure to give compound II (655 mg, 79.7% yield) as a yellow oil, which was used in the next step without further purification. 1 H NMR (400MHz, CDCl3): δppm3.44(s,4H),3.31(s,2H),2.78(t,J=7.1Hz,2H),2.68(t,J=6.9Hz,2H),1.88(br s, 1H), 1.69 (quin, J=7.03Hz, 2H), 1.44~1.50 (s, 27H).

[0238] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 904 mg, 4.72 mmol), and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N three times, then stirred under N at 25 °C for 3 h. LCMS showed the desired product. The reaction mixture was diluted with HO (10 mL) and extracted with 20 mL of EtOAc (2 x 10 mL). The organics were combined, washed with saturated brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 5:1 to 1:1) to give compound III (650 mg, 77.8% yield) as a yellow oil. 1 H NMR (400MHz, CDCl3): δppm3.90~3.95(s, 2H), 3.63(t,J=6.40Hz, 2H), 3.38~3.45(m, 6 H), 2.72(t, J=6.65Hz, 2H), 2.40(t, J=7.28Hz, 2H), 1.55~1.75(m, 8H), 1.44(s, 27H). C 27 H 50 Calculated mass of N2O8: 530.36; Found: 531.3 (M+H + ).

[0239] A mixture of compound III (5.5 g, 10.3 mmol) in HCl / dioxane (2 M, 55 mL) was stirred at 25 °C for 3 h. LCMS showed that compound III was completely consumed. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give the crude product, which was dissolved in CHCN (50 mL) and the volatiles were removed under vacuum. This process was repeated three times to give compound IV (2.05 g, 54.5% yield) as a white solid. 1H NMR (400MHz, D2O): δppm4.21(s, 1H), 4.07(d,J=4.5Hz, 4H), 3.99(s, 1H), 3.4 5~3.52(m, 3H), 3.42(t, J=6.5Hz, 1H), 3.32~3.38(m, 1H), 3.24~3.31(m, 1H), 2.37(t, J=7.4Hz, 1H), 2.24(t, J=7.4Hz, 1H), 1.99(dt, J=15.5, 7.53Hz, 1H), 1.85~1.94(m, 1H), 1.85~1.94(m, 1H), 1.39~1.56(m, 4H), 1.19~1.31(m, 2H).

[0240] A mixture of compound IV (500 mg, 1.05 mmol), intermediate A (2.02 g, 3.67 mmol), DIEA (813 mg, 6.30 mmol), EDCI (704 mg, 3.67 mmol), and HOBt (496 mg, 3.67 mmol) in DMF (10 mL) was degassed and purged with N three times, after which the mixture was stirred under a N atmosphere at 25 °C for 3 h. LCMS showed the desired product. The reaction mixture was quenched by the addition of H2O (10 mL) and extracted with DCM (10 mL x 2). The combined organics were extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated NaHCO3 solution and re-extracted with DCM (10 mL x 2). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide compound Va (570 mg, 0.281 mmol, 26.8% yield) as a white solid. 1 H NMR: (400MHz, CDCl3)ppmδ7.84~8.12(m,3H),6.85~7.15(m,2H),6.66~6.81(m,1H),5.36(br d,J=2.7Hz, 3H), 5.11~5.27(m, 3H), 4.63~4.85(m, 3H), 3.90~4.25(m, 18H), 3.37~3.75(m, 28H), 3.15~3.28(m, 4H), 2.64(br d. t, J=7.0Hz, 2H).

[0241] To a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL) was added diisopropylammonium tetrazolide (30.3 mg, 0.177 mmol), followed by the dropwise addition of 3-bis(diisopropylamino)phosphanyloxypropanenitrile (194 mg, 0.645 mmol) at ambient temperature under N2. The reaction mixture was stirred at 20-25 °C for 2 h. LCMS indicated that compound Va was completely consumed. After cooling to -20 °C, the reaction mixture was added to stirred brine / saturated aqueous NaHCO3 (1:1, 5 mL) at 0 °C. After stirring for 1 min, DCM (5 mL) was added. The layers were separated. The organics were washed with brine / saturated aqueous NaHCO3 (1:1, 5 mL), dried over Na2SO4, filtered, and concentrated to a volume of 1 mL. The remaining solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) with stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged, and the solid was collected. The solid was redissolved in 1 mL of DCM and precipitated by the addition of MTBE (20 mL). The solid was again separated by centrifugation. The collected solid was dissolved in anhydrous CH3CN. The volatiles were removed. This process was repeated two more times to obtain the GalNAc ligand phosphoramidite compound GLPA1 (153 mg, 84.4 μmol) as a white solid. 1 H NMR (400MHz, CDCl3):ppmδ7.71~8.06(m,2H),6.60~7.06(m,3H),5.37(b rd,J=3.0Hz,3H),5.18~5.32(m,3H),4.70~4.86(m,3H),3.92~4.25(m,18H),3.42~3.85(m,30H),3.25(m ,4H),2.59~2.75(m,4H),2.27~2.44(m,2H),2.15~2.20(s,9H)2.07(s,9H),1.96~2.03(m,18H),1.65(br s, 4H), 1.44(br d, J=7.28Hz, 2H), 1.14~1.24(m, 12H). 31 P NMR (CDCl3): ppm δ 147.15.

