Patatin-like phospholipase domain-containing 3(PNPLA3)iRNA composition and method of use thereof

Abstract

We provide treatment for individuals suffering from non-alcoholic fatty liver disease (NAFLD). [Solution] A double-stranded ribonucleic acid (dsRNA) for inhibiting intracellular PNPLA3 expression is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a complementary region to mRNA encoding patatin-like phospholipase domain 3 (PNPLA3), and the complementary region comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of a specific set of sequences.

Description

[Technical Field]

[0001] Related applications This application claims the interests of U.S. Provisional Patent Application No. 62 / 948,445, filed on 16 December 2019, and U.S. Provisional Patent Application No. 63 / 040,602, filed on 18 June 2020. The entire contents of each of the foregoing are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy prepared on December 3 is named 121301-10620_SL.txt and is 1,097,169 bytes in size. [Background technology]

[0003] The accumulation of excess triglycerides in the liver is known as hepatic steatohepatosis (or fatty liver) and is associated with adverse metabolic outcomes, including insulin resistance and dyslipidemia. Fatty liver is frequently found in individuals with excessive alcohol consumption and those with obesity, diabetes, or hyperlipidemia. However, non-alcoholic fatty liver disease (NAFLD) can develop in the absence of excessive alcohol consumption (>10g per day). NAFLD refers to a broad spectrum of liver disease that can progress from simple fatty liver (steatohepatosis) to non-alcoholic steatohepatitis (NASH) and cirrhosis (irreversible and progressive scarring of the liver). All stages of NAFLD generally involve the accumulation of fat (fatty infiltration) within hepatocytes (hepatocytes).

[0004] The NAFLD spectrum begins with its earliest stage, called simple fatty liver (steatosis), and progresses from there. Simple fatty liver involves the accumulation of fat (triglycerides) in liver cells without inflammation (hepatitis) or scarring (fibrosis). The next stage and severity within the NAFLD spectrum is NASH, which involves hepatitis in addition to the accumulation of fat in liver cells. Inflammatory cells destroy liver cells (hepatocyte necrosis), and NASH ultimately leads to scarring (fibrosis) of the liver, followed by irreversible and progressive scarring (cirrhosis). Cirrhosis caused by NASH is the final and most severe stage within the NAFLD spectrum.

[0005] In 2008, a whole-genome association study involving proton magnetic resonance spectroscopy of the liver to assess hepatic fat content identified a significant association between hepatic fat content and the patatin-like phospholipase domain 3 (PNPLA3) gene [see, for example, Romeo et al. (2008) Nat. Genet., 40(12):1461-1465]. Knock-in mouse studies have confirmed that the expression of a sequence polymorphism (rs738409, I148M) within PNPLA3 induces NAFLD, and that the accumulation of catalytically inactive PNPLA3 on lipid droplets is associated with triglyceride accumulation in the liver [Smagris et al. (2015) Hepatology, 61:108-118]. Specifically, the I148M mutant of PNPLA3 was associated with the promotion of fibrillation by activating the Hedgehog (Hh) signaling pathway, leading to activation and proliferation of hepatic stellate cells, as well as excessive development and deposition of the extracellular matrix [Chen et al. (2015) World J. Gastroenterol., 21(3):794-802]. [Overview of the project] [Problems that the invention aims to solve]

[0006] Currently, treatment for NAFLD is directed towards weight loss and management of any secondary conditions, such as insulin resistance or dyslipidemia. Currently, there are no approved pharmacological treatments for NAFLD. Therefore, there is a need for treatment for individuals with NAFLD. [Means for solving the problem]

[0007] The present invention provides an iRNA composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of a gene encoding a patatin-like phospholipase domain 3 (PNPLA3). The patatin-like phospholipase domain 3 (PNPLA3) may be intracellular PNPLA3, for example, intracellular PNPLA3 within a subject such as a human subject.

[0008] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by 0, 1, 2, or 3 nucleotides or less, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by 1, 2, or 3 nucleotides or less. In one embodiment, the dsRNA agent comprises at least one thermally unstable nucleotide modification, e.g., debasic modification; mismatch with opposing nucleotides in the double helix; and unstable sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA), or glycerol nucleic acid (GNA), for example, the antisense strand comprises at least one thermally unstable nucleotide modification.

[0009] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a complementary region to mRNA encoding patatin-like phospholipase domain-containing 3 (PNPLA3), and the complementary region comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50.

[0010] In one embodiment, the present invention comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 677~721;683~721;773~817;1185~1295;1185~1241;1202~1295;1202~1241;1255~1295;1738~1792;1901~1945;1920~1945;2108~2208;2108~2166;2108~2136;2121~2166;2121~2208;2169~2 The present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides or less from any one of the nucleotide sequences 208;2176-2208; or 2239-2265, and an antisense strand comprising at least 19 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0011] In one embodiment, the present invention comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 574-596; 677-699; 683-705; 699-721; 773-795; 795-817; 1185-1207; 1192-1214; 1202-1224; 1208-1230; 1209-1231; 1210-1232; 1211-1233; 1212-1234; 1213-1233; 1214-1234; 1214-1236; 1215-1237; 1216-1238; 12 19~1237;1219~1241;1255~1275;1256~1276;1257~1275;1257~1277;1258~1278;1259~1279;1260~1278;1260~1280;1261~1281;1262~1282;1263~1 283;1264~1282;1264~1284;1265~1285;1267~1285;1266~1286;1266~1288;1267~1285;1267~1287;1268~1290;1269~1289;1270~1290;1271~1291;1 272~1292;1273~1293;1274~1294;1275~1295;1631~1653;1738~1760;1739~1761;1740~1760;1740~1762;1741~1763;1744~1766;1746~1766;1750~ 1772; 1751-1773; 1752-1774; 1753-1775; 1754-1776; 1755-1777; 1756-1778; 1757-1779; 1758-1780; 1759-1781; 1760-1782; 1761-1783; 1762-1782; 1762~1784;1763~1785;1764~1786;1765~1787;1766~1786;1766~1788;1767~1787;1768~1788;1767~1789;1769~1789;1770~1788;1770~1790;1771~ 1791; 1772-1792; 1815-1837; 1901-1923; 1920-1942; 1923-1945; 2112-2130; 2169-2191; 2171-2191; 2176-2198; 2177-2199; 2178-2200; 2179-2201;This invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising one of the nucleotide sequences 2180~2202;2181~2203;2183~2205;2184~2206;2186~2208;2239~2261;2241~2263;2242~2264; or 2243~2265, and at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides or less, and the antisense strand comprises at least 19 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0012] In one embodiment, the antisense chain is AD-517197.2;AD-517258.2;AD-516748.2;AD-516851.2;AD-519351.2;AD-519754.2;AD-519828.2;AD-520018.2;AD-520035.2;AD-520062.2;AD-520064.2;AD-520065.2;AD-520067.2;AD-75289.2;AD-520069.2;AD-520099.2;AD-67575.7;AD-520101.2;AD-67605 .7;AD-1193323.1;AD-1193344.1;AD-1193350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-1193422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1; and AD-1193481.1, comprising at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that are 0, 1, 2, or 3 nucleotides or less different from any one of the nucleotide sequences of the antisense strand of a double hedged strand selected from the group consisting of .7;AD-1193323.1;AD-1193344.1;AD-1193350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-1193422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1; and AD-1193481.1.

[0013] In one embodiment, the antisense chains are AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, AD-519350.1, AD-519753.2, AD-519932.1, AD-519935.2 , AD-520018.6, AD-517837.2, AD-805635.2, AD-519329.2, AD-520063.2, AD-519757.2, AD-805631.2, AD-51691 7.2, AD-516828.2, AD-518983.2, AD-805636.2, AD-519754.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-5200 53.4, AD-519667.2, AD-519773.2, AD-519354.2, AD-520060.4, AD-520061.4, AD-1010733.2, AD-1010735.2;AD -1193323.1;AD-1193344.1;AD-1193350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-11 A double helix containing one of the nucleotide sequences of the antisense strand of a double helix selected from the group consisting of 93422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1; and AD-1193481.1, and at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, or 20, that differ by 0, 1, 2, or 3 nucleotides or less.

[0014] In one embodiment, the present invention comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises nucleotides 1200-1250, 1205-1250, 11210-1250, 1200-1245, 1200-1240, 1200-1235, 1200-1237, 1205-1237, 1210-1232, 1212-1237, 1212-1234, 1250-1300, 1255-1300, 1260-1300, 1250-1295, 1250-1390, 1250-1285, 1250- The present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences 1282, 1255-1282, 1260-1282, 1262-1300, 1262-1295, 1262-1390, and 1262-1282, wherein the antisense strand comprises at least 15 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO: 2.

[0015] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides or less from any one of the nucleotide sequences of the antisense strand, selected from the group consisting of AD-519345, AD-1193350, AD-1193365, AD-1193437; and AD-519347.

[0016] In one embodiment, the antisense strand is a double hemisphere and comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of the antisense strand of AD-519351 by 0, 1, 2, or 3 nucleotides or less.

[0017] In one embodiment, the antisense strand is a double hemisphere and comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of the antisense strand of AD-1193350 by 0, 1, 2, or 3 nucleotides or less.

[0018] In one embodiment, the antisense strand is a double hemisphere and comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of the antisense strand of AD-1193365 by 0, 1, 2, or 3 nucleotides or less.

[0019] In one embodiment, the antisense strand is a double hemisphere and comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of the antisense strand of AD-1193437 by 0, 1, 2, or 3 nucleotides or less.

[0020] In one embodiment, the antisense strand is a double hemisphere and comprises at least 15, for example, 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from the nucleotide sequence of the antisense strand of AD-519347 by 0, 1, 2, or 3 nucleotides or less.

[0021] In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

[0022] In one embodiment, substantially all of the nucleotides in the sense strand are modified; substantially all of the nucleotides in the antisense strand are modified; or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified.

[0023] In one embodiment, all nucleotides of the sense strand are modified; all nucleotides of the antisense strand are modified; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.

[0024] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, lock nucleotides, unlock nucleotides, conformation-fixed nucleotides, restricted ethyl nucleotides, debasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramides, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, thermally unstable nucleotides, glycol modified nucleotides (GNAs), and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof.

[0025] In one embodiment, the nucleotide modification is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol; and combinations thereof.

[0026] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, and vinyl phosphonate nucleotides; and combinations thereof.

[0027] In another embodiment, at least one of the modifications on the nucleotide is a thermally unstable nucleotide modification.

[0028] In one embodiment, the thermally unstable nucleotide modification is selected from the group consisting of debasic modification; mismatch with opposing nucleotides in a double helix; and unstable sugar modification, 2'-deoxy modification, acyclic nucleotides, unlocked nucleic acids (UNAs), and glycerol nucleic acids (GNAs).

[0029] In some embodiments, the modified nucleotide includes a short sequence of 3'-terminal deoxythymine nucleotides (dT).

[0030] In some embodiments, the modifications on the nucleotide are 2'-O-methyl modifications, GNA modifications, and 2'-fluoro modifications.

[0031] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate nucleotide linkage. In some embodiments, the dsRNA agent comprises six to eight phosphorothioate nucleotide linkages. In one embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at the 3' end of one strand. The strand may be an antisense strand. In another embodiment, the strand is a sense strand. In a related embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at the 5' end of one strand. The strand may be an antisense strand. In another embodiment, the strand is a sense strand. In another embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at both the 5' and 3' ends of one strand. The strand may be an antisense strand. In another embodiment, the strand is a sense strand.

[0032] The double-stranded region can be 19–30 nucleotide pairs long; 19–25 nucleotide pairs long; 19–23 nucleotide pairs long; 23–27 nucleotide pairs long; or 21–23 nucleotide pairs long.

[0033] In one embodiment, each chain is independently 30 nucleotides or less in length.

[0034] In one embodiment, the sense strand is 21 nucleotides long, and the antisense strand is 23 nucleotides long.

[0035] The complementary region can be at least 17 nucleotides long; between 19 and 23 nucleotides long; or 19 nucleotides long.

[0036] In one embodiment, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides.

[0037] In one embodiment, the dsRNA agent further comprises a ligand.

[0038] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0039] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0040] In one embodiment, the ligand is one or more GalNAc derivatives linked via monovalent, divalent, or trivalent branched linkers.

[0041] In one embodiment, the ligand is

[0042] [ka] That is the case.

[0043] In one embodiment, the dsRNA agent is shown in the following diagram.

[0044] [ka] It is conjugated with the ligand shown in and X is either O or S.

[0045] In one embodiment, X is O.

[0046] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage.

[0047] In one embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at the 3' end of one chain, for example, on the antisense or sense chain.

[0048] In another embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at the 5' end of one chain, for example, the antisense strand or the sense strand.

[0049] In one embodiment, the phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage is located at both the 5' and 3' ends of a single chain. In one embodiment, the chain is an antisense chain.

[0050] In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the double helix is ​​an AU base pair.

[0051] The present invention also provides cells containing any of the dsRNA agents of the present invention, and a pharmaceutical composition comprising any of the dsRNA agents of the present invention.

[0052] The pharmaceutical composition of the present invention may contain the dsRNA agent in a non-buffered solution, for example, physiological saline or water; or it may contain the dsRNA agent in a buffer solution, for example, a buffer solution containing acetate, citrate, procramine salt, carbonate, or phosphate, or any combination thereof; or in phosphate-buffered physiological saline (PBS).

[0053] In one embodiment, the present invention provides a method for inhibiting the expression of the intracellular patatin-like phospholipase domain-containing 3 (PNPLA3) gene. The method comprises contacting cells with either the dsRNA of the present invention or the pharmaceutical composition of the present invention, thereby inhibiting the expression of the intracellular PNPLA3 gene.

[0054] In one embodiment, the cells are cells from within a subject having a patatin-like phospholipase domain 3 (PNPLA3)-related disorder, such as a patatin-like phospholipase domain 3 (PNPLA3)-related disorder, selected from the group consisting of, for example, human subjects, such as fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD).

[0055] In one embodiment, contacting cells with a dsRNA agent inhibits PNPLA3 expression by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0056] In one embodiment, inhibiting PNPLA3 expression reduces the PNPLA3 protein level in the serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0057] In one embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced expression of patatin-like phospholipase domain 3 (PNPLA3). The method comprises administering to the subject a therapeutically effective amount of either the dsRNA of the present invention or the pharmaceutical composition of the present invention, thereby treating the subject having a disorder that would benefit from reduced PNPLA3 expression.

[0058] In another embodiment, the present invention provides a method for preventing at least one symptom in a subject having a disorder that benefits from reduced expression of patatin-like phospholipase domain 3 (PNPLA3). The method comprises administering to a subject a prophylactic effective amount of either the dsRNA of the present invention or the pharmaceutical composition of the present invention, thereby preventing at least one symptom in a subject having a disorder that benefits from reduced expression of PNPLA3.

[0059] In one embodiment, the disorder is a patatin-like phospholipase domain-containing 3 (PNPLA3)-related disorder, which is selected from the group consisting of fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD).

[0060] In one embodiment, the PNPLA3-related disorder is NAFLD.

[0061] In one embodiment, the subject is a human being.

[0062] In one embodiment, the dsRNA agent is administered to the subject in a dose of approximately 0.01 mg / kg to approximately 50 mg / kg.

[0063] In one embodiment, the dsRNA agent is administered subcutaneously to the subject.

[0064] In one embodiment of the present invention, the method includes further determining the PNPLA3 level in a sample derived from the subject.

[0065] In one embodiment, the level of patatin-like phospholipase domain-containing 3 (PNPLA3) in the target sample is the level of patatin-like phospholipase domain-containing 3 (PNPLA3) protein in a blood sample or serum sample.

[0066] In certain embodiments, the method of the present invention further comprises administering a further therapeutic agent to a subject. In further embodiments, the further therapeutic agent is selected from the group consisting of HMG-CoA reductase inhibitors, fibrates, bile acid chelating agents, niacin, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transport protein (CETP) inhibitors, microsomal triglyceride transport protein (MTTP) inhibitors, cholesterol modulators, bile acid modulators, peroxisome proliferation-activated receptor (PPAR) agonists, gene-based therapies, complex vasoprotective agents, glycoprotein Ilb / IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors, monocyte chemotactic protein (MCP) I inhibitors, or fish oil.