[0242] The GalNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure except that intermediate B was used. 1 H NMR (400MHz, CDCl3):ppmδ7.94~8.18(m,1H),7.69(br s,1H),6.66~7.10(m,3H),5.35(d,J=3.5Hz,3H),5.07~5.25(m,3H),4.76~4 .86(m, 3H), 4.01~4.31(m, 10H), 3.91~4.01(m, 8H), 3.74~3.86(m, 4H), 3.52~ 3.71(m, 30H), 3.42~3.50(m, 6H), 3.15~3.25(m, 4H), 2.52~2.70(m, 4H), 2.2 2~2.45(m, 2H), 2.15~2.22(s, 9H), 2.06(s, 9H), 1.95~2.03(m, 18H), 1.77(br s, 2H), 1.58~1.66(m, 4H), 1.40(m, 2H), 1.08~1.24(m, 12H). 31 P NMR (CDCl3): ppm δ 147.12.

[0243] To prepare GLPA15, the following scheme 3 was followed.

[0244] [ka] Scheme 3 Starting from secondary amine compound I (compound II in Scheme 2), Cbz protection was introduced to give compound II. The tert-butyl group of compound II was removed by acid treatment to give triacid compound III. Amide coupling of compound III with intermediate A gave compound IV. The Cbz protecting group of compound IV was removed by hydrogenation to give secondary amine compound V, which was reacted with glutaric anhydride to give carboxyl compound VI. Compound VI was reacted with piperidin-4-ol under amide coupling reaction conditions to give compound VII. Phosphoramidite compound GLPA15 was synthesized by treating compound VII with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole. 1 H NMR (400MHz in DMSO-d6): δppm8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64~3.97(m,12H),3.55~3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17~3.30(m,9H),3.07(br d, J=14.26Hz, 4H), 2.76(t, J=5.82Hz, 2H), 2.18~2.47(m, 6H), 2.10(s, 9H), 1 .99(s, 9H), 1.89(s, 9H), 1.78(s, 9H), 1.52~1.74(m, 6H), 1.12~1.19(m, 12H). 31P NMR (DMSO-d6): ppmδ145.25.

[0245] In one study, the method used to attach a targeting group containing GalNAc (also referred to herein as a GalNAc delivery compound) to the 5' end of the sense strand involved using a GalNAc phosphoramidite (GLPA1) in the final coupling step of solid-phase synthesis, using a synthetic process similar to the process used when oligonucleotide chain propagation is performed to add nucleotides to the 5' end of the sense strand.

[0246] Some studies have used a solid support (CPG) containing GLO-n as a method for attaching a GalNAc-containing targeting group to the 3'-end of the sense strand. Other studies have used a solid support (CPG) containing GLO-n as a method for attaching a GalNAc-containing targeting group to the 3'-end of the sense strand. Other studies have used a CPG solid support containing the GalNAc-containing targeting group via an ester bond, and then used the resulting CPG with the attached GalNAc-containing targeting group during synthesis of the sense strand, resulting in the attachment of a GalNAc-containing targeting group to the 3'-end of the sense strand.

[0247] Example 3 Phosphoramidite Compound 2 [ka] Scheme 4 DMTrCl (232 g, 684 mmol, 1.0 equiv.) in pyridine (400 mL) was added to a solution of isomannide compound A (100 g, 684 mmol, 1.0 equiv.) in pyridine (600 mL), and the mixture was stirred at 25 °C for 12 h. LC-MS analysis showed that compound A was completely consumed and one main peak with the desired mass was detected. The resulting reaction mixture was diluted with water (500 mL) and extracted with DCM (500 mL × 2). The combined organic phases were washed with brine (500 mL), dried over NaSO, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (DCM / MeOH = 100 / 1 to 50 / 1, 0.1% EtN) to give compound B (150 g, 48.9% yield) as a yellow solid. 1 H NMR:EC4783-404-P1B1_C(400MHz, DMSO-d6)δppm7.46(br d, J=7.63Hz, 2H)7.28~7.37(m, 6H)7.19~7.25(m, 1H)6.90(br d. t, J=8.13Hz, 1H)3.05(t, J=8.44Hz, 1H)2.85(br t, J = 7.50 Hz, 1H).

[0248] A solution of compound B (80.0 g, 178 mmol, 1.0 equiv) in DCM (800 mL) was added dropwise to 2H-tetrazole (0.45 M, 436 mL, 1.1 equiv) at 25 °C under a N atmosphere, and then compound C (80.6 g, 267 mmol, 85.0 mL, 1.5 equiv) in DCM (200 mL) was added dropwise to the mixture. The reaction mixture was stirred at 25 °C for 1.0 h. LC-MS showed that compound B was completely consumed and one main peak with the desired mass was detected. The resulting reaction mixture was cooled to -20 °C, poured into ice-cold saturated NaHCO (500 mL), extracted with DCM (500 mL × 3), and the combined organic layers were washed with saturated NaHCO / brine (1:1, 300 mL / 300 mL), dried over NaSO, and concentrated in vacuo (35 °C) to give a residue (100 mL). The residue was purified by column chromatography (AlO, DCM / MeOH (100 / 1 to 50 / 1, 0.1% EtN) to give compound 2 (77 g, 119 mmol, 66.5% yield) as a white solid. 1 H NMR: EC4783-423-P1B1_C (400MHz, DMSO-d6) δppm7.22 (br d, J=7.50Hz, 2H) 7.05~7.14 (m, 6H) 6.96~7.02 (m, 1H) 6.67 (br dd, J=8.82, 1.81Hz, 4H) 3.95~4.07(m, 2H) 3.73~3.83(m, 1H) 3.62~3.72(m, 2H) 3.48~3.53(m , 6H) 3.27~3.37(m, 3H) 3.11(s, 6H) 2.82(td, J=8.54, 2.31Hz, 1H) 2.47~2.63(m, 3H) 2.28(br d, J=1.63Hz, 3H)0.82~1.00(m, 13H).