[0067] The present invention also provides a kit comprising either the dsRNA of the present invention or a pharmaceutical composition of the present invention, and, as appropriate, instructions for use. [Brief explanation of the drawing]

[0068] [Figure 1]Figure 1 is a graph showing human PNPLA3 mRNA levels in mice (n=3 per group) 7 or 14 days after a single subcutaneous administration of 3 mg / kg or 10 mg / kg of the indicated dsRNA double-stranded molecule. The human PNPLA3 mRNA levels are shown compared to the control level detected by PBS treatment. [Figure 2] Figure 2 is a graph showing the human PNPLA3 mRNA levels in mice (n=3 per group) 7 days after administration of a single subcutaneous dose of the indicated dsRNA double helix, as described in Example 3. The human PNPLA3 mRNA levels are shown in comparison to the control level detected by PBS treatment. [Figure 3] Figure 3 is a graph showing the human PNPLA3 mRNA levels in mice (n=3 per group) 7 days after a single subcutaneous administration of 10 mg / kg of the indicated dsRNA double-stranded molecule. The human PNPLA3 mRNA levels are shown in comparison to the control level detected by PBS treatment. [Figure 4] Figure 4 is a graph showing the human PNPLA3 mRNA levels in mice (n=3 per group) 7 days after administration of the indicated dsRNA double helix, either as described in Example 3, with a single dose of 10 mg / kg or multiple doses of 10 mg / kg administered subcutaneously. The human PNPLA3 mRNA levels are shown in comparison to the control level detected by PBS treatment. [Figure 5] Figure 5 is a graph showing the human PNPLA3 mRNA levels in mice (n=3 per group) 7 days after a single subcutaneous administration of 10 mg / kg of the indicated dsRNA double-stranded molecule. The human PNPLA3 mRNA levels are shown in comparison to the control level detected by PBS treatment. [Figure 6]Figure 6 is a graph showing the human PNPLA3 mRNA levels in mice (n=3 per group) 7 days after a single subcutaneous administration of 10 mg / kg of the indicated dsRNA double-stranded molecule. The human PNPLA3 mRNA levels are shown in comparison to the control level detected by PBS treatment. [Figure 7] Figure 7 is a graph showing the human PNPLA3 mRNA levels in mice (n=3 per group) 7 days after a single subcutaneous administration of 10 mg / kg of the indicated dsRNA double-stranded molecule. The human PNPLA3 mRNA levels are shown in comparison to the control level detected by PBS treatment. [Modes for carrying out the invention]

[0069] This invention provides iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the patatin-like phospholipase domain-containing 3 (PNPLA3) gene. The gene may be intracellular, for example, an intracellular gene within a target such as a human subject. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (patatin-like phospholipase domain-containing 3 (PNPLA3) gene) in mammals.

[0070] The iRNAs of the present invention are designed to target the human patatin-like phospholipase domain-containing 3 (PNPLA3) gene, including portions of the gene that are conserved within the patatin-like phospholipase domain-containing 3 (PNPLA3) orthologs of other mammalian species. Not intended to be limited to theory, the aforementioned properties and combinations or partial combinations of specific target sites or specific modifications in these iRNAs are thought to confer improved efficacy, stability, efficacy, persistence, and safety to the iRNAs of the present invention.

[0071] Accordingly, the present invention provides a method for treating and preventing patatin-like phospholipase domain-containing 3 (PNPLA3)-related disorders, such as fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD), using an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PNPLA3 gene.

[0072] The iRNA of the present invention is at most about 30 nucleotides or less in length, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, and includes an RNA strand (antisense strand) having a region substantially complementary to at least a portion of the mRNA transcript of the PNPLA3 gene.

[0073] In certain embodiments, one or both strands of the double-stranded RNAi agent of the present invention are substantially complementary to at least a portion of the mRNA transcript of the PNPLA3 gene and comprise a region of at least 19 consecutive nucleotides, with a maximum length of 66 nucleotides, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides. In some embodiments, such an iRNA agent having a longer antisense strand may preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, in which case the sense strand and antisense strand form a double helix of 18-30 consecutive nucleotides.

[0074] The use of the iRNAs of the present invention enables targeted degradation of the mRNA of the corresponding gene (PNPLA3 gene) in mammals. Using in vitro assays, the inventors have confirmed that iRNAs targeting the PNPLA3 gene result in significant inhibition of PNPLA3 gene expression as a result of potent mediation of RNAi. Therefore, methods and compositions containing these iRNAs are useful for treating subjects with PNPLA3-related disorders, such as fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD).

[0075] Accordingly, the present invention provides methods and combination therapies for treating subjects having disorders that benefit from inhibition or reduction of PNPLA3 gene expression, such as patatin-like phospholipase domain-containing 3 (PNPLA3) related diseases, including fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD), using iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PNPLA3 gene.

[0076] The present invention also provides a method for preventing at least one symptom in subjects having disorders that benefit from inhibition or reduction of PNPLA3 gene expression, such as fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). For example, in subjects having NAFLLD, the method of the present invention can alleviate at least one symptom in the subject, such as fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider angiography, yellowing of the skin and eyes (jaundice), itching, solidification of body fluids and swelling of the legs (edema), abdominal swelling (ascites), and mental confusion.

[0077] The following "Modes for Carrying Out the Invention" disclose how to prepare and use compositions containing iRNA to inhibit the expression of the PNPLA3 gene, as well as compositions, uses, and methods for treating subjects who would benefit from the inhibition and / or reduction of PNPLA3 gene expression, for example, subjects who are susceptible to or diagnosed with PNPLA3-related disorders.

[0078] I. Definition To make the present invention easier to understand, we will first define certain terms. In addition, please note that whenever parameter values ​​or ranges of values ​​are enumerated, the values ​​and the intermediate ranges between the enumerated values ​​are also intended to be part of the present invention.

[0079] In this specification, the articles “a” and “an” are used to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, “element” means one or more elements, e.g., multiple elements.

[0080] In this specification, the term "including" is used interchangeably with the phrase "including, but not limited to," which means "including, but not limited to."

[0081] In this specification, the term "or" is used interchangeably with the term "and / or" unless the context explicitly indicates otherwise. For example, "sense strand or antisense strand" is understood as "sense strand or antisense strand, or sense strand and antisense strand."

[0082] The term “approximately” is used herein to mean that a value is within the range of typical tolerances in the art. For example, “approximately” may be understood to mean approximately two standard deviations from the mean. In a particular embodiment, “approximately” means ±10%. In a particular embodiment, “approximately” means ±5%. When “approximately” precedes a set of numbers or ranges, it is understood that “approximately” may modify each of the set of numbers or ranges of numbers.

[0083] The term “at least” preceding a number or range of numbers is understood to include all subsequent numbers or integers that may logically be included, as is evident from the number adjacent to the term “at least” and the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, “at least 19 nucleotides out of a 21-nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated characteristic. When “at least” precedes a range of numbers or numbers, it is understood that “at least” may modify each of the numbers or ranges of numbers.

[0084] As used herein, “less than or equal to” or “less than” is understood, as logically pertinent from the context, to be the value adjacent to the phrase and any value or integer logically below it, up to zero. For example, a double helix with “less than or equal to 2 nucleotides” has 2, 1, or 0 nucleotides in its protrusions. When “less than or equal to” follows a set of numbers or ranges, it is understood that “less than or equal to” can modify each of the sets of numbers or ranges of numbers. As used herein, ranges include both upper and lower limits.

[0085] The detection methods used herein may include determining that the amount of analyte present is below the detection level of the method.

[0086] If there is a discrepancy between the indicated target site and the nucleotide sequence of the sense or antisense strand, the indicated sequence takes precedence.

[0087] In the event of any discrepancy between a sequence and its corresponding site on a transcript, or between it and other sequences, the nucleotide sequences listed herein shall prevail.

[0088] In this specification, the term "patatin-like phospholipase domain-containing 3," used interchangeably with the term "PNPLA3," refers to a well-known gene that encodes a triacylglycerol lipase that mediates the hydrolysis of triacylglycerols within adipocytes.

[0089] Exemplary nucleotide and amino acid sequences of PNPLA3 include, for example, GenBank accession number: NM_025225.2 [Human (Homo sapiens) PNPLA3; SEQ ID NO: 1; Reverse complement, SEQ ID NO: 2]; GenBank accession number: NM_054088.3 [Mouse (Mus musculus) PNPLA3; SEQ ID NO: 3; Reverse complement, SEQ ID NO: 4]; GenBank accession number: NM_001282324.1 [Rat (Rattus norvegicus) PNPLA3; SEQ ID NO: 5; Reverse complement, SEQ ID NO: 6]; GenBank accession number: XM_005567051.1 [Cynomolgus monkey (Macaca fascicularis) PNPLA3; SEQ ID NO: 7; Reverse complement, SEQ ID NO: 8]; GenBank accession number: XM_001109144.2 [Rhesus monkey (Macaca It can be found in [mulatta)PNPLA3;SEQ ID NO: 9; Reverse complement, SEQ ID NO: 10]; and GenBank accession number: XM_005567052.1 [cynomolgus monkey PNPLA3;SEQ ID NO: 11; Reverse complement, SEQ ID NO: 12].

[0090] Further examples of PNPLA3 mRNA sequences are readily available through public databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0091] Further information about PNPLA3 can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=pnpla3.

[0092] The full contents of each of the aforementioned GenBank accession numbers and gene database numbers as of the filing date of this application are incorporated herein by reference.

[0093] As used herein, the term PNPLA3 also refers to mutations in the PNPLA3 gene, including variants presented in SNP databases. Numerous sequence mutations within the PNPLA3 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=pnpla3, whose full content as of the filing date of this application is incorporated herein by reference).

[0094] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the PNPLA3 gene, which includes mRNA that is the product of RNA processing of the primary transcript. The target portion of the sequence will be at least sufficiently long to be used as a substrate for iRNA-led cleavage at or near this portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the PNPLA3 gene. In one embodiment, the target sequence is located within the protein-coding region of PNPLA3.

[0095] The target sequence may be approximately 19 to 36 nucleotides in length, for example, preferably approximately 19 to 30 nucleotides. For example, the target sequence may be approximately 19 to 30 nucleotides, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length. In certain embodiments, the target sequence is 19–23 nucleotides in length, and may also be 21–23 nucleotides in length. Intermediate ranges and lengths between those listed above are also assumed to be part of this disclosure.

[0096] As used herein, the term “sequence-containing chain” refers to an oligonucleotide chain containing a sequence described by a sequence referred to using standard nucleotide nomenclature.

[0097] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or surrogate substitutions (see, for example, Table 1), which are further detailed below. Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of oligonucleotides containing such substitutions. For example, to the extent that, a nucleotide containing inosine as its base can base-pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be replaced, for example, with inosine-containing nucleotides within the nucleotide sequences of the dsRNAs characterized in the present invention. In another example, adenine and cytosine at any position within an oligonucleotide may be replaced by guanine and uracil, respectively, to form GU fluctuation base pairs with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the present invention.

[0098] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant,” as used interchangeably herein, refer to agents containing RNA that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as defined herein. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, for example, inhibits the expression of the PNPLA3 gene within cells, for example, within subjects such as mammalian subjects.

[0099] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, the target mRNA sequence of PNPLA3, to direct the cleavage of the target RNA. Without being bound by theory, it is thought that long double-stranded RNA introduced into a cell is degraded into siRNA by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic 2-base 3' overhang [Bernstein, et al., (2001) Nature 409:363]. Subsequently, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one embodiment, the present invention relates to a single-stranded RNA (siRNA) that is generated in a cell and facilitates the formation of a RISC complex that performs silencing of a target gene, namely the PNPLA3 gene. Accordingly, in this specification, the term "siRNA" is also used to refer to the iRNA described above.

[0100] In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are typically 15–30 nucleotides long and are chemically modified. The design and testing of single-stranded siRNAs are described in full in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883–894, each of which is incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs that have been chemically modified in the manner described herein or in the manner described in Lima et al., (2012) Cell 150:883–894.

[0101] In certain embodiments of the present invention, the “iRNA” for use in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred herein to as “double-stranded RNA agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two, antiparallel, substantially complementary nucleic acid strands, and is referred to as having “sense” and “antisense” orientations with respect to the target RNA, i.e., the PNPLA3 gene. In certain embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0102] Generally, the majority of nucleotides in each chain of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each chain or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, “iRNA” may include ribonucleotides with chemical modifications; iRNA may include substantial modifications in multiple nucleotides. As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of functional groups or atoms from nucleoside linkages, sugar moieties, or nucleobases. Modifications suitable for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by “iRNA” or “RNAi agent” as used herein and in the claims.

[0103] In certain embodiments of this disclosure, the incorporation of deoxyribonucleotides when present in an RNAi agent is considered to constitute modified nucleotides.

[0104] The double-stranded region can be of any length, enabling the specific degradation of the desired target RNA via the RISC pathway, approximately 19–36 base pairs in length, for example, approximately 19–30 base pairs, for example, approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, approximately 19–30, 1 The lengths may range from 9 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In certain embodiments, the double-stranded region is 19 to 21 base pairs long, for example, 21 base pairs long. Intermediate ranges and lengths between those listed above are also assumed to be part of this disclosure.

[0105] The two strands forming a double helix structure may be different parts of a single large RNA molecule, or they may be separate RNA molecules. When the two strands are part of a single large molecule and are therefore connected by an uninterrupted nucleotide strand that forms a double helix structure between the 3' end of one strand and the corresponding 5' end of the other, the connecting RNA strands are referred to as a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, a hairpin loop may be 10 nucleotides or less. In some embodiments, a hairpin loop may be 8 unpaired nucleotides or less. In some embodiments, a hairpin loop may be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may be 4 to 8 nucleotides.

[0106] If the two substantially complementary strands of a dsRNA are contained within separate RNA molecules, these molecules do not need to be covalently linked, but may be. When the two strands are covalently linked by means other than an uninterrupted nucleotide chain, forming a double-strand structure between the 3' end of one strand and the corresponding 5' end of the other, the linkage structure is called a "linker." RNA strands may have the same number of nucleotides or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest dsRNA strand minus any protrusions present in the double helix. In addition to the double-strand structure, RNAi may contain one or more nucleotide protrusions. In one embodiment of an RNAi agent, at least one strand contains a 3' protrusion of at least one nucleotide. In another embodiment, at least one strand contains a 3' protrusion of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent contains a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand contains a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent contain an overhang of at least one nucleotide.

[0107] In a particular embodiment, the iRNA agent of the present invention is a dsRNA in which each strand comprises 19 to 23 nucleotides and interacts with a target RNA sequence, such as the PNPLA3 gene, to direct the cleavage of the target RNA.

[0108] In some embodiments, the iRNA of this disclosure is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a target mRNA sequence of PNPLA3, to direct the cleavage of the target RNA.

[0109] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA can contain at least one nucleotide overhang; alternatively, an overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain a deoxynucleotide / nucleoside, a nucleotide / nucleoside analog, or consist of these. An overhang may be located on the sense strand, on the antisense strand, or on any combination thereof. Furthermore, a nucleotide overhang may be located at the 5' end of the antisense or sense strand of a dsRNA, at the 3' end, or at both ends.

[0110] In one embodiment, the antisense strand of the dsRNA has a protrusion of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at its 3' or 5' end. In another embodiment, one or more nucleotides within the protrusion are replaced by a thiophosphate nucleoside.

[0111] In one particular embodiment, the antisense strand of the dsRNA has a protrusion of 1 to 10 nucleotides at its 3' or 5' end, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides within the protrusion are replaced by a thiophosphate nucleoside.

[0112] In certain embodiments, the antisense strand of dsRNA has a protrusion of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at its 3' or 5' end. In certain embodiments, the protrusion on the sense strand or the antisense strand, or both, may include lengths exceeding 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the elongation protrusion is located on the sense strand of the double helix. In certain embodiments, the elongation protrusion is located on the 3' end of the sense strand of the double helix. In certain embodiments, the elongation protrusion is located on the 5' end of the sense strand of the double helix. In certain embodiments, the elongation protrusion is located on the antisense strand of the double helix. In certain embodiments, the elongation protrusion is located on the 3' end of the antisense strand of the double helix. In certain embodiments, the elongation projection is located on the 5' end of the antisense strand of the double helix. In certain embodiments, one or more nucleotides within the elongation projection are replaced by a thiophosphate nucleoside. In certain embodiments, the projection includes a self-complementary moiety such that the projection can form a hairpin structure that is stable under physiological conditions.

[0113] "Smooth" or "blunt-ended" means that there are no unpaired nucleotides at this end of a double-stranded RNA agent, i.e., there are no nucleotide overhangs. A "blunt-ended" double-stranded RNA agent is double-stranded along its entire length, i.e., there are no nucleotide overhangs at any end of the molecule. The RNAi agents of the present invention include RNAi agents that do not have nucleotide overhangs at one end (i.e., agents with one overhanging end and one blunt end), or RNAi agents that do not have nucleotide overhangs at either end. Such molecules are very often double-stranded along their entire length.

[0114] The terms "antisense strand" or "guide strand" refer to an iRNA strand, such as a dsRNA strand, that contains a region substantially complementary to the target sequence, such as PNPLA3 mRNA.

[0115] As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., the PNPLA3 nucleotide sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in an internal region of the molecule or in a terminal region. Generally, the most acceptable mismatches are located in terminal regions ranging from 5' or 3' ends of the iRNA to 5, 4, or 3 nucleotides. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches within the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the sense strand, for example, the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches within the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, within the range of 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, within the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) are not within the seed region.

[0116] Therefore, the RNAi agents described herein may contain one or more mismatches with the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In a particular embodiment, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch may be restricted to the range of the last five nucleotides from the 5' or 3' end of the complementarity region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to the PNPLA3 gene region generally does not contain any mismatches within the range of 13 nucleotides in the middle. Methods described herein, or methods known in the art, may be used to determine whether RNAi agents containing a mismatch with a target sequence are effective in inhibiting the expression of the PNPLA3 gene. Investigating the effectiveness of mismatched RNAi agents in inhibiting PNPLA3 gene expression is particularly important when it is known that specific complementary regions within the PNPLA3 gene have polymorphic sequence variations within the population.

[0117] As used herein, the terms “sense strand” or “passenger strand” refer to an iRNA strand containing an antisense strand region and a substantially complementary region, as defined herein.

[0118] As used herein, “substantially all modified nucleotides” means that most of the nucleotides are modified but not completely modified, and may include not more than five, four, three, two, or one unmodified nucleotides.

[0119] As used herein, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where cleavage occurs. In some embodiments, the cleavage region is located on either end of a cleavage site and contains three bases directly adjacent to the cleavage site. In some embodiments, the cleavage region is located on either end of a cleavage site and contains two bases directly adjacent to the cleavage site. In some embodiments, the cleavage site occurs specifically at the site where nucleotides 10 and 11 of the antisense strand are bound, and the cleavage region contains nucleotides 11, 12, and 13.

[0120] As used herein, and unless otherwise indicated, the term “complementarity” refers, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, to the ability of an oligonucleotide or polynucleotide containing a second nucleotide sequence to hybridize with that oligonucleotide or polynucleotide under certain conditions to form a double-stranded structure, as understood by those skilled in the art. Such conditions may be strict, including, for example, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, and subsequent washing for 12–16 minutes at 50°C or 70°C [see, for example, “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press]. Other conditions, such as physiologically relevant conditions that may be encountered within living organisms, may also be applicable. Those skilled in the art will be able to determine the set of conditions most appropriate for testing the complementarity of the two sequences, depending on the final application of the nucleotides to be hybridized.

[0121] Complementary sequences within iRNA, for example, complementary sequences within dsRNA as described herein, include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence, spanning the full length of one or both nucleotide sequences. In this specification, such sequences may be referred to as “fully complementary” in light of each other. However, where in this specification the first sequence is referred to as “substantially complementary” in light of the second sequence, the two sequences may be fully complementary and, while retaining their ability to hybridize in their final application, e.g., under conditions most relevant to inhibiting gene expression via the RISC pathway, they may generally form one or more mismatched base pairs, not exceeding five, four, three, or two, during hybridization for double helixes up to 30 base pairs. However, if two oligonucleotides are designed to form one or more single-stranded protrusions during hybridization, such protrusions shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing a 21-nucleotide sequence comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, can also be described as "perfectly complementary" for the purposes described herein.

[0122] The “complementary” sequences used herein may also include, or may be entirely formed from, non-Watson-Crick base pairs, or base pairs formed from non-natural and modified nucleotides, insofar as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairings or Hoogstein base pairings.

[0123] In this specification, the terms “complementarity,” “fully complementary,” and “substantially complementary” may be used in relation to base matching between the sense strand and antisense strand of a dsRNA, or between the antisense strand of a double-stranded RNA agent and a target sequence, as understood from the context of their use.

[0124] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding the PNPLA3 gene). For example, a polynucleotide is complementary to at least a portion of PNPLA3 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the PNPLA3 gene.

[0125] Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target PNPLA3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and include a continuous nucleotide sequence that is at least 80% complementary over its entire length, such as being about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to an equivalent region of any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, or about 99% complementary to any one of the fragments of SEQ ID NOs: 1, 3, 5, 7, 9, or 11.

[0126] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence fragment, and throughout their entire length, they are nucleotides 677-721;683-721;773-817;1185-1295;1185-1241;1202-1295;1202-1241;1255-1295;1738-1792;1901-1945;1920-1945;21 It comprises a sequence of nucleotides that is at least 80% complementary to the SEQ ID NO: SEQ ID NO: 08~2208;2108~2166;2108~2136;2121~2166;2121~2208;2169~2208;2176~2208; or 2239~2265, such as being approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% complementary to the SEQ ID NO: 1.

[0127] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence fragment, and throughout their entire length, they are nucleotides 574-596; 677-699; 683-705; 699-721; 773-795; 795-817; 1185-1207; 1192-1214; 1202-1224; 1208-1230; 1209-1231; 1210-1232; 1211-1233; 1212-1234; 1213-1233; 1214-1234; 1214-1236; 1215~1237;1216~1238;1219~1237;1219~1241;1255~1275;1256~1276;1257~1275;1257~1277;1258~1278;1259~1279;1260~1278;1260~1280;1261~ 1281;1262~1282;1263~1283;1264~1282;1264~1284;1265~1285;1267~1285;1266~1286;1266~1288;1267~1285;1267~1287;1268~1290;1269~1289; 1270~1290;1271~1291;1272~1292;1273~1293;1274~1294;1275~1295;1631~1653;1738~1760;1739~1761;1740~1760;1740~1762;1741~1763;1744~ 1766;1746~1766;1750~1772;1751~1773;1752~1774;1753~1775;1754~1776;1755~1777;1756~1778;1757~1779;1758~1780;1759~1781;1760~1782; 1761~1783;1762~1782;1762~1784;1763~1785;1764~1786;1765~1787;1766~1786;1766~1788;1767~1787;1768~1788;1767~1789;1769~1789;1770~ 1788; 1770-1790; 1771-1791; 1772-1792; 1815-1837; 1901-1923; 1920-1942; 1923-1945; 2112-2130; 2169-2191; 2171-2191; 2176-2198; 2177-2199;The sequence contains a continuous nucleotide sequence that is at least 80% complementary to the SEQ ID NO: 2178~2200;2179~2201;2180~2202;2181~2203;2183~2205;2184~2206;2186~2208;2239~2261;2241~2263;2242~2264; or 2243~2265, such as being approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% complementary to the SEQ ID NO: 1 fragment.

[0128] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence fragment and, throughout their entire length, nucleotides 1200-1250, 1205-1250, 11210-1250, 1200-1245, 1200-1240, 1200-1235, 1200-1237, 1205-1237, 1210-1232, 1212-1237, 1212-1234, 1250-1300, 1255-1300, 126 The sequence includes a continuous nucleotide sequence that is at least 80% complementary to the SEQ ID NO:

[0129] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and comprise a continuous nucleotide sequence that is at least approximately 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, or to any one fragment of the sense strand nucleotide sequence in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, such as approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% complementary.

[0130] In one embodiment, the RNAi agent of the present disclosure comprises an antisense polynucleotide and a substantially complementary sense strand, wherein the antisense polynucleotide is identical to the target PNPLA3 sequence, in which case the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least approximately 80% complementary, such as approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% complementary to an equivalent region of the nucleotide sequence of SEQ ID NOs. 2, 4, 6, 8, 10, or 12, or to any one of the fragments of SEQ ID NOs. 2, 4, 6, 8, 10, or 12.

[0131] In some embodiments, the iRNA of the Disclosure comprises an antisense polynucleotide and a substantially complementary sense strand, the antisense polynucleotide being complementary to a target PNPLA3 sequence, in which case the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least approximately 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, or any fragment of the antisense strand nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, such as approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% complementary.

[0132] In a particular embodiment, the sense strand and antisense strand are double strands, AD-517197.2;AD-517258.2;AD-516748.2;AD-516851.2;AD-519351.2;AD-519754.2;AD-519828.2;AD-520018.2;AD-520035.2;AD-520062.2;AD-520064.2;AD-520065.2;AD-520067.2;AD-75289.2;AD-520069.2;AD-520099 Select one of the following: .2;AD-67575.7;AD-520101.2;AD-67605.7, AD-1193323.1;AD-1193344.1;AD-1193350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-1193422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1; and AD-1193481.1.

[0133] In some embodiments, the sense strand and antisense strand are double strands: AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, AD-519350.1 , AD-519753.2, AD-519932.1, AD-519935.2, AD-520018.6, AD-517837.2, AD-805635.2, AD-519329.2 , AD-520063.2, AD-519757.2, AD-805631.2, AD-516917.2, AD-516828.2, AD-518983.2, AD-805636.2 , AD-519754.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-520053.4, AD-519667.2, AD-519773.2 , AD-519354.2, AD-520060.4, AD-520061.4, AD-1010733.2, AD-1010735.2, AD-1193323.1; Select one of the following: 44.1;AD-1193350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-1193422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1; and AD-1193481.1.

[0134] In some embodiments, the sense strand and antisense strand are selected from any one of the following double strands: AD-519345.1;AD-1193350.1;AD-1193365.1;AD-1193437.1; and AD-519347.1.

[0135] In some embodiments, the sense and antisense strands are derived from a double strand, AD-519351.

[0136] In one embodiment, the sense chain and antisense chain are derived from AD-1193350, which is a double hemisphere.

[0137] In one embodiment, the sense strand and antisense strand are derived from a double strand, AD-1193365.

[0138] In one embodiment, the sense chain and antisense chain are derived from a double hemisphere, AD-1193437.

[0139] In one embodiment, the sense chain and antisense chain are derived from a double hemisphere of AD-519347.

[0140] Generally, “iRNA” includes ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications used in a dsRNA molecule are encompassed by “iRNA” as used herein and in the claims.

[0141] In certain embodiments of this disclosure, the incorporation of deoxyribonucleotides when present in an RNAi agent is considered to constitute modified nucleotides.

[0142] In one embodiment of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence in the target mRNA. The single-stranded antisense oligonucleotide may inhibit translation stoichiometrically by base pairing with mRNA, or it may inhibit translation by physically interfering with the translation mechanism; see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule can be about 14 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may contain a sequence of at least about 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides derived from any one of the antisense sequences described herein.

[0143] As used herein, the phrase “bringing iRNA, such as dsRNA, into contact with cells” includes bringing cells into contact by any possible means. Bringing cells into contact with iRNA includes bringing cells into contact with iRNA in vitro or in vivo. Contact may be direct or indirect. For example, iRNA may be brought into physical contact with cells by the person performing the method, or alternatively, iRNA may be placed in a situation that allows it to subsequently come into contact with cells, or a situation that causes it to subsequently come into contact with cells.

[0144] Cell contact in vitro can be achieved, for example, by incubating cells with iRNA. Cell contact in vivo may be achieved, for example, by injecting iRNA into or near the tissue where the cells are located, or by injecting the iRNA into another region, such as the bloodstream or subcutaneous space, so that the drug subsequently reaches the tissue where the cells to be contacted are located. For example, the iRNA may contain a ligand, such as GalNAc, that directs the iRNA to a target site, such as the liver, and may be coupled with it. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may also be contacted with iRNA in vitro and then transplanted into a target.

[0145] In certain embodiments, contacting cells with iRNA includes "introducing iRNA into cells" or "delivering iRNA into cells" by facilitating or resulting in uptake or absorption into cells. Absorption or uptake of iRNA may occur via spontaneous diffusion or active intracellular processes, or by auxiliary agents or devices. Introduction of iRNA into cells may be in vitro or in vivo. For example, for in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described below herein or are known in the art.

[0146] The terms “lipid nanoparticles” or “LNPs” refer to vesicles containing a lipid layer that encapsulate a pharmaceutically active molecule, such as a nucleic acid molecule, iRNA, or a plasmid on which iRNA is transcribed. LNPs are described, for example, in full in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, which are incorporated herein by reference.

[0147] As used herein, “Subject” refers to animals such as primates (humans, non-human primates, e.g., monkeys and chimpanzees), mammals (including cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds, that endogenously or heterologously express the target gene. In some embodiments, the subject is a human being treated for or evaluated for a disease or disorder that would benefit from reduced PNPLA3 expression as described herein; a human being at risk of a disease or disorder that would benefit from reduced PNPLA3 expression; a human being having a disease or disorder that would benefit from reduced PNPLA3 expression; or a human being treated for a disease or disorder that would benefit from reduced PNPLA3 expression. In some embodiments, the subject is a female human being. In other embodiments, the subject is a male human being. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0148] As used herein, the terms “treating” or “treatment” refer to a beneficial or desired outcome, such as the reduction of at least one sign or symptom of PNPLA3-related disorder in a subject. Treatment also includes the reduction of one or more signs or symptoms associated with undesirable PNPLA3 expression; a decrease in the degree of undesirable PNPLA3 activation or stabilization; and improvement or mitigation of undesirable PNPLA3 activation or stabilization. “Treatment” may also mean an extension of survival compared to the expected survival in the absence of treatment. In the context of levels or symptoms of PNPLA3 or disease markers in a subject, the term “reduction” refers to a statistically significant reduction of such levels. A reduction may be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. In certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction is at least 50% of a disease marker, e.g., a protein level or a gene expression level. In the context of PNPLA3 levels in a subject, “reduction” is preferably a drop to a level that is acceptable as within the normal range for individuals without such disorder. In certain embodiments, “reduction” means a reduction in the difference between the level of a marker or symptom for a subject suffering from the disease and a level that is acceptable as within the normal range for the individual, e.g., the level of weight loss between an obese individual and an individual with a weight that is acceptable as within the normal range.

[0149] Where used herein to refer to diseases, disorders, or conditions that can be treated or improved by reducing the expression of the PNPLA3 gene, “prevention” or “prevention of” means that the subject is likely to develop symptoms associated with undesirable or excessive PNPLA3 expression, such as elevated protein levels in the Hedgehog signaling pathway, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). For example, the likelihood of developing NAFLD is reduced if, for example, an individual with one or more risk factors for NAFLD does not develop NAFLD, or develops NAFLD of a less severe degree compared to a population with the same risk factors but who does not receive the treatment described herein. The absence of symptoms of a disease, disorder, or condition, or a reduction in the incidence of symptoms associated with such disease, disorder, or condition (e.g., at least about 10% on a clinically acceptable scale for the disease or disorder), or delayed symptom presentation (e.g., on a daily, weekly, monthly, or yearly basis), are considered effective preventive measures.

[0150] As used herein, the terms “patatin-like phospholipase domain-containing PNPLA3-related disease” or “PNPLA3-related disease” refer to diseases or disorders caused by or associated with the expression of the PNPLA3 gene or the production of the PNPLA3 protein. The term “PNPLA3-related disease” includes diseases, disorders, or conditions that benefit from reduced PNPLA3 gene expression, replication, or PNPLA3 protein activity. Non-limiting examples of PNPLA3-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), Liber cirrhosis, accumulation of fat in the liver, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). In another embodiment, PNPLA3-related disease is non-alcoholic fatty liver disease (NAFLD). In another embodiment, PNPLA3-related disease is non-alcoholic steatohepatitis (NASH). In another embodiment, PNPLA3-related disease is cirrhosis. In another embodiment, PNPLA3-related disease is insulin-resistant. In another embodiment, PNPLA3-related disease is not insulin-resistant. In one embodiment, PNPLA3-related disease is obese.