[0249] Phosphoramidite Compound 1 [ka] Scheme 5 To a solution of compound B (500 mg, 1.11 mmol, 1.0 equiv) in DCM (5.0 mL) was added compound D (607 mg, 3.34 mmol, 3.0 equiv) and DIEA (432 mg, 3.34 mmol, 582 μL, 3.0 equiv) under a N atmosphere at 0-5 °C, and the mixture was stirred at 25 °C for 1.0 h. LC-MS showed that compound B was completely consumed, and several new peaks were observed on LC-MS, with ~70.9% of the desired compound being detected. The resulting reaction mixture was cooled to -20 °C, poured into cold (0-5 °C) saturated NaHCO3 (5.0 mL), extracted with DCM (5.0 mL × 2), and the combined organic layers were washed with cold (0-5 °C) saturated NaHCO3 / brine (1:1, 5.0 mL / 5.0 mL), dried over Na2SO4, and concentrated in vacuo to give a residue (~5 mL). The residue was purified by column chromatography (alkali Al2O3, petroleum ether / ethyl acetate (10:1 to 5:1, 0.1% Et3N) to give compound 1 (280 mg, 471 μmol, 42.3% yield) as a white solid. 1 H NMR: EC10615-49-P1N (400MHz, DMSO-d6) δppm7.44 (br d, J=7.63Hz, 2H), 7.31 (br t, J=7.94Hz, 6H), 7.18~7.26(m, 1H), 6.89(brd, J=8.00Hz, 4H), 4.08~4.13(m, 1H), 3.95~4 .03(m, 1H), 3.84~3.93(m, 1H), 3.77~3.83(m, 1H), 3.74(s, 6H), 3.43~3.53(m, 3H), 3.38(br d, J=6.75Hz, 1H), 2.94~3.04(m, 1H), 2.70~2.85(m, 1H), 1.09~1.15(m, 12H), 1.07(br s, 3H).

[0250] Other phosphoramidites can be prepared according to the procedures described herein and / or prior art such as, but not limited to, US Pat. No. 4,262,220 and WO 02 / 36743.

[0251] Example 4 Preparation of a solid support containing phosphoramidite monomers of the present invention [ka] [ka] represents the amine methyl polyethylene macroporous resin carrier moiety.

[0252] Scheme 6 Under nitrogen protection, dichloromethane (19.50 kg) was added to a 50 L glass kettle and stirring was initiated. The temperature was controlled at 20-30 °C, and DMTr(2H) ...)))))))))))). The system was maintained at 20-30 °C for 18 hours, and a sample was taken to terminate the reaction. Saturated sodium bicarbonate solution (22.50 kg) was added to the reaction system, and the mixture was stirred for 10-20 minutes, allowing the layers to separate. The organic phase was separated, and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to yield a residue that formed a gray to off-white solid (1.83 kg).

[0253] N,N-Dimethylformamide (23.50 kg) was added to a 100 L glass kettle and stirred. The temperature was controlled at 20-30°C. Under nitrogen protection, the products of the previous step, O-benzotriazole tetramethylurea hexafluorophosphate (0.33 kg) and N,N-diisopropylethylamine (0.13 kg), were added to the 100 L glass kettle through a solids feed funnel, stirred for 10-30 minutes, and then discharged into a 50 L zinc barrel for use. Macroporous amine methyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Science and Technology Co., Ltd., batch number HA2X1209, loading capacity 0.48 mmol / g) was added to the aforementioned 100 L solid-phase synthesis reactor through a solid feed funnel. The temperature was controlled at 20-30 °C, and N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction solution in the zinc barrel from the previous step were added to the solid-phase synthesis reactor. The system was subjected to an adiabatic reaction, and the solid loading was > The loading was tracked up to 250 µmol / g, and the loading detection method was UV. The system was filtered under nitrogen pressure, and the filter cake was washed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg). The filter cake was left in the kettle. CAP.A (50% acetonitrile and 50% acetic anhydride, 4.40 kg + 4.42 kg + 4.30 kg) and CAP.B (20% pyridine and 30% N-methylimidazole and 50% acetonitrile, 4.40 kg + 4.40 kg + 4.47 kg) were added to an 80 L glass kettle and stirred for 3-8 minutes before use. This procedure was repeated three times, capped, and acetonitrile (18.00 kg + 18.00 kg + 18.00 kg + 17.50 kg + 17.50 kg) was added to the solid-phase synthesis kettle. Nitrogen was blown in for 10 to 30 minutes, and then the mixture was filter-pressed. This procedure was repeated four times, and the filter cake was purged with nitrogen in the solid-phase synthesis kettle for 2 to 4 hours, after which it was transferred to a 50 L filter press tank. The temperature was controlled at 15 to 30°C, and drying was continued. After drying, a yellow to white solid product weighing 3.516 kg was obtained.

[0254] Example 5 Synthesis of PNPLA3 RNAi Agent The PNPLA3 RNAi agent duplexes shown in Tables 2-3 above were synthesized according to the following general procedure.