[0151] In one embodiment, the PNPLA3-associated disease is non-alcoholic fatty liver disease (NAFLD). The term "non-alcoholic fatty liver disease," as used interchangeably with "NAFLD" herein, refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 mg (gm) of alcohol per day. NAFLD is the most common liver disease in the United States and is generally associated with insulin-resistant / type 2 diabetes and obesity. NAFLD manifests as steatosis, steatohepatitis, cirrhosis, and, in some cases, hepatocellular carcinoma. For a comprehensive review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153–1163, the full contents of which are incorporated herein by reference.

[0152] As used herein, “therapeutic dose” is intended to include the amount of RNAi agent that, when administered to a subject with PNPLA3-related disease, is sufficient to treat the disease (e.g., by reducing, improving, or maintaining the symptoms of the existing disease, or one or more symptoms of the disease). “Therapeutic dose” may vary depending on the RNAi agent, how the drug is administered, the disease and its severity and medical history, age, weight, family history, genetic composition, the type of prior or concomitant treatment, if any, and other individual characteristics of the subject being treated.

[0153] As used herein, “prophylactic effective dose” is intended to include the amount of RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject with PNPLA3-related disorder. Disease improvement includes slowing the course of the disease or reducing the severity of delayed-onset disease. The “prophylactic effective dose” may vary depending on the RNAi agent, how the drug is administered, the degree of disease risk and medical history, age, weight, family history, genetic composition, the type of prior or concomitant treatment, if any, and other individual characteristics of the subject being treated.

[0154] The “therapeutic effective dose” or “preventive effective dose” also includes the amount of RNAi agent that produces some desired effect in a reasonable benefit / risk ratio applicable to any treatment. The iRNA used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0155] In this specification, the term "pharmaceutically acceptable" is used to mean a compound, material, composition, or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is commensurate with a reasonable benefit-to-risk ratio.

[0156] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid [e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or steric acid], or a solvent encapsulation material involved in the transfer or transport of a compound of interest from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Such carriers are known in the art. pharmaceutically acceptable carriers include carriers for administration by injection.

[0157] As used herein, the term “sample” includes similar bodily fluids, cells, or tissues isolated from the subject, as well as collections of bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples derived from tissue, organs, or localized areas. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may originate from the liver (e.g., the whole liver or a specific segment of the liver, or a specific type of cell within the liver, such as hepatocytes). In some embodiments, “sample derived from subject” refers to urine obtained from the subject. “Sample derived from subject” may also refer to blood from the subject, or serum or plasma derived from blood.

[0158] II. The iRNA of the present invention The present invention provides an iRNA that inhibits the expression of the PNPLA3 gene. In a preferred embodiment, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the PNPLA3 gene in cells of a target, such as cells inside mammals, such as humans, that are prone to developing PNPLA3-related disorders, such as hypertriglyceridemia. The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed in the expression of the PNPLA3 gene. The complementary region is about 19 to 30 nucleotides long (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides long). When contacted with cells expressing the PNPLA3 gene, the iRNA inhibits the expression of the PNPLA3 gene (e.g., human, primate, non-primate, or rat PNPLA3 gene) by at least about 50%, as assayed by protein-based methods such as PCR or branched DNA (bDNA)-based methods, or immunofluorescence analysis using methods such as Western blotting or flow cytometry. In a preferred embodiment, inhibition of expression is determined by qPCR as presented in the examples herein, for example, for siRNA at a concentration of 10 nM in a suitable biological cell line prepared in this example. In a preferred embodiment, inhibition of expression in vivo is determined by knockdown of the human gene at the lowest level of RNA expression in rodents expressing the human gene, such as mice expressing a human target gene or AAV-infected mice, when administered, for example, as a single dose at 3 mg / kg.

[0159] A dsRNA contains two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary, and generally fully complementary, to the target sequence. The target sequence may be derived from the mRNA sequence formed during the expression of the PNPLA3 gene. The other strand (the sense strand) contains a region that is complementary to the antisense strand so that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere in this specification and as is known in the art, the complementary sequence of the dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, contrasted with a complementary sequence on a separate oligonucleotide.

[0160] Generally, double-stranded structures are 15-30 base pairs long, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 The lengths are 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain preferred embodiments, the double-stranded structure is 18–25 base pairs long, for example, 18–25, 18–24, 18–23, 18–22, 18–21, 18–20, 19–25, 19–24, 19–23, 19–22, 19–21, 19–20, 20–25, 20–24, 20–23, 20–22, 20–21, 21–25, 21–24, 21–23, 21–22, 22–25, 22–24, 22–23, 23–25, 23–24, or 24–25 base pairs long, for example, 19–21 base pairs long. Intermediate ranges and lengths between those listed above are also assumed to be part of this disclosure.

[0161] Similarly, the region complementary to the target sequence is 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 1 Lengths of 9-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, for example, lengths of 19-23 nucleotides or 21-23 nucleotides. Intermediate ranges and lengths between those listed above are also assumed to be part of this disclosure.

[0162] In some embodiments, the double-stranded structure is 19–30 base pairs long. Similarly, the region complementary to the target sequence is 19–30 nucleotides long.

[0163] In some embodiments, dsRNAs are about 19–23 nucleotides long, or about 25–30 nucleotides long. Generally, dsRNAs are long enough to be used as substrates for Dicer enzymes. For example, it is well known in the art that dsRNAs longer than about 21–23 nucleotides can be used as substrates for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is very often part of a larger RNA molecule, an mRNA molecule. Where applicable, the “part” of the mRNA target is a continuous sequence of mRNA targets of sufficient length to allow it to serve as a substrate for RNAi-driven cleavage (i.e., cleavage via the RISC pathway).

[0164] Those skilled in the art will also recognize that the double-stranded region is the main functional portion of dsRNA, for example, a double-stranded region of about 19 to about 30 base pairs, for example, about 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Therefore, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs, insofar as it is processed into a functional double helix of 15-30 base pairs, for example, to target the desired RNA for cleavage, is a dsRNA. Thus, those skilled in the art will recognize that, in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting the expression of the PNPLA3 gene is not generated in the target cell by cleavage of a large dsRNA.

[0165] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, for example, 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. Overhangs may be located on the sense strand, on the antisense strand, or on any combination thereof. Furthermore, nucleotide overhangs may be located on the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0166] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared individually. Then, the components of the strands are annealed. The individual strands of the siRNA compound can be prepared using liquid-phase organic synthesis, solid-phase organic synthesis, or both. Organic synthesis has the advantage that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be prepared using liquid-phase organic synthesis, solid-phase organic synthesis, or both.

[0167] In one embodiment, the dsRNA of the present invention comprises a sense sequence and an antisense sequence, which are at least two nucleotide sequences. The sense strand is selected from the sequence group presented in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, and the corresponding antisense strand of the sense strand is selected from the sequence group presented in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence produced in the expression of the PNPLA3 gene. Thus, in this embodiment, the dsRNA would contain two oligonucleotides, one of which is described as a sense strand in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, and a second oligonucleotide described as the corresponding antisense strand of the sense strand in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50.

[0168] In one particular embodiment, a substantially complementary sequence of dsRNA is contained in a separate oligonucleotide. In another embodiment, a substantially complementary sequence of dsRNA is contained in a single oligonucleotide.

[0169] In a particular embodiment, the sense strand or antisense strand is a double strand, AD-517197.2;AD-517258.2;AD-516748.2;AD-516851.2;AD-519351.2;AD-519754.2;AD-519828.2;AD-520018.2;AD-520035.2;AD-520062.2;AD-520064.2;AD-520065.2;AD-520067.2;AD-75289.2;AD-520069.2;AD-520099.2;AD-67 Selected from one of the following sense or antisense strands: 575.7;AD-520101.2;AD-1193323.1;AD-1193344.1;AD-1193350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-1193422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1;AD-1193481.1; or AD-67605.7.

[0170] In some embodiments, the sense strand or antisense strand is a double strand, AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, AD-519350.1, AD- 519753.2, AD-519932.1, AD-519935.2, AD-520018.6, AD-517837.2, AD-805635.2, AD-519329.2, AD-52 0063.2, AD-519757.2, AD-805631.2, AD-516917.2, AD-516828.2, AD-518983.2, AD-805636.2, AD-5197 54.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-520053.4, AD-519667.2, AD-519773.2, AD-519354 .2, AD-520060.4, AD-520061.4, AD-1010733.2, AD-1010735.2, AD-1193323.1;AD-1193344.1;AD-1193 Selected from one of the sense or antisense strands: 350.1;AD-1193365.1;AD-1193379.1;AD-1193407.1;AD-1193421.1;AD-1193422.1;AD-1193429.1;AD-1193437.1;AD-1193443.1;AD-1193471.1; or AD-1193481.1.

[0171] In some embodiments, the sense strand or antisense strand is selected from one of the following double strands: AD-519345.1;AD-1193350.1;AD-1193365.1;AD-1193437.1; and AD-519347.1.

[0172] In some embodiments, the sense or antisense strand is selected from the sense or antisense strand of AD-519351, which is a double strand.

[0173] In one embodiment, the sense strand or antisense strand is selected from the sense strand or antisense strand of AD-1193350, which is a double strand.

[0174] In one embodiment, the sense strand or antisense strand is selected from the sense strand or antisense strand of AD-1193365, which is a double strand.

[0175] In one embodiment, the sense strand or antisense strand is selected from the sense strand or antisense strand of AD-1193437, which is a double strand.

[0176] In one embodiment, the sense strand or antisense strand is selected from the sense strand or antisense strand of AD-519347, which is a double strand.

[0177] For example, the sequences in Tables 3, 5, 7, 9, 11, 21, 24, 27, 30, 32, 36, and 50 are not described as modified or conjugated sequences, but it will be understood that the RNA of the RNAi of this disclosure, for example, the dsRNA of the present invention, may include any one of the sequences explicitly shown in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, which are unmodified, unconjugated, modified in a manner different from those described herein, or conjugated. In other words, the present invention includes the dsRNAs of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, which are unmodified, unconjugated, or modified or conjugated as described herein.

[0178] Those skilled in the art are aware that dsRNAs having a double-strand structure of approximately 20–23 base pairs, for example, 21 base pairs, are said to be particularly effective in inducing RNA interference [Elbashir et al., EMBO 2001, 20:6877-6888]. However, other researchers have found that shorter or longer RNA double-strand structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the embodiments described above, depending on the properties of the oligonucleotide sequences presented in any one of Tables 2–11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, the dsRNAs described herein may, at a minimum, consist of at least one strand of 21 nucleotides in length. Shorter double helices having one of the sequences in Table 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, with only a few nucleotides subtracted from one or both ends, can reasonably be expected to be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having at least 19, 20, or more consecutive nucleotide sequences derived from one of the sequences in Table 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, and which differ in their ability to inhibit PNPLA3 gene expression by no more than approximately 5, 10, 15, 20, 25, or 30% of the dsRNA including the full-length sequence, are assumed to be within the scope of the present invention.

[0179] In addition, the RNAs presented in Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50 identify RISC-mediated cleavage-sensitive sites in the PNPLA3 transcript. Thus, the present invention further features iRNAs that target the interior of one of these sites. The iRNAs used herein are said to target the interior of a specific site in the RNA transcript if the iRNA promotes cleavage of the transcript at any location within this particular site. Such iRNAs generally consist of at least about 19 consecutive nucleotides derived from one of the sequences presented in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50, coupled to further nucleotide sequences taken from a region adjacent to a selected sequence within the PNPLA3 gene.

[0180] III. Modified iRNA of the present invention In certain embodiments, the RNA of the RNAi of this disclosure, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of the RNAi of this disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present invention, substantially all of the nucleotides of the RNAi of this disclosure are modified. In other embodiments of the present invention, all or substantially all of the nucleotides of the iRNA are modified, i.e., there are no more than five, four, three, two, or one unmodified nucleotides in the iRNA chain.

[0181] The nucleic acids featured in the present invention may be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, reverse linkage) or 3'-terminal modifications (conjugation, DNA nucleotide, reverse linkage, etc.); base modifications, e.g., replacement with stabilizing bases, unstable bases, or bases that base-pair with an expanded repertoire of partners, base removal (debasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; or skeletal modifications including modification or replacement of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified skeleton or RNA not containing natural nucleoside linkages. RNAs with modified skeletons include, in particular, RNAs that do not have a phosphorus atom in their skeleton. For the purposes of this specification, and as may be referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton may also be considered oligonucleosides. In some embodiments, modified iRNAs will have a phosphorus atom in their internucleoside skeleton.

[0182] The modified RNA backbone includes, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl, and other alkylphosphonates, phosphinates, phosphoramidates, thionophosphoramides, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, which have the usual 3'-5' linkage, as well as analogs having the 2'-5' linkage, and analogs having opposite polarity where pairs of adjacent nucleoside units are 3'-5' linked to 5'-3' or 2'-5' linked to 5'-2'. Various salt forms, mixed salt forms, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment of the present invention, the dsRNA agent is in sodium salt form. In a particular embodiment, when the dsRNA agent of the present invention is in sodium salt form, the sodium ion is present in the agent as a counterion to substantially all of the phosphodiester groups and / or phosphorothioate groups present in the agent. A drug in which substantially all of the phosphodiester and / or phosphorothioate linkages have a sodium counterion contains not more than five, four, three, two, or one phosphodiester and / or phosphorothioate linkages that do not have a sodium counterion. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, the sodium ion is present in the agent as a counterion to all of the phosphodiester groups and / or phosphorothioate groups present in the agent.

[0183] Representative U.S. patents teaching the preparation of the phosphorus-containing linkage described above are incorporated herein by reference in their entirety: U.S. Patents No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5,264,423; No. 5,276,019; and No. 5,2 No. 78,302; No. 5,286,717; No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; Same No. 5,466,677; Same No. 5,476,925; Same No. 5,519,126; Same No. 5,536,821; Same No. 5,541,316; Same No. 5,550,111; Same No. 5,563,2 No. 53; No. 5,571,799; No. 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6, 169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423 ; Nos. 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; including, but not limited to, U.S. Reissue Patent No. 39464.

[0184] Among these, modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by nucleoside linkages of short alkyl or cycloalkyl groups, nucleoside linkages of mixed heteroatoms and alkyl or cycloalkyl groups, or nucleoside linkages of one or more short chains with heteroatoms or heterocycles. Modified RNA skeletons include modified RNA skeletons having morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane skeletons; sulfide skeletons, sulfoxide skeletons, and sulfone skeletons; formacetyl skeletons and thioformacetyl skeletons; methyleneformacetyl skeletons and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino skeletons and methylenehydrazino skeletons; sulfonate skeletons and sulfonamide skeletons; amide skeletons; and skeletons having mixed N, O, S, and CH2 components.

[0185] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides are incorporated herein by reference in their entirety: U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466, This includes, but is not limited to, No. 677; No. 5,470,967; No. 5,489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439.

[0186] RNA mimetic compounds suitable for use in the iRNAs presented herein are envisioned in which both the sugar and nucleoside linkages of the nucleotide unit, i.e., the backbone, are replaced by novel groups. The base unit is maintained for hybridization with a suitable nucleic acid targeting compound. One such oligomeric compound in which the RNA mimetic has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly bonded to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in iRNAs according to the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0187] Some embodiments of the present invention include RNA with a phosphorothioate backbone and oligonucleosides with a heteroatom backbone, in particular including -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene(methylimino) backbone or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-], and the amide backbone of U.S. Patent No. 5,602,240, as referenced above. In some embodiments, the RNA featured herein has a morpholino backbone structure of U.S. Patent No. 5,034,506, as referenced above.

[0188] Modified RNAs can also contain one or more substituted sugar moieties. iRNAs, for example, the dsRNAs characterized herein, at the 2'-position, can include one of the following: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl [wherein alkyl, alkenyl, and alkynyl can be substituted or unsubstituted, C1-C 10 alkyl, or C2-C 10 alkenyl and alkynyl]. Exemplary suitable modifications can 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 [wherein n and m are from 1 to about 10]. In other embodiments, the dsRNA at the 2'-position can be C1-C 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkaryl groups, aralkyl groups, O-alkaryl groups or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, insertants, groups for improving the pharmacokinetic properties of iRNA, or groups for improving the pharmacodynamic properties of iRNA, and one of other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy[2'-O-CH2CH2OCH3] (also known as 2'-O-(2-methoxyethyl) or 2'-MOE), i.e., an alkoxy-alkoxy group. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2. Further exemplary modifications include 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers within these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0189] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA RNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide, or within 2'-5' ligated dsRNA, and at the 5' position of the 5' terminal nucleotide. iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching such modified sugar structures include U.S. Patents No. 4,981,957; No. 5,118,800; No. 5,319,080; No. 5,359,044; No. 5,393,878; No. 5,446,137; No. 5,466,786; No. 5,514,785; and No. 5,514,785, all of which are jointly owned with this application. This includes, but is not limited to, Nos. 519,134; Nos. 5,567,811; Nos. 5,576,427; Nos. 5,591,722; Nos. 5,597,909; Nos. 5,610,300; Nos. 5,627,053; Nos. 5,639,873; Nos. 5,646,265; Nos. 5,658,873; Nos. 5,670,633; and Nos. 5,700,920. The entire contents of each of the aforementioned are incorporated herein by reference.

[0190] iRNA may also include modifications or substitutions of nucleobases (often simply referred to as “bases” in the art). As used herein, “unmodified” nucleobases or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine, 6-methyl derivatives and other alkyl derivatives, adenine and guanine, 2-propyl derivatives and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothimine, 5-uracil (pseudolacil), 4-thiouracil, 8-haloadenine and 8-haloguanine, 8-aminoadenine and 8-aminoguanine, 8-thioladenine and 8- This includes other synthetic and natural nucleobases such as thiolguanine, 8-thioalkyladenine and 8-thioalkylguanine, 8-hydroxyladenine analogs (anal) and 8-hydroxylguanine analogs, other 8-substituted adenines and 8-substituted guanines, 5-halouracil and 5-halocytosine, in particular 5-bromouracil and 5-bromocytosine, 5-trifluoromethyluracil and 5-trifluoromethylcytosine, and other 5-substituted uracils and 5-substituted cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine (daazaadenine), and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain nucleobases among these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, as well as N-2 substituted purines, N-6 substituted purines, and 0-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is more specifically an exemplary base substitution when combined with 2'-O-methoxyethyl sugar modification.

[0191] Representative U.S. patents that teach the preparation of certain modified nucleobases among those mentioned above, as well as other modified nucleobases, are incorporated herein by reference in their entirety: U.S. Patents No. 3,687,808; No. 4,845,205; No. 5,130,30; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,7 No. 11; No. 5,552,540; No. 5,587,469; No. 5,594,121; No. 5,596,091; No. 5,614,617 No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; This includes, but is not limited to, the following publications: No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088.

[0192] The RNA of iRNA can also be modified to include one or more lock nucleic acids (LNAs). A lock nucleic acid is a nucleotide that has a modified ribose moiety, including an additional crosslink connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose within the 3' internal structural conformation. The addition of lock nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193].

[0193] In some embodiments, the RNA of the iRNA may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge between two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of a sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4' carbon and the 2' carbon of the sugar ring. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety that includes an additional bridge connecting the 2' carbon and the 4' carbon. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose within the 3'-side internal structural conformation. The addition of lock nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in polynucleotides of the present invention include, without limitation, nucleosides comprising a bridge between the 4' atom and the 2' atom of a ribosyl ring. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides comprising a 4'-2' bridge.Examples of such 4'-2' bridged bicyclic nucleosides include 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "restricted ethyl" or "cEt"), and 4'-CH(CH2OCH3)-O-2' (and their analogues; see, for example, U.S. Patent No. 7,399,845); and 4'-C(CH3)(CH3)-O-2' (and their analogues; see, for example, U.S. Patent No. 8,278,283). See also: 4'-CH2-N(OCH3)-2' (and analogues thereof; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' [wherein R is H, C1-C12 alkyl, or a protecting group] (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogues thereof; see, e.g., U.S. Patent No. 8,278,426). All of the contents of each of the preceding sections are incorporated herein by reference.

[0194] Further representative U.S. patents and U.S. patent publications teaching the preparation of lock nucleic acid nucleotides are incorporated herein by reference in their entirety, including: U.S. Patent No. 6,268,490; U.S. Patent No. 6,525,191; U.S. Patent No. 6,670,461; U.S. Patent No. 6,770,748; U.S. Patent No. 6,794,499; U.S. Patent No. 6,998,484; U.S. Patent No. 7,053,207; U.S. Patent No. 7,034, This includes, but is not limited to, No. 133; No. 7,084,125; No. 7,399,845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; US2008 / 0039618; and US2009 / 0012281.

[0195] For example, any of the aforementioned bicyclic nucleosides having one or more sugar stereochemical configurations (see WO99 / 14226), including α-L-ribofuranose and β-D-ribofuranose, can be prepared.

[0196] The RNA of the iRNA may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” refers to a restricted nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S conformation, which is referred herein to as “S-cEt”.

[0197] The iRNAs of this disclosure may also include one or more “conformation-fixed nucleotides” (“CRNs”). A CRN is a nucleotide analog with a linker connecting the C2' carbon to the C4' carbon of ribose, or a linker connecting the C3 carbon to the C5' carbon of ribose. The CRN locks the ribose ring into a stable conformation, increasing its hybridization affinity to mRNA. The linker is long enough to position the oxygen in a position optimal for stability and affinity, resulting in reduced puckering of the ribose ring.

[0198] Representative publications teaching the preparation of certain CRNs among those mentioned above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT Publication No. WO2013 / 036868, the entire contents of which are incorporated herein by reference.

[0199] In some embodiments, the iRNAs of this disclosure comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA are unlocked acyclic nucleic acids in which one of the sugar bonds is removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference).

[0200] Representative U.S. publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publications 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0201] Potential stabilization modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3''-phosphate, the reverse base dT (idT), and other stabilization modifications. Disclosures regarding these modifications can be found in PCT Publication WO2011 / 005861.

[0202] Other modifications to the nucleotides of the iRNAs of this disclosure include 5' phosphates or 5' phosphate mimetics on the antisense iRNA strand, such as 5' terminal phosphates or phosphate mimetics. Suitable phosphate mimetics are disclosed, for example, in their entirety in U.S. Patent Publication 2012 / 0157511, which is incorporated herein by reference.

[0203] A. Modified iRNA containing the motif of the present invention In certain embodiments of the present invention, the double-stranded RNA agents of the present invention include, for example, chemically modified agents disclosed in WO2013 / 075035, the entire contents of each of which are incorporated herein by reference. WO2013 / 075035, in particular, presents the introduction of a motif consisting of three identical modifications on three consecutive nucleotides at or near a cleavage site into the sense or antisense strand of a dsRNAi agent. In some embodiments, the sense and antisense strands of the dsRNAi agent may be fully modified in other ways. The introduction of these motifs, if present, disrupts the modification pattern of the sense or antisense strand. The dsRNAi agent may be conjugated, for example, with a GalNAc derivative ligand on the sense strand.

[0204] More specifically, gene silencing activity of the dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNA agent were completely modified such that they had one or more motifs consisting of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0205] Accordingly, the present invention provides a double-stranded RNA agent capable of inhibiting the expression of a target gene (i.e., the PNPLA3 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, for example, 17-30 nucleotides long, 25-30 nucleotides long, 27-30 nucleotides long, 19-25 nucleotides long, 19-23 nucleotides long, 19-21 nucleotides long, 21-25 nucleotides long, or 21-23 nucleotides long.

[0206] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "dsRNAi agent." The double-stranded region of the dsRNAi agent may be, for example, 27–30 nucleotide pairs long, 19–25 nucleotide pairs long, 19–23 nucleotide pairs long, 19–21 nucleotide pairs long, 21–25 nucleotide pairs long, or 21–23 nucleotide pairs long. In another example, the double-stranded region may be selected from lengths of 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0207] In certain embodiments, a dsRNAi agent may contain one or more overhang regions or capping groups at one or both 3' ends, 5' ends, or both ends of a strand. The overhangs may independently be 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. In certain embodiments, the overhang region may include the elongated overhang region presented above. The overhang may result from one strand being longer than the other, or from two strands of equal length being attached end strands. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be linked by further bases, for example, to form a hairpin, or by other non-base linkers.

[0208] In certain embodiments, the nucleotides within the overhang region of the dsRNAi agent may be modified nucleotides or unmodified nucleotides, each independently including, but not limited to, 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0209] For example, TT can be a protruding sequence for any end on any strand. The protrusion may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence altogether.

[0210] 5' or 3' overhangs on the sense strand, antisense strand, or both strands of a dsRNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides with a phosphorothioate between them, in which case the two nucleotides may be the same or different. In some embodiments, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In some embodiments, this 3' overhang is located within the antisense strand. In some embodiments, this 3' overhang is located within the sense strand.

[0211] dsRNAi agents may contain a single overhang that can enhance RNAi interference activity without affecting their overall stability. For example, the single-strand overhang may be located at the 3' end of the sense strand, or alternatively, at the 3' end of the antisense strand. The RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of a dsRNAi agent has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Not wanting to be bound by theory, the asymmetry between the blunt end at the 5' end of the antisense strand and the overhang at the 3' end of the antisense strand is favorable for loading the guide strand into the RISC process.

[0212] In a particular embodiment, the dsRNAi agent is a 19-nucleotide-length double-ended bluntmer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 of the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0213] In another embodiment, the dsRNAi agent is a 20-nucleotide-length double-ended bluntomer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 of the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0214] In yet another embodiment, the dsRNAi agent is a 21-nucleotide-length double-ended bluntomer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 of the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0215] In a particular embodiment, the dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 of the 5' end; and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end, with one end of the RNAi agent being blunt-ended and the other end containing a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0216] If the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide links between the three terminal nucleotides, where two of the three nucleotides are the overhanging nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent also has two phosphorothioate internucleotide links between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In a particular embodiment, any nucleotide in the sense and antisense strands of the dsRNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In a particular embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro within an alternating motif. The dsRNAi agent may further contain a ligand (preferably GalNAc3).

[0217] In a particular embodiment, the dsRNAi agent comprises a sense strand and an antisense strand, the sense strand being 25–30 nucleotides long, starting with a 5' terminal nucleotide (position 1), and containing at least 8 ribonucleotides at positions 1–23 of the first strand; the antisense strand being 36–66 nucleotides long, starting with a 3' terminal nucleotide, and containing at least 8 ribonucleotides so as to form a double helix at positions 1–23 of the sense strand; in this case, at least 3' terminal nucleotides of the antisense strand are unpaired with the sense strand, and at the 3' end, up to 6 consecutive nucleotides are unpaired with the sense strand, thereby forming a single-stranded 3' overhang of 1–6 nucleotides; the 5' end of the antisense strand is unpaired with 10–30 consecutive nucleotides The sense strand contains a conjugate nucleotide, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantial double-stranded region between the sense strand and the antisense strand; the antisense strand is sufficiently complementary to the target RNA over a length of at least 19 ribonucleotides of the antisense strand so as to reduce the expression of the target gene when the double-stranded nucleic acid is introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, in which case at least one of the motifs is located at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site of the strand.

[0218] In a particular embodiment, the dsRNAi agent comprises a sense strand and an antisense strand, the dsRNAi agent comprising a first strand at least 25 nucleotides long and at most 29 nucleotides long, and a second strand at most 30 nucleotides long, with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand having a double-stranded region at its 3' end that is 1 to 4 nucleotides longer than the first strand and at least 25 nucleotides long, and the second strand being sufficiently complementary to the target mRNA over a length of at least 19 nucleotides of the second strand so that when the RNAi agent is introduced into mammalian cells, it reduces the expression of the target gene, in which case cleavage of the dsRNAi agent by Dicer preferentially results in an siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. The dsRNAi agent may further contain a ligand.

[0219] In a particular embodiment, the sense strand of the dsRNAi agent contains at least one motif from three identical modifications on three consecutive nucleotides, in which case one of the motifs is located at a cleavage site within the sense strand.

[0220] In certain embodiments, the antisense strand of the dsRNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, in which case one of the motifs is located at or near a cleavage site in the antisense strand.

[0221] For dsRNAi agents having a double-stranded region of 19–23 nucleotides in length, the cleavage sites on the antisense strand are typically near positions 10, 11, and 12 at the 5' end. Thus, motifs with three identical modifications can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand, depending on the count from the first nucleotide at the 5' end of the antisense strand, or from the count from the first paired nucleotide within the double-stranded region at the 5' end of the antisense strand. The cleavage sites within the antisense strand can also vary depending on the length from the 5' end of the double-stranded region of the dsRNAi agent.

[0222] The sense strand of a dsRNAi agent may contain at least one motif from three identical modifications on three consecutive nucleotides at the cleavage site; the antisense strand may have at least one motif from three identical modifications on three consecutive nucleotides at or near the cleavage site. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide overlap; that is, at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0223] In some embodiments, the sense strand of a dsRNAi agent may contain one or more motifs consisting of three identical modifications on three consecutive nucleotides. The first motif may be located at or near a cleavage site on the strand, and the other motifs may be wing modifications. As used herein, the term “wing modification” refers to a motif located in a different part of the strand, separated from the motif at or near the cleavage site on the same strand. Wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are directly adjacent to each other, their chemical properties are significantly different from each other; if the motifs are separated by one or more nucleotides (than), their chemical properties may be the same or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may be located at one end relative to the first motif, either at or near the cleavage site, or on either side of the read motif.

[0224] Similar to the sense strand, the antisense strand of a dsRNAi agent may contain one or more motifs consisting of three identical modifications on three consecutive nucleotides, at least one of which is located at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications in alignment, similar to the wing modifications that may be present on the sense strand.

[0225] In some embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0226] In other embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0227] If the sense strand and antisense strand of a dsRNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0228] If the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, the sense and antisense strands may be aligned such that two modifications each, originating from one strand, are located at one end of the double-stranded region and have one, two, or three nucleotide duplicates; two modifications each, originating from one strand, are located at the other end of the double-stranded region and have one, two, or three nucleotide duplicates; and two modifications each, originating from one strand, are located on each side of the read motif and have one, two, or three nucleotide duplicates within the double-stranded region.

[0229] In some embodiments, any nucleotides in the sense and antisense strands of a dsRNAi agent, including nucleotides that are part of a motif, may be modified. Each nucleotide may be modified by one or more alterations of one or both of the unbound phosphate oxygens, or one or more of the bound phosphate oxygens; alterations of components of the ribose sugar, e.g., the 2'-hydroxyl on the ribose sugar; overall replacement of the phosphate moiety by a "dephosphorylation" linker; modification or replacement of naturally occurring bases; and replacement or modification of the ribose-phosphate backbone, either the same or different modifications.

[0230] Since nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate groups, or unbound oxygen atoms within phosphate groups, occur at repeating positions within the nucleic acid. In some cases, modifications may occur at all target positions within the nucleic acid, but in many cases, this is not the case. For example, modifications may occur only at the 3' or 5' end, or only within the terminal region, for example, at the terminal nucleotide, or at the last two, three, four, five, or ten nucleotides of the chain. Modifications can occur in the double-stranded region, the single-stranded region, or both. Modifications may occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at the unbound O-position may occur at one or both ends, only within the terminal region, for example, at the terminal nucleotide, or at the last two, three, four, five, or ten nucleotides of the chain, or within the double-chain and single-chain regions, particularly at the ends. The 5' end or both ends may be phosphorylated.