[0255] The sequences of the sense and antisense strands of siRNA were synthesized on an oligonucleotide synthesizer using an established solid-phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain propagation is achieved through a four-step cycle of deprotection, condensation, capping, and an oxidation or sulfurization step for each nucleotide addition. Synthesis was performed on a solid support made of controlled pore glass (CPG, 1000A). Monomeric phosphoramidites may be commercially purchased or may be the phosphoramidite compounds described in Example 3 and WO 2016 / 028649. The phosphoramidite compounds herein may be attached to the 3' end as monomeric phosphoramidites and further attached to the CPG solid support. When attached to the 5' end, the phosphoramidite compound may be used in a final coupling reaction and, if desired, can be further conjugated to a target ligand.

[0256] Phosphoramidites bearing GalNAc ligand clusters (non-limiting examples: GLPA1, GLPA2, and GLPA15) were synthesized according to the procedures in Examples 1-2 herein. For siRNAs used in in vitro screening (Table 2), synthesis was performed at a 2 μmol scale, and for siRNAs used in in vivo testing (Table 3), synthesis was performed at a 5 μmol or larger scale. When a GalNAc ligand (non-limiting example: GLO-0) was attached to the 3' end of the sense strand, a CPG solid support with a GalNAc ligand attached was used. When a GalNAc ligand (non-limiting example: GLS-5 or GLS-15) was attached to the 5' end of the sense strand, a GalNAc phosphoramidite (non-limiting example: GLPA1, GLPA2, or GLPA15) was used in the final coupling reaction.

[0257] The sense and antisense strands were synthesized by solid-phase synthesis in a four-step cycle, as detailed below. The 4,4'-dimethoxytrityl protecting group (DMT) was deprotected using 3% trichloroacetic acid (TCA) in dichloromethane or 10% dichloroacetic acid (DCA) in toluene. 5-Ethylthio-1H-tetrazole was used as the activator in the coupling step. Capping was performed with CapA (acetic anhydride in acetonitrile) / CapB (pyridine / NMI / acetonitrile) (v / v, 1:1). I2 in Py / HO and phenylacetyl disulfide (PADS) in pyridine / MeCN or xanthan gum hydride (DDTT) in pyridine were used for the oxidation and sulfurization reactions, respectively.

[0258] After the final solid-phase synthesis step, the solid-support-bound oligomer was cleaved and the protecting groups removed by treatment with a 1:1 volume solution of 40% methylamine (by weight) and 28% ammonium hydroxide in water. Solid-support-bound oligomers containing monomeric phosphomimetic moieties were optionally treated with MeCN:TMSI:pyridine (50:2:2, v / v / v) prior to C&D (cleavage and protection). For the synthesis of siRNAs used in in vitro screening, the crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc and ice-cold EtOH was added to precipitate the single-stranded product as the sodium salt, which was used for annealing without further purification. For the synthesis of multitarget molecules used in in vivo testing, the crude single-stranded product was further purified by ion-pair reverse-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving in 1.0 M NaOAc and precipitating with ice-cold EtOH. Equimolar amounts of complementary sense and antisense oligonucleotides were annealed in water to form double-stranded siRNA products, which were lyophilized to yield soft white solids.

[0259] Example 6 In vitro screening of PNPLA3 siRNA duplexes Hep3B cells were trypsinized to an appropriate density and seeded into 96-well plates. Simultaneously with seeding, cells were transfected with test or control siRNA using Lipofectamine RNAiMax (Invitrogen-13778-150) according to the manufacturer's recommended protocol. siRNAs were tested in triplicate at two concentrations (0.2 nM and 1.0 nM).

[0260] Day 0, psiCHECK™-2 vector transfection (1 plate) (1) Transfer 2.5 μg of psiCHECK™-2 vector plasmid to an RNASE-free Eppendorf tube (Solution Mix #1). (2) Add trypsin to detach Hep3B cells in one flask, count the cell number using a Vi-Cell counter, and adjust the cell density to 1 x 10^5 / mL.

[0261] (3) Transfer 7.5 μL of Fugene-HD to the Solution Mix #1 tube and mix.

[0262] (4) Add the solution from step 3 to the cell suspension, mix, and dispense the suspension into a 96-well plate (100 μL / well).

[0263] Day 1, siRNA transfection (1) Dilute Lipofectamine® RNAiMAX Reagent with Opti-MEM® medium.

[0264] (2) Dilute siRNA with RNA-free water to make a 12x stock.

[0265] (3) Mix equal amounts of diluted RNAiMax and siRNA, and incubate the mixture at room temperature for 15 minutes to allow complex formation.

[0266] (4) Add 45 μL / well of the compound Lipofectamine® RNAiMAX (Opti-MEM) mixture to 225 μL / well of DMEM fresh medium, discard the supernatant from the assay plate, and add 120 μL / well of the mixed compound to the 96-well plate.

[0267] (5) No-compound control wells were defined as cells transfected with the psiCHECK™-2 vector and without siRNA treatment, and blank controls were wells with cells only.

[0268] Day 2, Dual-Glo® Luciferase Assay (1) Add the reagent to the assay plate and wait 10 minutes for cell lysis to occur.

[0269] (2) Transfer 100 μL of the cell lysate to a plate and measure firefly luminescence.

[0270] (3) Add 50 μL of Dual-Glo® Stop&Glo® Reagent to the assay plate, mix, wait 10 minutes, and then measure Renilla luminescence.