[0231] For example, to enhance stability, the protrusion may contain a specific base, or a modified nucleotide or nucleotide surrogate may be contained within a single-chain protrusion, e.g., within the 5' protrusion or the 3' protrusion, or both. For example, it may be desirable to contain a purine nucleotide within the protrusion. In some embodiments, all or part of the bases within the 3' or 5' protrusion may be modified, for example, by modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of a ribose sugar, e.g., the use of a 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modified deoxyribonucleotide instead of a ribosaccharide within the nucleobase, and modifications within the phosphate group, e.g., the use of phosphorothioate modifications. The protrusion does not need to be homologous to the target sequence.

[0232] In some embodiments, each residue in the sense and antisense chains is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The chains may contain more than one modification. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0233] At least two distinct modifications are typically present on the sense and antisense chains. These two modifications may be 2'-O-methyl modifications, 2'-fluoro modifications, or other modifications.

[0234] In a particular embodiment, N a or N b This includes modifications in alternating patterns. As used herein, the term “alternating motif” refers to a motif having one or more modifications present on an alternating nucleotide of a single chain. An alternating nucleotide may refer to one modification every two nucleotides, or one modification every three nucleotides, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be “ABABABABABAB···”, “AABBAABBAABB···”, “AABAABAABAAB···”, “AAABAAABAAAB···”, “AAABBBAAABBB···”, or “ABCABCABCABC···”.

[0235] The types of modifications contained within an alternating motif may be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0236] In some embodiments, the RNAi agent of the present invention includes a modification pattern for alternating motifs on the sense strand that is shifted compared to the modification pattern for alternating motifs on the antisense strand. The shift may be such that the modified nucleotides on the sense strand correspond to a different modified nucleotide group on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand within a dsRNA double helix, within the double helix region, the alternating motifs on the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motifs on the antisense strand may begin with "BABABA" from 5' to 3' of the strand. As another example, within the double helix region, the alternating motifs on the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motifs on the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand, so that there is a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0237] In some embodiments, the dsRNAi agent includes an alternating motif pattern of 2'-O-methyl and 2'-F modifications on the sense strand, which has a shift compared to the initial alternating motif pattern of 2'-O-methyl and 2'-F modifications on the antisense strand; that is, 2'-O-modified nucleotides on the sense strand base-pair with 2'-F-modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0238] The introduction of one or more motifs into the sense or antisense strand through three identical modifications on three consecutive nucleotides disrupts the initial modification pattern present in the sense or antisense strand. This disruption of the modification pattern in the sense or antisense strand, achieved by introducing one or more motifs into the sense or antisense strand through three identical modifications on three consecutive nucleotides, can enhance gene silencing activity against the target gene.

[0239] In some embodiments, when a motif consisting of three identical modifications on three consecutive nucleotides is introduced into any of the chain, the modifications of the nucleotides adjacent to the motif are different from the modifications of the motif. For example, a portion of the sequence containing the motif is "···N a YYYN b ..."[In the sequence, "Y" represents the modification of a motif by three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification to a nucleotide adjacent to the motif "YYY", and is a modification different from the modification of Y, in this case, N a and N b This may be the same modification or different modifications. Alternatively, if a wing modification exists, N a or N b It may or may not exist.

[0240] iRNA may further contain at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. Modification by phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage may occur at any nucleotide at any position on the sense strand, antisense strand, or both strands. For example, modification by internucleotide linkage may occur at any nucleotide on the sense strand or antisense strand; each modification by internucleotide linkage may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both modifications by internucleotide linkage in an alternating pattern. The alternating pattern of modifications by internucleotide linkage on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of modifications by internucleotide linkage on the sense strand may have a shift compared to the alternating pattern of modifications by internucleotide linkage on the antisense strand. In one embodiment, the double-stranded RNAi agent contains 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand includes two phosphorothioate nucleotide linkages at its 5' end and two phosphorothioate nucleotide linkages at its 3' end, and the sense strand includes at least two phosphorothioate nucleotide linkages at its 5' or 3' end.

[0241] In some embodiments, the dsRNAi agent includes modifications within the overhang region by phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages. For example, the overhang region may contain two nucleotides having a phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage between the two nucleotides. The nucleotide linkage modification may also be made to link the overhang nucleotide to a terminal paired nucleotide within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked via phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages, but further phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages may be made to link the overhang nucleotide to a paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate nucleotide linkages between three terminal nucleotides, in which case two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. These three nucleotides at the ends may be located at the 3' end of the antisense strand, at the 3' end of the sense strand, at the 5' end of the antisense strand, or at the 5' end of the antisense strand.

[0242] In some embodiments, the two-nucleotide overhang is located at the 3' end of the antisense strand, and there are two phosphorothioate nucleotide links between the three terminal nucleotides, in which case two of the three nucleotides are the overhanging nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In addition, the dsRNAi agent may have two phosphorothioate nucleotide links between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0243] In one embodiment, the dsRNAi agent includes mismatch(s) with a target within the double helix, or a combination thereof. Mismatches may occur within the protruding region or within the double helix region. Base pairs may be ranked based on their tendency to promote dissociation or dissolution [for example, regarding the free energy of association or dissociation of a particular pair, the simplest method is to consider the pair in individual pair bases, but the next-neighbor method or similar analysis may also be used]. With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or non-canonical pairings (described elsewhere in this specification) are preferred over canonical (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over canonical pairings.

[0244] In a particular embodiment, the dsRNAi agent independently comprises at least one of the first one, two, three, four, or five base pairs in the 5'-terminal duplex region of the antisense strand, selected from a group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairings or pairings other than canonical pairings, or pairings containing universal bases at the 5'-terminus of the duplex, to facilitate the dissociation of the antisense strand.

[0245] In a particular embodiment, the nucleotide at position 1 within the 5'-terminal double-stranded region of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs within the 5'-terminal double-stranded region of the antisense strand is an AU base pair. For example, the first base pair within the 5'-terminal double-stranded region of the antisense strand is an AU base pair.

[0246] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxythymine (dT), or the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). For example, a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, may be present at the 3' end of the sense strand, the antisense strand, or both strands.

[0247] In a particular embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) [In formula: i and j are each independently either 0 or 1; p and q are each independently between 0 and 6; each N a Each sequence independently contains 0 to 25 modified nucleotides, and each sequence represents an oligonucleotide sequence containing at least two nucleotides with different modifications; each N b Each represents an oligonucleotide sequence containing 0 to 10 modified nucleotides independently; each n p and each n q Each represents an overhanging nucleotide independently; Here, N b and Y do not have the same modifier; XXX, YYY, and ZZZ each independently represent a single motif consisting of three identical modifications on three consecutive nucleotides. Preferably, all YYY nucleotides are 2'-F modified nucleotides. It can be represented by:

[0248] In some embodiments, N a or N b This includes modifications using alternating patterns.

[0249] In some embodiments, the YYY motif is present at or near the cleavage site of the sense strand. For example, if the dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may be present at or near the cleavage site of the sense strand (e.g., it may be present at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) by counting from the first nucleotide at the 5' end; or by counting at the first paired nucleotide within the double-stranded region at the 5' end.

[0250] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the sense chain is given by the following equation: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be represented by:

[0251] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a These can independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0252] If the sense chain is expressed as equation (Ic), then Nb This represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a These can independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0253] If the sense chain is represented as expression (Id), then each N b This independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. a These can independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0254] Each of X, Y, and Z may be the same as or different from the others.

[0255] In other embodiments, i is 0, j is 0, and the sense chain is given by the expression: 5'n p -N a -YYY-N a -n q 3'(Ia) It can be represented by:

[0256] If the sense chain is represented by equation (Ia), then each N a These can independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0257] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -np '3'(II) [In formula: k and l are, independently, either 0 or 1; p' and q' are each independently between 0 and 6; each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two nucleotides with different modifications; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and each n q ' independently represents an overhanging nucleotide; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif consisting of three identical modifications on three consecutive nucleotides. It can be represented by:

[0258] In some embodiments, N a 'or N b ' includes modifications using alternating patterns.

[0259] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, if the dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, by counting from the first nucleotide at the 5' end; or by counting at the first paired nucleotide within the double-stranded region at the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, 13.

[0260] In a particular embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0261] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0262] Thus, the antisense strand has the following formula: 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’ (IIb); 5’n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’ (IIc); or 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N b ’-X’X’X’-N a ’-n p’ 3’ (IId) and can be represented by.

[0263] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0264] When the antisense strand is represented as formula (IIc), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0265] If the antisense chain is expressed as equation (IId), then each N b ' independently represents oligonucleotide sequences containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, N b It is 0, 1, 2, 3, 4, 5, or 6.

[0266] In other embodiments, k is 0, l is 0, and the antisense chain is given by the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia) It can be represented by:

[0267] If the antisense chain is represented by equation (IIa), then each N a The ' ' independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X', Y', and Z' may be the same as or different from the others.

[0268] Each nucleotide in the sense and antisense strands can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0269] In some embodiments, the sense strand of the dsRNAi agent may contain a YYY motif located at positions 9, 10, and 11 of the strand, which may be determined by counting from the first nucleotide at the 5' end, or by counting at the first paired nucleotide within the 5'-terminal double-stranded region, when the double-stranded region is 21nt; Y represents a 2'-F modification. The sense strand may also contain an XXX or ZZZ motif as a wing modification at the opposite end of the double-stranded region; XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification, respectively.

[0270] In some embodiments, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand, which may be determined by counting from the first nucleotide at the 5' end, or by counting at the first paired nucleotide within the 5'-terminal double-stranded region; where Y' represents a 2'-O-methyl modification. The antisense strand may also contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification, respectively.

[0271] Each sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0272] Therefore, the dsRNAi agent for use in the method of the present invention may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the iRNA double helix is ​​given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -nq 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) [In formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two nucleotides with different modifications; each N b and each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Here, each of these may or may not exist, for each n p ',n p , n q ', and n q Each represents an overhanging nucleotide independently; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif consisting of three identical modifications on three consecutive nucleotides. It can be represented by:

[0273] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0274] An exemplary combination of the sense strand and antisense strand that form the iRNA double helix is ​​given by the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5' (IIId) Includes.

[0275] When a dsRNAi agent is represented by formula (IIIa), each N a These independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0276] When a dsRNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a These independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0277] When a dsRNAi agent is represented by formula (IIIc), each N b , N b ' independently represents oligonucleotide sequences containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a These independently represent oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0278] When a dsRNAi agent is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a , N a ' independently represents oligonucleotide sequences containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b、 and N b Each of these independently includes modifications in an alternating pattern.

[0279] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0280] If a dsRNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one of the Y nucleotides may form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides may form a base pair with the corresponding Y' nucleotide; or all three of the Y nucleotides may form a base pair with the corresponding Y' nucleotides.

[0281] If a dsRNAi agent is represented by formula (IIIb) or (IIId), then at least one of the Z nucleotides may form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides may form a base pair with the corresponding Z' nucleotide; or all three of the Z nucleotides may form a base pair with the corresponding Z' nucleotides.

[0282] If a dsRNAi agent is represented by formula (IIIc) or (IIId), then at least one of the X nucleotides may form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides may form a base pair with the corresponding X' nucleotide; or all three of the X nucleotides may form a base pair with the corresponding X' nucleotide.

[0283] In certain embodiments, modifications on the Y nucleotide are different from modifications on the Y' nucleotide, modifications on the Z nucleotide are different from modifications on the Z' nucleotide, or modifications on the X nucleotide are different from modifications on the X' nucleotide.

[0284] In a particular embodiment, if the dsRNAi agent is represented by formula (IIId), then N aThe modification is a 2'-O-methyl modification or a 2'-fluoro modification. In other embodiments, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl modifications or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage. In yet another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl modifications or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via phosphorothioate linkage, and the sense strand is conjugated with one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described below). In other embodiments, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl modifications or 2'-fluoro modifications, n p '>0 and at least one n p The nucleotide is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated with one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0285] In some embodiments, when the dsRNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl modifications or 2'-fluoro modifications, n p '>0 and at least one n p The nucleotide is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated with one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0286] In some embodiments, the dsRNAi agent is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), in which case the double helixes are linked by a linker. The linker may be cleaved or not. The multimer may further contain ligands. Each double helix may target the same gene or two different genes; each double helix may target the same gene at two different target sites.

[0287] In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six, or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), in which case the double helixes are linked by linkers. The linkers may be cleavage-type or non-cleavage-type. The multimer may further contain ligands. Each double helix may target the same gene or two different genes; each double helix may target the same gene at two different target sites.

[0288] In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be ligated to each other and conjugated with a ligand at one or both of their 5' and 3' ends. Each agent may target the same gene or two different genes; each agent may target the same gene at two different target sites.

[0289] In a particular embodiment, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 6 nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluoro modifications in the sense strand, for example, 4 nucleotides with 2'-fluoro modifications and 2 nucleotides with 2'-fluoro modifications in the antisense strand.

[0290] In other embodiments, the RNAi agent of the present invention may contain a very small number of nucleotides containing 2'-fluoro modifications, for example, two or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain two, one, or zero nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent may contain two nucleotides with 2'-fluoro modifications, for example, zero nucleotides with 2-fluoro (2-fluoro) modifications in the sense strand, and two nucleotides with 2'-fluoro modifications in the antisense strand.

[0291] Various publications describe multimeric iRNAs that can be used in the methods of the present invention. The entire contents of each of these publications are incorporated herein by reference: WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520. Includes.

[0292] In certain embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modification of the RNAi agent described herein. In exemplary embodiments, the vinyl phosphonate of the Disclosure has the following structure:

[0293] [ka] The vinyl phosphonate of the Disclosure may be conjugated to the antisense or sense strand of the dsRNA of the Disclosure. In a particular preferred embodiment, the vinyl phosphonate of the Disclosure is conjugated to the antisense strand of the dsRNA, which may be conjugated at the 5' end of the antisense strand of the dsRNA.

[0294] Vinyl phosphate modification is also envisioned for the compositions and methods of the present disclosure. An exemplary vinyl phosphate structure is:

[0295] [ka] That is the case.

[0296] As will be described in more detail below, iRNAs containing the conjugation of one or more carbohydrate moieties can optimize one or more properties of the iRNA. In many cases, the carbohydrate moieties will be conjugated to modified subunits of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA may be replaced by a non-carbohydrate (preferably cyclic) carrier to which another moiety, such as a carbohydrate ligand, is conjugated. In this specification, a ribonucleotide subunit in which the ribose sugar of a subunit is thus replaced is referred to as a ribose-substituted modified subunit (RRMS). The cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms may be carbon atoms, or it may be a heterocyclic ring system, i.e., one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, and sulfur. The cyclic carrier may be a monocyclic ring system, and may contain two or more rings, e.g., a fusion ring. The cyclic carrier may be a fully saturated ring system, and may contain one or more double bonds.

[0297] The ligand may be conjugated to the polynucleotide via a carrier. The carrier comprises (i) at least one “skeletal junction,” preferably two “skeletal junctions,” and (ii) at least one “tethering junction.” As used herein, “skeletal junction” refers to a functional group, e.g., a hydroxyl group, or generally a bond of the carrier that is available for and suitable for incorporation into the ribonucleic acid skeleton, e.g., a phosphate skeleton, or a modified phosphate skeleton, e.g., a sulfur-containing skeleton. In some embodiments, a “tethering junction” (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (an atom significantly different from the atom resulting in the skeletal junction) that is a component of the cyclic carrier and connects the selected moiety. The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected by interposing a tether to the cyclic carrier. Therefore, cyclic carriers often contain functional groups, such as amino groups, or generally, bond to the constituent rings that are suitable for the incorporation or tethering of other chemical entities, such as ligands.

[0298] The iRNA may be conjugated to a ligand via a carrier, in which case the carrier may be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, and decalin; preferably, the acyclic group is a serinol skeleton or a diethanolamine skeleton.