[0271] (4) Calculate the relative expression Data analysis Ratio of sample well = (sample Renilla luminescence - background blank) / (sample firefly luminescence - background blank) Ratio of compound-free control wells = (control Renilla luminescence - background blank) / (control sample firefly luminescence - background blank) Inhibition rate (%) = 100 - (ratio of sample wells / average ratio of compound-free control) x 100% Table 4 shows experimental results of in vitro studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 2.

[0272] [Table 6-1] [Table 6-2] [Table 6-3]

[0273] Table 5 shows experimental results of in vitro studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 2.

[0274] [Table 7-1] [Table 7-2]

[0275] Example 7 In vivo testing of PNPLA3 siRNA duplexes On day 7 after siRNA administration, female C57BL / 6J mice (4 per group) were infected intravenously with an adeno-associated virus 8 (AAV8) vector encoding human PNPLA3 and luciferase genes. On day 1, mice received a single subcutaneous injection of 6 mg / kg PNPLA3 siRNA or saline. Blood samples were collected on days 1, 15, 22, and / or 29 before siRNA administration. Plasma samples were isolated and luciferase activity was measured according to the manufacturer's recommended protocol. Because human PNPLA3 expression levels correlate with luciferase expression levels, the percentage of remaining PNPLA3 was calculated as the ratio of luciferase signaling between post- and pre-administration plasma samples in the siRNA-treated group, normalized by the ratio of luciferase signaling between post- and pre-administration samples in the saline-treated group. The siRNA duplexes tested here, which had the modification patterns of the present invention, achieved significant knockdown of PNPLA3 mRNA and showed a longer duration of activity.

[0276] Table 6 shows experimental results of in vivo studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 3.

[0277] [Table 8]

[0278] Table 7 shows experimental results of in vivo studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 3.

[0279] [Table 9]

[0280] Table 8 shows experimental results of in vivo studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 3.

[0281] [Table 10]

[0282] Table 9 shows experimental results of in vivo studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 3.

[0283] [Table 11]

[0284] Example 8 In vivo testing of PNPLA3 siRNA agents in cynomolgus monkeys Eighteen healthy, untreated male cynomolgus monkeys (2-6 years old) were selected and randomly divided into six groups (3 per group). On day 0, saline or the test substance was subcutaneously injected once at 4 mg / kg. Liver biopsy samples were collected on days 0 (before administration), 21, and 42. PNPLA3 mRNA levels in liver tissue were measured using QPCR. The residual PNPLA3 mRNA levels in each group (normalized to the pre-administration level on day 0) are shown in Table 10.

[0285] Data analysis ΔCT = mean Ct of target gene - mean Ct of GAPDH ΔΔCT = ΔCT (sample) - ΔCT (before administration), mRNA relative expression = 2 -ΔΔCT Table 10 shows experimental results of in vivo studies using various PNPLA3 RNAi agents to inhibit PNPLA3 expression. The duplex sequences used correspond to those shown in Table 3.

[0286] [Table 12]

[0287] Equivalents While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision numerous other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is within the scope of the present invention, unless such features, systems, articles, materials, and / or methods are mutually inconsistent.

[0288] All definitions and definitions used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0289] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly stated to the contrary, should be understood to mean "at least one."

[0290] As used herein, the term "or" means "and / or" and is used interchangeably with the latter unless the context clearly excludes it. The phrase "and / or" as used in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are sometimes conjunctive and other times disjunctive. When there are more than one element and they are separated by a comma, the comma before "and / or" has the same meaning as "and / or" and denotes "and" or "or," respectively. Unless expressly stated to the contrary, other elements may optionally be present other than the elements specifically identified by the term "and / or," whether or not they are related to those specifically identified elements.

[0291] All references, patents, patent applications, and publications cited or referred to in this application are incorporated herein by reference in their entirety.

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3), the dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides, and the sense strand and the antisense strand can be partially, substantially, or fully complementary to each other, and optionally comprise a targeting ligand. dsRNA agents.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3), the dsRNA agent comprising a sense strand and an antisense strand, wherein nucleotide positions 2-18 of the antisense strand comprise a region of complementarity to a PNPLA3 RNA transcript, the region of complementarity comprising at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, and optionally comprising a target ligand. Double-stranded ribonucleic acid (dsRNA) agents.

3. the antisense strand comprises a region of complementarity to the PNPLA3 RNA transcript of at least 15, 16, 17, 18, or 19 contiguous nucleotides that differs by no more than three nucleotides from one of the antisense sequences listed in one of Tables 1-3; The dsRNA agent according to any one of claims 1 to 2.

4. The antisense strand of the dsRNA is at least substantially complementary to any one of the target regions of SEQ ID NO: 1 and provided in any one of Tables 1 to 3. The dsRNA agent according to any one of claims 1 to 3.

5. The antisense strand of the dsRNA is perfectly complementary to any one of the target regions of SEQ ID NO: 1 and provided in any one of Tables 1 to 3. The dsRNA agent according to any one of claims 1 to 3.

6. the dsRNA agent comprises a sense strand sequence set forth in any one of Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent; The dsRNA agent of any one of claims 1 to 5.

7. the dsRNA agent comprises a sense strand sequence set forth in any one of Tables 1-3, wherein the sense strand sequence is perfectly complementary to the antisense strand sequence in the dsRNA agent; The dsRNA agent of any one of claims 1 to 5.

8. the dsRNA agent comprises an antisense strand sequence set forth in any one of Tables 1-3. The dsRNA agent of any one of claims 1 to 7.

9. the dsRNA agent comprises a sequence set forth as a duplex sequence in any of Tables 1-3. The dsRNA agent of any one of claims 1 to 8.

10. the dsRNA agent comprises at least one modified nucleotide. The dsRNA according to any one of claims 1 to 9.

11. all or substantially all of the nucleotides of the antisense strand are modified nucleotides; The dsRNA agent of any one of claims 1 to 10.

12. the at least one modified nucleotide comprises a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seconucleotide mimic, a locked nucleotide, an unlocked nucleic acid nucleotide (UNA), a glycol nucleic acid nucleotide (GNA), a 2'-F-arabino nucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-Ome nucleotide, an inverted 2'-deoxy nucleotide, an isomannide nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a mophorino nucleotide, and a 3'-OMe nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a vinyl phosphonate, or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base containing nucleotide; 12. The dsRNA agent of claim 10 or 11.

13. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3), the dsRNA agent comprising a sense strand and an antisense strand, the sense strand being complementary to the antisense strand, and the antisense strand comprising a region complementary to a portion of an mRNA encoding PNPLA3, each strand being about 15 to about 30 nucleotides in length, the sense strand sequence being represented by formula (I): 【Chemistry 1】 During the ceremony, Each N' Ｆ represents a 2'-fluoro modified nucleotide, and each N' Ｎ１ , N' Ｎ２ , N' Ｎ３ , N' Ｎ４ , N' Ｎ５ , N' Ｎ６ , N' Ｎ７ , and Ｎ８ N' independently represents a modified or unmodified nucleotide, and each N' Ｌ independently represent a modified or unmodified nucleotide, but do not represent a 2'-fluoro modified nucleotide; m' and n' are each independently an integer from 0 to 7.

14. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3), the dsRNA agent comprising a sense strand and an antisense strand, the sense strand being complementary to the antisense strand, and the antisense strand comprising a region complementary to a portion of an mRNA encoding PNPLA3, each strand being about 18 to about 30 nucleotides in length, the antisense strand sequence being represented by formula (II): 【Chemistry 2】 During the ceremony, Each N Ｆ represents a 2'-fluoro modified nucleotide, and each N Ｍ１ , N Ｍ２ , N Ｍ３ , N Ｍ４ , N Ｍ５ , N Ｍ６ , N Ｍ７ , and N Ｍ８ independently represent a modified or unmodified nucleotide, preferably N Ｍ１ , N Ｍ２ , N Ｍ３ , N Ｍ６ , and N Ｍ７ each independently represents a 2'-fluoro modified nucleotide, and each N Ｌ independently represent a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide; and n is an integer from 0 to 7.

15. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of patatin-like phospholipase domain-containing 3 (PNPLA3), the dsRNA agent comprising a sense strand and an antisense strand, the sense strand and the antisense strand forming a dsRNA duplex, the sense strand being complementary to the antisense strand, the antisense strand comprising a region of complementarity to an mRNA encoding PNPLA3, the region of complementarity comprising at least 15 contiguous nucleotides, the dsRNA duplex being represented by formula (III): 【Transformation 3】 During the ceremony, Each N Ｆ and N' Ｆ independently represent a 2'-fluoro modified nucleotide, and each N Ｍ１ , N Ｍ２ , N Ｍ３ , N Ｍ４ , N Ｍ５ , N Ｍ６ , N Ｍ７ , N Ｍ８ , N' Ｎ１ , N' Ｎ２ , N' Ｎ３ , N' Ｎ４ , N' Ｎ５ , N' Ｎ６ , N' Ｎ７ , and N' Ｎ８ each independently represents a modified or unmodified nucleotide, and each N Ｌ and N' Ｌ independently represent a modified or unmodified nucleotide, but do not represent a 2'-fluoro modified nucleotide; m', n', and n are each independently an integer from 0 to 7.

16. the dsRNA agent includes an E-vinyl phosphonate nucleotide at the 5' end of the guide strand. The dsRNA agent of any one of claims 1 to 14.

17. The antisense strand sequence may be represented by formula (II'): 【Chemistry 4】 During the ceremony, Each N Ｆ represents a 2'-fluoro modified nucleotide, and each N Ｍ１ , N Ｍ２ , N Ｍ３ , N Ｍ４ , N Ｍ５ , N Ｍ６ , N Ｍ７ , and N Ｍ８ independently represent a modified or unmodified nucleotide, preferably N Ｍ１ , N Ｍ２ , N Ｍ３ , N Ｍ６ , and N Ｍ７ each independently represents a 2'-fluoro modified nucleotide, and each N Ｌ independently represent a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide; N Ｚ represents a nucleotide containing a vinyl phosphonate, and n is an integer from 0 to 7; 17. The dsRNA agent of claim 16.

18. The sense strand and the antisense strand form a dsRNA duplex, wherein the sense strand is complementary to the antisense strand, and the antisense strand comprises a region of complementarity to an mRNA encoding PNPLA3, the region of complementarity comprising at least 15 consecutive nucleotides, and the dsRNA duplex is represented by formula (III'): 【Transformation 5】 During the ceremony, Each N Ｆ and N' Ｆ independently represent a 2'-fluoro modified nucleotide, and each N Ｍ１ , N Ｍ２ , N Ｍ３ , N Ｍ４ , N Ｍ５ , N Ｍ６ , N Ｍ７ , N Ｍ８ , N' Ｎ１ , N' Ｎ２ , N' Ｎ３ , N' Ｎ４ , N' Ｎ５ , N' Ｎ６ , N' Ｎ７ , and N' Ｎ８ each independently represents a modified or unmodified nucleotide, and each N Ｌ and N' Ｌ independently represent a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide; N Ｚ represents a nucleotide containing a vinyl phosphonate, and m', n', and n are each independently an integer from 0 to 7; 17. The dsRNA agent of claim 16.