[0299] i. Thermal instability modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating a thermal instability modification within the seed region of the antisense strand (i.e., at positions 2–9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. dsRNAs with an antisense strand containing at least one double-stranded thermal instability modification within the first nine nucleotide positions, counting from the 5' end of the antisense strand, have been found to reduce off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) double-stranded thermal instability modification within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more double-stranded thermal instability modifications are located within positions 2–9, or preferably 4–8, from the 5' end of the antisense strand. In some further embodiments, the double-stranded thermal instability modification(s) are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some even further embodiments, the double-stranded thermal instability modification(s) are located at position 7 from the 5' end of the antisense strand. The term “thermal instability modification(s)” includes modifications(s) that reduce the overall melting temperature (Tm) of dsRNA [preferably modifications(s) that result in a Tm 1, 2, 3, or 4 degrees lower than that of dsRNA without such modifications]. In some embodiments, the double-stranded thermal instability modification(s) are located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0300] The iRNA agent contains a sense strand and an antisense strand, each having 14 to 40 nucleotides. The RNAi agent is given by formula (L):

[0301] [ka] [In formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently nucleotides containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.] It can be represented by:

[0302] C1 is a thermally unstable nucleotide located opposite the seed region of the antisense strand (i.e., at positions 2–8 of the 5' end of the antisense strand). For example, C1 is located on the sense strand, pairing with a nucleotide at positions 2–8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 of the 5' end of the sense strand. The C1 nucleotide may harbor thermally unstable modifications, including debasic modifications; mismatches with opposing nucleotides in the double helix; and sugar modifications such as 2'-deoxy modifications, or acyclic nucleotides, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 may i) mismatch with opposing nucleotides in the antisense strand; ii)

[0303] [ka] A debasic modification selected from the group consisting of; and iii)

[0304] [ka] , and

[0305] [ka] [In the formula, B is a modified nucleobase or an unmodified nucleobase, and R 1 and R 2 R3 is independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The C1 has a thermally unstable modification selected from the group consisting of sugar modifications selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; at least one nucleobase in the mismatch pair may be a 2'-deoxynucleobase. In one example, the thermally unstable modification in C1 is GNA or

[0306] [ka] That is the case. T1, T1', T2', and T3 each independently represent a nucleotide containing a modification that imparts a steric bulk to the nucleotide that is less than or equal to the steric bulk of the 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effects of nucleotide modifications are known to those skilled in the art. The modifications may be modifications at the 2' position of the ribose sugar of the nucleotide, or modifications to a non-ribose nucleotide, acyclic nucleotide, or nucleotide backbone, which are similar to or equivalent to modifications at the 2' position of the ribose sugar, and impart a steric bulk to the nucleotide that is less than or equal to the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA. n 1 , n 3 , and q1 These are independently 4 to 15 nucleotides in length. n 5 , q 3 , and q 7 Each nucleotide is independently 1 to 6 nucleotides long. n 4 , q 2 , and q 6 Each is independently 1 to 3 nucleotides in length; alternatively, n 4 It is 0. q 5 Each nucleotide is independently 0 to 10 nucleotides in length. n 2 and q 4 Each nucleotide is independently 0 to 3 nucleotides long.

[0307] Alternatively, n 4 Its length is between 0 and 3 nucleotides.

[0308] In one embodiment, n 4 n can be 0. In one example, n 4 is 0, and q 2 and q 6 In another example, n 4 is 0, and q 2 and q 6 It is 1, with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0309] In one embodiment, n 4 , q 2 , and q 6 Each of these values ​​is 1.

[0310] In one embodiment, n2 , n 4 , q 2 , q 4 , and q 6 Each of these values ​​is 1.

[0311] In one embodiment, C1 is located at the 14th to 17th position from the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides long. 4 In one embodiment, C1 is located at the 15th position of the 5' end of the sense strand.

[0312] In one embodiment, T3' begins at position 2 of the 5' end of the antisense chain. In one example, T3' is located at position 2 of the 5' end of the antisense chain, q 6 It is equal to 1.

[0313] In one embodiment, T1' begins at position 14 of the 5' end of the antisense chain. In one example, T1' is located at position 14 of the 5' end of the antisense chain, q 2 It is equal to 1.

[0314] In an exemplary embodiment, T3' starts from position 2 of the 5' end of the antisense chain, and T1' starts from position 14 of the 5' end of the antisense chain. In one example, T3' starts from position 2 of the 5' end of the antisense chain, q 6 is equal to 1, T1' starts at position 14 of the 5' end of the antisense chain, q 2 It is equal to 1.

[0315] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (i.e., not counting the T1' and T3' nucleotides).

[0316] In one embodiment, T1' is located at position 14 of the 5' end of the antisense chain. In one example, T1' is located at position 14 of the 5' end of the antisense chain, q 2Modifications at the 2' position, or at non-ribose, acyclic, or skeletal positions, result in a steric bulk of less than 2'-OMe-ribose.

[0317] In one embodiment, T3' is located at position 2 of the 5' end of the antisense chain. In one example, T3' is located at position 2 of the 5' end of the antisense chain, q 6 Modifications at the 2' position, or at non-ribose, acyclic, or skeletal positions, result in a steric bulk of 2'-OMe-ribose or less.

[0318] In one embodiment, T1 is located at the cleavage site of the sense strand. In one example, when the sense strand is 19-22 nucleotides long, T1 is located at position 11 of the 5' end of the sense strand, n 2 is 1. In an exemplary embodiment, T1 is located at the sense strand cleavage site at position 11 of the 5' end of the sense strand, when the sense strand is 19-22 nucleotides long, and n 2 It is 1.

[0319] In one embodiment, T2' begins at position 6 of the 5' end of the antisense chain. In one example, T2' is located at positions 6-10 of the 5' end of the antisense chain, q 4 It is 1.

[0320] In an exemplary embodiment, T1 is located at a cleavage site on the sense strand, for example, at position 11 of the 5' end of the sense strand, when the sense strand is 19-22 nucleotides long. 2 is 1; T1' is at position 14 of the 5' end of the antisense chain, q 2 is equal to 1, the modification to T1' is at the 2' position of the ribose sugar, or at a non-ribose, acyclic or skeletal position, and is not sterically bulky as 2'-OMe-ribose; T2' is at the 6-10 position of the 5' end of the antisense chain, q 4 is 1; T3' is at position 2 of the 5' end of the antisense chain, q 6This is equal to 1, and the modification to T3' is at the 2' position, or in a non-ribose, acyclic, or skeletal position, resulting in a steric bulk of 2'-OMe-ribose or less.

[0321] In one embodiment, T2' begins at position 8 of the 5' end of the antisense chain. In one example, T2' begins at position 8 of the 5' end of the antisense chain, q 4 The answer is 2.

[0322] In one embodiment, T2' begins at position 9 of the 5' end of the antisense chain. In one example, T2' is located at position 9 of the 5' end of the antisense chain, q 4 It is 1.

[0323] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0324] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0325] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1.

[0326] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0327] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1.

[0328] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0329] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1.

[0330] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0331] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and may be accompanied by at least two further TTs at the 3' end of the antisense chain.

[0332] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; may have at least two additional TTs at the 3' end of the antisense strand; has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand); has two phosphorothioate nucleotide linkage modifications within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand); and has two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0333] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1.

[0334] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end).

[0335] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1.

[0336] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0337] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1.

[0338] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; the sense strand has modifications by two phosphorothioate nucleotide linkages within positions 1-5 (counting from the 5' end of the sense strand), the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 (counting from the 5' end of the antisense strand), and the antisense strand has modifications by two phosphorothioate nucleotide linkages within positions 18-23 (counting from the 5' end of the antisense strand).

[0339] RNAi agents may contain a phosphorus-containing group at the 5' end of the sense or antisense strand. The phosphorus-containing group at the 5' end may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl(

[0340] [ka] ) may be. If the phosphorus-containing group at the 5' terminus is a vinyl phosphonate (5'-VP), then 5'-VP is a 5'-E-VP isomer (i.e., trans-vinyl phosphate,

[0341] [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate,

[0342] [ka] ), or a mixture thereof.

[0343] In one embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the sense strand. In another embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the antisense strand.

[0344] In one embodiment, the RNAi agent contains 5'-P. In one embodiment, the RNAi agent contains 5'-P within the antisense strand.

[0345] In one embodiment, the RNAi agent contains 5'-PS. In one embodiment, the RNAi agent contains 5'-PS within the antisense strand.

[0346] In one embodiment, the RNAi agent contains 5'-VP. In one embodiment, the RNAi agent contains 5'-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-Z-VP in the antisense strand.

[0347] In one embodiment, the RNAi agent contains 5'-PS2. In one embodiment, the RNAi agent contains 5'-PS2 in the antisense strand.

[0348] In one embodiment, the RNAi agent contains 5'-PS2. In one embodiment, the RNAi agent contains 5'-deoxy-5'-C-malonyl in the antisense strand.

[0349] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1. RNAi agents also include 5'-PS.

[0350] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q6 is 1, B4' is 2'-OMe, and q 7 It is 1. RNAi agents also contain 5'-P.

[0351] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0352] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1. RNAi agents also contain 5'-PS2.

[0353] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-deoxy-5'-C-malonyl.

[0354] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-P.

[0355] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-PS.

[0356] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0357] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-PS2.

[0358] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0359] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1. RNAi agents also contain 5'-P.

[0360] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0361] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0362] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1. RNAi agents also contain 5'-PS2.

[0363] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-deoxy-5'-C-malonyl.

[0364] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). RNAi agents also contain 5'-P.

[0365] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). RNAi agents also contain 5'-PS.

[0366] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 This is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0367] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). RNAi agents also contain 5'-PS2.

[0368] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0369] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. RNAi agents also contain 5'-P.

[0370] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. RNAi agents also include 5'-PS.

[0371] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0372] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. dsRNAi RNA (dsRNAi RNA) also includes 5'-PS2.

[0373] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-deoxy-5'-C-malonyl.

[0374] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-P.

[0375] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-PS.

[0376] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0377] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-PS2.

[0378] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0379] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. RNAi agents also contain 5'-P.

[0380] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. RNAi agents also include 5'-PS.

[0381] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination of these.

[0382] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1. RNAi agents also contain 5'-PS2.

[0383] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-deoxy-5'-C-malonyl.

[0384] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-P.

[0385] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-PS.

[0386] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7It is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modifications by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modifications by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0387] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 It is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also contain 5'-PS2.

[0388] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; within positions 1 to 5 of the sense strand (counting from the 5'-end of the sense strand), there is a modification with a phosphorothioate internucleotide linkage, at positions 1 and 2 of the antisense strand (counting from the 5'-end of the antisense strand), there is a modification with a phosphorothioate internucleotide linkage, and within positions 18 to 23 of the antisense strand (counting from the 5'-end of the antisense strand), there is a modification with a phosphorothioate internucleotide linkage. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0389] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0390] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; with modifications by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and with modifications by two phosphorothioate nucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0391] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it is modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0392] In one embodiment, the 5'-VP is located at the 5' end of the antisense chain, and the targeting ligand is located at the 3' end of the sense chain.

[0393] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it is modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0394] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2’-OMe, and n 3 is 7, and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q 3 is 4, T2’ is 2’-F, and q 4 is 2, B3’ is 2’-OMe or 2’-F, and q 5 is 5, T3’ is 2’-F, and q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; within positions 1 to 5 of the sense strand (counting from the 5’ end of the sense strand), there is a modification with a phosphorothioate internucleotide linkage, at positions 1 and 2 of the antisense strand (counting from the 5’ end of the antisense strand), there is a modification with a phosphorothioate internucleotide linkage, and within positions 18 to 23 of the antisense strand (counting from the 5’ end of the antisense strand), there is a modification with a phosphorothioate internucleotide linkage. The RNAi agent also includes 5’-deoxy-5’-C-malonyl and a targeting ligand. In one embodiment, 5’-deoxy-5’-C-malonyl is at the 5’ end of the antisense strand, and the targeting ligand is at the 3’ end of the sense strand.

[0395] In one embodiment, B1 is 2’-OMe or 2’-F, and n 1 is 8, T1 is 2’F, and n 2 is 3, B2 is 2’-OMe, and n 3 is 7, and n 4 is 0, B3 is 2’-OMe, and n 5 is 3, B1’ is 2’-OMe or 2’-F, and q 1 is 9, T1’ is 2’-F, and q 2 is 1, B2’ is 2’-OMe or 2’-F, and q3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0396] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; with modification by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), with modification by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0398] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0399] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0400] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0401] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; it is modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0402] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0403] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; it is modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0404] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0405] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0406] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; it is modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0407] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0408] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; it is modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0409] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; modified by two phosphorothioate nucleotide linkages within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), modified by two phosphorothioate nucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and modified by two phosphorothioate nucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0410] In a particular embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20 (counting from the 5' end) A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23, and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). Antisense chain having Includes; In this case, the dsRNA agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0411] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0412] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, 10, and 12-21, 2'-F modifications at positions 7 and 9, and deoxynucleotides (e.g., dT) at position 11 (counting from the 5' end); and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23, and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0413] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, 10, 12, 14, and 16-21, and 2'-F modifications at positions 7, 9, 11, 13, and 15; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0414] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-9 and 12-21, and 2'-F modifications at positions 10 and 11; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0415] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0416] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21, and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 25 nucleotides; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has four nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0417] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0418] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 21 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, and 12-21, and 2'-F modifications at positions 7 and 9-11; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0419] In another specific embodiment, the RNAi agent of the present invention is (a) a sense chain, (i) Length of 19 nucleotides; (ii) An ASGPR ligand conjugated to the 3' terminus, comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19, and 2'-F modifications at positions 5 and 7-9; and (iv) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end). A sense chain having; (b) an antisense chain, (i) Length of 21 nucleotides; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) Phosphothioate nucleotide ligations between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end) Antisense chain having Includes; In this case, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0420] In a particular embodiment, the iRNA for use in the method of the present invention is an agent selected from any one of the agents listed in Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50. These agents may further include ligands.

[0421] III. iRNA conjugated to a ligand Another modification of the RNAi RNA in this disclosure involves chemically linking one or more ligands, moieties, or conjugates to the iRNA that enhance its activity, intracellular distribution, or, for example, its uptake into cells. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556).In other embodiments, the ligand may be cholic acid [Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060], thioethers, such as beryl-S-tritylthiol [Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770], thiocholesterol [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, e.g., dihexadecyl-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 chains or polyethylene glycol chains [Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973], or adamantane acetate [Manoharan et al., Tetrahedron Lett., [1995, 36:3651-3654], palmityl moiety [Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237], or octadecylamine moiety or hexylaminocarbonyloxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937].

[0422] In certain embodiments, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In preferred embodiments, the ligand results in enhanced affinity to selective targets, such as molecules, cells or cell types, compartments, such as cellular or organ compartments, tissues, organs, or regions within the body, compared to molecular species in which such ligand is absent. Preferred ligands do not participate in the pairing of double helixes within double-stranded nucleic acids.

[0423] Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be synthetic polymers, such as recombinant or synthetic molecules including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide mimetic polyamines, dendrimer polyamines, arginine, amidine, protoamines, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helix peptides.

[0424] The ligand may also include a targeting group, such as a cell-targeting agent or tissue-targeting agent, such as an antibody that binds to a specified cell type, such as a lectin, glycoprotein, lipid, or protein, such as kidney cells. The targeting group may be tyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucinous carbohydrate, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamate, polyasparate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic. In a particular embodiment, the ligand is a polyhydric galactose, such as N-acetylgalactosamine.

[0425] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), heterocyclic aromatic carbohydrates (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules [e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl) The compounds include fluoroglycerides, dimethoxytrityl, or phenoxazine, and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino acids, mercaptoside, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic rings), dinitrophenyl, HRP, or AP.

[0426] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity to a colligand, or antibodies, such as antibodies that bind to a specified cell type, such as hepatocytes. Ligands can also include hormones and hormone receptors. Ligands can also include non-peptide molecular species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, or polyvalent fucose. Ligands can also be, for example, lipopolysaccharides, p38MAP kinase activators, or NF-κB activators.

[0427] A ligand can be a drug that can increase the uptake of an iRNA agent into cells by disrupting the cytoskeleton, for example, by disrupting cellular microtubules, microfilaments, or intermediate filaments. Examples of such drugs may be taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlaquinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoservin.

[0428] In some embodiments, ligands conjugated to iRNAs, as described herein, act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein-binding agents, PEGs, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins, and therefore, short oligonucleotides containing multiple phosphorothioate linkages in their backbone, such as oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable for use as ligands (e.g., PK-modulating ligands) in the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0429] The ligand-conjugated iRNA of the present invention can be synthesized using an oligonucleotide possessing a reactive pendant functional group (described below), which is derived from the conjugation of the linking molecule to the oligonucleotide. This reactive oligonucleotide can be directly reacted with a commercially available ligand, a synthetic ligand possessing one of various protecting groups, or a ligand to which the linking portion is conjugated.