19. N Ｚ is a vinylphosphonate modified nucleotide, preferably N Ｚ is the structure 【Transformation 6】 VPu* having The dsRNA agent of any one of claims 16 to 18.

20. the dsRNA agent includes at least one phosphorothioate internucleoside linkage. The dsRNA agent of any one of claims 1 to 19.

21. the sense strand comprises at least one phosphorothioate internucleoside linkage; The dsRNA agent of any one of claims 1 to 19.

22. the antisense strand comprises at least one phosphorothioate internucleoside linkage; The dsRNA agent of any one of claims 1 to 19.

23. The sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages, preferably 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages introduced at the 5'-end, 3'-end, or both ends of the sense strand. The dsRNA agent of any one of claims 20 to 22.

24. The antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages, preferably 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages are introduced at the 5'-end, 3'-end, or both ends of the antisense strand; The dsRNA agent of any one of claims 20 to 22.

25. all or substantially all of the nucleotides of the sense strand and the antisense strand are modified nucleotides; The dsRNA agent of any one of claims 1 to 24.

26. The modified sense strand is a modified sense strand sequence set forth in one of Tables 2-3. The dsRNA agent of any one of claims 1 to 25.

27. The modified antisense strand is a modified antisense strand sequence set forth in one of Tables 2-3. The dsRNA agent of any one of claims 1 to 25.

28. the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is between 16 and 23 nucleotides in length; The dsRNA agent of any one of claims 1 to 27.

29. the complementary region is 19 to 21 nucleotides in length; The dsRNA agent of any one of claims 1 to 28.

30. Each strand is 30 nucleotides or less in length; 30. The dsRNA agent of any one of claims 1 to 29.

31. Each strand is no longer than 25 nucleotides in length. The dsRNA agent of any one of claims 1 to 30.

32. Each strand is no longer than 23 nucleotides. The dsRNA agent of any one of claims 1 to 31.

33. the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting or linking groups. The dsRNA agent of any one of claims 1 to 32.

34. the one or more targeting or linking groups are conjugated to the sense strand; 34. The dsRNA agent of claim 33.

35. the targeting group or linking group comprises N-acetylgalactosamine (GalNAc); 35. The dsRNA agent of claim 33 or 34.

36. The targeting group has the structure:

36. The dsRNA agent of any one of claims 33 to 35. Table 1-1 Table 1-2 Table 1-3 Table 1-4

37. the dsRNA agent comprises a targeting group conjugated to the 5' end of the sense strand. The dsRNA agent of any one of claims 1 to 36.

38. the dsRNA agent comprises a targeting group conjugated to the 3' end of the sense strand. The dsRNA agent of any one of claims 1 to 36.

39. the antisense strand comprises one inverted abasic residue at the 3' end; The dsRNA agent of any one of claims 1 to 38.

40. the sense strand comprises one or two inverted abasic residues at the 3'-end or / and 5'-end, or one or two iman residues at the 3'-end or / and 5'-end; The dsRNA agent of any one of claims 1 to 38.

41. the dsRNA agent has two blunt ends. The dsRNA agent of any one of claims 1 to 40.

42. At least one strand comprises a 3' overhang of at least one nucleotide; The dsRNA agent of any one of claims 1 to 40.

43. At least one strand comprises a 3' overhang of at least two nucleotides; The dsRNA agent of any one of claims 1 to 40.

44. 44. A composition comprising the dsRNA agent of any one of claims 1-43.

45. further comprising a pharmaceutically acceptable carrier, 45. The composition of claim 44.

46. further comprising one or more additional therapeutic agents, 46. ​​The composition of claim 45.

47. The composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.

47. The composition of claim 46.

48. The composition is formulated for subcutaneous administration or formulated for intravenous (IV) administration.

38. The composition of claim 37.

49. 37. A cell comprising the dsRNA agent of any one of claims 1 to 36.

50. the cell is a mammalian cell, optionally a human cell; 43. The cell of claim 42.

51. 1. A method for inhibiting expression of the PNPLA3 gene in a cell, comprising: (i) preparing a cell comprising an effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 43 or a composition according to any one of claims 44 to 48; method.

52. (ii) further comprising inhibiting expression of the PNPLA3 gene in the cells prepared in claim 51(i) by maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the PNPLA3 gene.

52. The method of claim 51.

53. the cell is present in a subject and the dsRNA agent is administered subcutaneously to the subject.

52. The method of claim 51.

54. the cell is present in a subject and the dsRNA agent is administered to the subject by IV administration.

52. The method of claim 51.

55. further comprising assessing inhibition of the PNPLA3 gene after administering the dsRNA agent to the subject, wherein said assessing means comprises: (i) determining one or more physiological characteristics of a PNPLA3-associated disease or condition in said subject; (ii) comparing the determined physiological characteristic with a baseline pre-treatment physiological characteristic for the PNPLA3-associated disease or condition and / or a control physiological characteristic for the PNPLA3-associated disease or condition; The comparison indicates one or more of the presence or absence of inhibition of expression of the PNPLA3 gene in the subject.

55. The method of claim 53 or 54.

56. The physiological characteristic determined is one or more of PNPLA3 mRNA level, PNPLA3 protein level, fat level and / or lipid droplet level in the liver, or the number or extent of amyloid deposits in the subject.

56. The method of claim 55.

57. a decrease in one or more of the subject's PNPLA3 mRNA level, the subject's PNPLA3 protein level, and the number or extent of amyloid deposits in the subject indicates a decrease in PNPLA3 gene expression in the subject.

57. The method of claim 56.

58. A method for inhibiting expression of the PNPLA3 gene in a subject, comprising administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 43 or a composition according to any one of claims 44 to 48. method.

59. the dsRNA agent is administered subcutaneously to the subject.

59. The method of claim 58.

60. the dsRNA agent is administered to the subject by IV administration; 59. The method of claim 58.

61. The method further includes assessing inhibition of the PNPLA3 gene after administration of the dsRNA agent, wherein the means for assessing comprises: (i) determining one or more physiological characteristics of a PNPLA3-associated disease or condition in said subject; (ii) comparing the determined physiological characteristic with a baseline pre-treatment physiological characteristic for the PNPLA3-associated disease or condition and / or a control physiological characteristic for the PNPLA3-associated disease or condition; The comparison indicates one or more of the presence or absence of inhibition of expression of the PNPLA3 gene in the subject.

61. The method of any one of claims 58 to 60.

62. The physiological characteristic determined is one or more of PNPLA3 mRNA level, PNPLA3 protein level, fat level and / or lipid droplet level in the liver, or number or extent of amyloid deposits.

62. The method of claim 61.

63. a decrease in one or more of the subject's PNPLA3 mRNA level, the subject's PNPLA3 protein level, and / or the number or extent of amyloid deposits indicates a decrease in PNPLA3 gene expression in the subject.

63. The method of claim 62.

64. A method for treating a disease or condition associated with the presence of PNPLA3 protein, comprising administering to a subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 43, or a composition according to any one of claims 44 to 48, to inhibit expression of the PNPLA3 gene. method.

65. the disease or condition is one or more of liver disease, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), cirrhosis, fat accumulation in the liver, inflammation of the liver, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, alcoholic liver disease, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, or non-alcoholic fatty liver disease (NAFLD); 65. The method of claim 64.

66. further comprising administering an additional therapeutic regimen to the subject.

65. The method of claim 64.

67. The additional treatment regimen comprises administering to the subject one or more PNPLA3 antisense polynucleotides of the present invention, administering to the subject a non-PNPLA3 dsRNA therapeutic agent, and behavioral modification in the subject.

67. The method of claim 66.

68. the non-PNPLA3 dsRNA therapeutic agent is one or more of an HMG-CoA reductase inhibitor, a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, an acyl-CoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol regulator, a bile acid regulator, a peroxisome proliferator-activated receptor (PPAR) agonist, a gene-based therapy, a combination vasoprotectant, a glycoprotein IIb / IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor, a squalene synthase inhibitor, a monocyte chemoattractant protein (MCP)-I inhibitor, or a fish oil; 68. The method of claim 67.

69. the dsRNA agent is administered subcutaneously to the subject.

65. The method of claim 64.

70. the dsRNA agent is administered to the subject by IV administration; 65. The method of claim 64.

71. further comprising determining the efficacy of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject.

71. The method of any one of claims 64 to 70.

72. The means for determining the effectiveness of the treatment in the subject comprises: (i) determining one or more physiological characteristics of the PNPLA3-associated disease or condition in the subject; (ii) comparing the determined physiological characteristic with a baseline pre-treatment physiological characteristic for the PNPLA3-related disease or condition; The comparison indicates one or more of the presence or absence of administration of the double-stranded ribonucleic acid (dsRNA) agent to the subject and the level of efficacy.

72. The method of claim 71.

73. The physiological characteristic determined is PNPLA3 mRNA level, PNPLA3 protein level, fat level and / or lipid droplet level in the liver, or the number or extent of amyloid deposits in the subject.

73. The method of claim 72.

74. A decrease in one or more of PNPLA3 mRNA levels, PNPLA3 protein levels, or the number or extent of amyloid deposits in the subject indicates the effectiveness of administering the double-stranded ribonucleic acid (dsRNA) agent to the subject.

73. The method of claim 72.

75. 47. A method of reducing the level of PNPLA3 protein in a subject compared to the subject's baseline pre-treatment level of PNPLA3 protein, comprising administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1-43, or a composition of any one of claims 44-48, to reduce the level of PNPLA3 gene expression. method.

76. the dsRNA agent is administered subcutaneously to the subject or administered IV to the subject; 76. The method of claim 75.

77. 47. A method of altering the physiological characteristics of a PNPLA3-associated disease or condition in a subject compared to the subject's baseline pre-treatment physiological characteristics of the PNPLA3-associated disease or condition, comprising administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1-43 or a composition of any one of claims 44-48, thereby altering the physiological characteristics of the PNPLA3-associated disease or condition in the subject. method.

78. the dsRNA agent is administered subcutaneously to the subject or administered IV to the subject; 78. The method of claim 77.

79. The physiological characteristic is one or more of PNPLA3 mRNA level, PNPLA3 protein level, fat level and / or lipid droplet level in the liver, or the number or extent of amyloid deposits in the subject.

78. The method of claim 77.

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