[0430] The oligonucleotides used in the conjugates of the present invention can be conveniently and predictably prepared by well-known solid-phase synthesis methods. Apparatus for such synthesis is available from several distributors, including, for example, Applied Biosystem® (Foster City, Calif.). In addition, or alternatively, any other method known in the art for such synthesis may be used. It is also known to prepare other oligonucleotides, such as phosphorothioate derivatives and alkylated derivatives, using similar techniques.

[0431] In the ligand-conjugate iRNA and ligand molecule possessing a sequence-specific linking nucleoside according to the present invention, the oligonucleotide and oligonucleoside can be assembled on a suitable DNA synthesizer using a standard nucleotide precursor or nucleoside precursor, or a nucleotide conjugate precursor or nucleoside conjugate precursor that already possesses a linking portion, or a ligand-nucleotide conjugate precursor or ligand-nucleoside conjugate precursor that already possesses a ligand molecule, or a ligand-containing component other than a nucleoside.

[0432] When using nucleotide conjugate precursors that already possess a linking site, typically, the synthesis of a sequence-specific linking nucleoside is completed, and then the ligand molecule is reacted with the linking site to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linking nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0433] A. Lipid conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues, such as target tissues in the body other than the kidneys. For example, the target tissue may be liver tissue, including the parenchymal cells of the liver. Other molecules that can bind to HSA may also be used as ligands. For example, naproxen or aspirin may be used. Lipid or lipid-based ligands may (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or target cell membranes, or (c) be used to modulate binding to serum proteins, such as HSA.

[0434] Lipid-based ligands can be used to inhibit, for example, control the binding of conjugates to target tissues. For instance, a lipid or lipid-based ligand that strongly binds to HSAs is less likely to be targeted to the kidneys and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that weakly binds to HSAs can be used to target conjugates to the kidneys.

[0435] In certain embodiments, a lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate is distributed to tissues other than the kidney. However, it is preferable that the affinity is not so strong that the HSA-ligand binding cannot be dissolved.

[0436] In other embodiments, the lipid-based ligand either weakly or not binds at all to the HSA so that the conjugate is preferably distributed to the kidney. Other portions that target renal cells may also be used instead of or in addition to the lipid-based ligand.

[0437] In another embodiment, the ligand is a portion taken up by target cells, e.g., proliferating cells, e.g., a vitamin. These are particularly useful for treating disorders characterized by undesirable cell proliferation, e.g., by malignant or non-malignant cells, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B (B vitamins), e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as hepatocytes. Also included are HSA and low-density lipoprotein (LDL).

[0438] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, preferably a helix-type cell permeabilizer. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or antenopedia. If the agent is a peptide, the agent can be modified, including the use of peptidyl mimes, invertomers, non-peptide linkages or pseudopeptide linkages, and D-amino acids. The helix agent preferably has a lipophilic phase and an oleophobic phase, preferably an alpha-helix agent.

[0439] Ligands may be peptides or peptide mimetics. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules capable of folding into defined three-dimensional structures, similar to natural peptides. The conjugation of peptides and peptide mimetics to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cellular recognition and absorption. The peptide or peptide mimetic portion may be approximately 5 to 50 amino acids long, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0440] Peptides or peptide mimetic compounds can be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, a restricting peptide, or a cross-linked peptide. In another alternative example, the peptide moiety may contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is recombinant fibroblast growth factor (RFGF) having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). A hydrophobic MTS-containing RFGF analog [e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 15)] may also be a targeting moiety. The peptide moiety can be a “delivery” peptide capable of carrying large polar molecules across the cell membrane, including peptides, oligonucleotides, and proteins. For example, sequences derived from the HIV Tat protein [GRKKRRQRRRPPQ (SEQ ID NO: 16)] and the Drosophila Antennapedia protein [RQIKIWFQNRRMKWKK (SEQ ID NO: 17)] have been found to be capable of functioning as delivery peptides. Peptides or peptide mimetic compounds can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or OBOC (one-bead-one-compound) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). An example of a peptide or peptide mimetic compound tethered to a dsRNA agent via monomeric units incorporated for the purpose of targeting cells is the arginine-glycine-aspartate (RGD) peptide or RGD mimetic. The peptide portion can range in length from approximately 5 to 40 amino acids. The peptide portion may have structural modifications, such as structural modifications that increase stability or direct conformational properties. Any of the structural modifications described below may be used.

[0441] The RGD peptides for use in the compositions and methods of the present invention may be linear or cyclic and may be modified to facilitate targeting of specific tissues, for example, by glycosylation or methylation. RGD-containing peptides and peptide mimetic compounds may include D-amino acids as well as synthetic RGD mimetic compounds. In addition to RGD, other parts that target integrin ligands may also be used. Preferred conjugates of these ligands target PECAM-1 or VEGF.

[0442] "Cell-permeable peptides" are capable of permeating cells, such as microbial cells like bacterial or fungal cells, or mammalian cells like human cells. Microbial cell-permeable peptides can be, for example, α-helix linear peptides [e.g., LL-37 or ceropin P1], disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenesins), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides can also include nuclear localization signals (NLS). For example, cell-permeable peptides can be bipartite amphiphilic peptides such as MPGs derived from fusion peptide domains, such as the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0443] C. Carbohydrate Conjugate In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for compositions suitable for in vivo therapeutic use, as well as for in vivo nucleic acid delivery, as described herein. As used herein, “carbohydrate” means a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen atom, nitrogen atom, or sulfur atom bonded to each carbon atom; or a compound, each having as part a carbohydrate portion consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen atom, nitrogen atom, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about four, five, six, seven, eight, or nine monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. A specific monosaccharide includes C5 and sugars greater than C5 (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0444] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide.

[0445] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate is used as a ligand to target iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to hepatocytes by being used, for example, as a ligand to the asialoglycoprotein receptor in hepatocytes (e.g., hepatocytes).

[0446] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be conjugated via a linker, for example, a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated with an iRNA agent (for example, to the 3' end of the sense strand) via a linker, for example, a linker described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated with an iRNA agent (for example, to the 5' end of the sense strand) via a linker, for example, a linker described herein.

[0447] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a tetravalent linker.

[0448] In certain embodiments, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative conjugated to an iRNA agent. In certain embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently conjugated to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0449] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of a single large molecule, linked by an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently contain GalNAc or a GalNAc derivative, joined via a monovalent linker. The hairpin loop may also be formed by an elongation projection within one of the strands of the double helix.

[0450] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of a single large molecule, linked by an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently contain GalNAc or a GalNAc derivative, joined via a monovalent linker. The hairpin loop may also be formed by an elongation projection within one of the strands of the double helix.

[0451] In one embodiment of the present invention, the carbohydrate conjugate for use in compositions and methods is:

[0452] [ka] [ka] [ka] [ka] [ka] [In the formula, Y is either O or S, and n is between 3 and 6] (Formula XXIV);

[0453] [ka] [In the formula, Y is either O or S, and n is between 3 and 6] (Formula XXV);

[0454] [ka]

[0455] [ka] [In the formula, X is either O or S] (Formula XXVII);

[0456] [ka]

[0457] [ka] [ka] It is selected from the group consisting of the following.

[0458] In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is

[0459] [ka] These include N-acetylgalactosamine.

[0460] In some embodiments, the RNAi agent is represented by the following diagram [wherein X is O or S]

[0461] [ka] It is joined to the carbohydrate conjugate via the linker shown in [the diagram].

[0462] In some embodiments, the RNAi agent is as defined in Table 1, as follows:

[0463] [ka] As shown, it is conjugated with L96.

[0464] Another representative carbohydrate conjugate for use in the embodiments described herein is:

[0465] [ka] (Formula XXXVI) [wherein if one of X or Y is an oligonucleotide, the other is hydrogen] This includes, but is not limited to, these.

[0466] In some embodiments, suitable ligands are ligands disclosed in WO2019 / 055633, the full contents of which are incorporated herein by reference. In one embodiment, the ligand has the following structure:

[0467] [ka] Includes.

[0468] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a trivalent linker.

[0469] In one embodiment of the present invention, the double-stranded RNAi agent comprises one or more GalNAc or GalNAc derivatives conjugated to an iRNA agent. The GalNAc may be conjugated to any nucleotide via a linker on the sense strand or the antisense strand. The GalNAc may be conjugated to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 3' end of the antisense strand. In one embodiment, the GalNAc is conjugated to the 3' end of the sense strand, for example, via a trivalent linker.

[0470] In other embodiments, each double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAcs or GalNAc derivatives, each independently conjugated to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of linkers, for example, monovalent linkers.

[0471] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of a single large molecule, linked by an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing a plurality of unpaired nucleotides, then each unpaired nucleotide within the hairpin loop may independently contain GalNAc or a GalNAc derivative, joined via a monovalent linker.

[0472] In some embodiments, the carbohydrate conjugate further comprises one or more further ligands described above, such as but not limited to PK modulators or cell-permeable peptides.

[0473] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in PCT Publications WO2014 / 179620 and WO2014 / 179627, the entire contents of each of these, which are incorporated herein by reference.

[0474] D. Linker In some embodiments, the conjugates or ligands described herein may be linked to the iRNA oligonucleotide by a variety of linkers, which may be cleavage-type or non-cleavage-type.

[0475] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, for example, an organic part that covalently joins two parts of a compound.Linkers are typically direct bonds, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or substituted alkyl or unsubstituted alkyl, substituted alkenyl or unsubstituted alkenyl, substituted alkynyl or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkyl, alkenylheteroaryl Alkenyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl hetero(herero)cyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl The atomic chain includes, but is not limited to, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl, and comprises an atomic chain in which one or more methylene groups are interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted heterocycle [wherein R8 is hydrogen, acyl, aliphatic, or substituted aliphatic].In one embodiment, the linker is approximately 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0476] A cleavable linker is a linker that is sufficiently stable outside the cell but, upon entering the target cell, is cleaved, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable linker is cleaved at a rate of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 times, or more, or at least 100 times, in the target blood or under a second set of reference conditions (e.g., conditions that mimic or represent intracellular conditions) or under a first set of reference conditions (e.g., conditions that mimic or represent conditions found in blood or serum).

[0477] Severable linkers are sensitive to severing agents, such as pH, redox potential, or the presence of degradable molecules. Generally, severing agents are found more frequently, at higher levels, or with higher activity inside cells than in serum or blood. Examples of such degrading agents include redox agents selected for specific substrates, or redox agents without substrate specificity, which include, for example, mercaptans present in cells that can degrade redox-severable linkers by reduction; esterases; endosomes; or agents that can create an acidic environment, such as an acidic environment resulting in a pH of 5 or less; and redox agents containing oxidases or reductases or reducing agents, such as general acids, peptidases (which may be substrate-specific), and enzymes that can hydrolyze or degrade acid-severable linkers by acting as phosphatases.

[0478] Scleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is somewhat lower, ranging from approximately 7.1 to 7.3. Endosomes have a highly acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers may cleave at a favorable pH, thereby possessing scleavable linking groups that release cationic lipids from intracellular ligands or into desired cellular compartments.

[0479] Linkers may contain cleavage-type linking groups that are cleaved by specific enzymes. The type of cleavage-type linking group incorporated into the linker may depend on the targeting cell. For example, hepatic targeting ligands may be linked to cationic lipids via linkers containing ester groups. Since hepatocytes are rich in esterases, the linker will be cleaved more efficiently within hepatocytes than in esterase-unriched cell types. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0480] Linkers containing peptide bonds can be used to target peptidase-rich cell types, such as hepatocytes and synovial cells.

[0481] Generally, the suitability of a candidate cleavage ligator can be assessed by examining the ability of a degrading agent (or degrading condition) to cleave the candidate ligator. It may also be desirable to examine the candidate cleavage ligator's resistance to cleavage in blood or in contact with other non-target tissues. Therefore, when the first condition is selected to indicate cleavage within target cells, and the second condition is selected to indicate cleavage in other tissues or in body fluids, such as blood or serum, the relative sensitivity to cleavage between the first and second conditions can be determined. Assessment can be performed in cell-free systems, cells, cell cultures, organ cultures or tissue cultures, or in whole animals. It may be useful to perform an initial assessment under cell-free or culture conditions and then confirm it with a further assessment in whole animals. In a preferred embodiment, a useful candidate compound is cleaved in a cell (or under in vitro conditions selected to mimic intracellular conditions) at a rate at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0482] i. Redox-cut type coupling base In certain embodiments, the cleavage-type linking group is a redox-cleavage-type linking group that is cleaved upon reduction or oxidation. An example of a reductive-cleavage-type linking group is a disulfide linking group (-SS-). Methods described herein can be considered to determine whether a candidate cleavage-type linking group is a suitable “reductive-cleavage-type linking group” or whether it is suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate substance may be assessed by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate observed intracellularly, for example, in a target cell. The candidate substance may also be assessed under conditions selected to mimic blood or serum conditions. In one embodiment, the candidate compound is cleaved by about 10% at most in blood. In other embodiments, useful candidate compounds are degraded intracellularly (or under in vitro conditions selected to mimic intracellular conditions) at a rate of at least approximately 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or approximately 100 times faster than in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using standard enzyme kinetic assays under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0483] ii. Phosphate-based cleavage-type linking groups In other embodiments, the cleavage linker includes a phosphate-based cleavage linking group. The phosphate-based cleavage linking group is cleaved by agents that decompose or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells include enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidate linking groups can be assessed using methods similar to those described above.

[0484] iii. Acid-cleavable linking group In other embodiments, the cleavage linker includes an acid-cleavage linking group. An acid-cleavage linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments, the acid-cleavage linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by a drug such as an enzyme that can act as a general acid. Within cells, specific low-pH organelles, such as endosomes and lysosomes, provide a cleavage environment for acid-cleavage linking groups. Examples of acid-cleavage linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavage group may have the general formula: -C=NN-, C(O)O, or -OC(O). Preferred embodiments are those in which the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidate linking groups can be assessed using methods similar to those described above.

[0485] iv. Ester-based linking groups In other embodiments, the cleavage linker comprises an ester-based cleavage linking group. The ester-based cleavage linking group is cleaved by intracellular enzymes such as esterases and amidases. Examples of ester-based cleavage linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkylylene groups. The ester-based cleavage linking group has the general formula: -C(O)O- or -OC(O)-. These candidate linking groups can be assessed using methods similar to those described above.

[0486] v. Peptide-based cleaving groups In yet another embodiment, the cleavage linker comprises a peptide-based cleavage linker. The peptide-based cleavage linker is cleaved by enzymes such as peptidases and proteases in cells. The peptide-based cleavage linker is a peptide bond formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavage linker does not contain an amide group [-C(O)NH-]. The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage linker is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavage linker has the general formula: -NHCHRAC(O)NHCHRBC(O)- [wherein RA and RB are the R groups of two adjacent amino acids]. These candidate linking groups can be assessed using methods similar to those described above.

[0487] In some embodiments, the iRNAs of this disclosure are conjugated with carbohydrates via linkers. Non-limiting examples of carbohydrate conjugations of iRNAs with linkers of the compositions and methods of the present invention include:

[0488] [ka] [ka] (Formula XLIV) [wherein if one of X or Y is an oligonucleotide, the other is hydrogen] This includes, but is not limited to, these.

[0489] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives conjugated via a divalent or trivalent branched linker.

[0490] In one embodiment of the present invention, dsRNA is a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XLV) to (XLVI):

[0491] [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent a number between 0 and 20 for each occurrence, in which case the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each of these is, independently, for each occurrence, non-existent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Independently, for each occurrence, are non-existent, alkylene, and substituted alkylene, in which case one or more methylene are O, S, S(O), SO2, N(R) NIt may be interrupted or terminated by one or more of the following: C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each of these independently determines, for each occurrence, non-existence, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R) a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO,

[0492] [ka] , or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L5Cは Each instance independently represents a ligand; that is, a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a It is conjugated with H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are given by formula (XLIX):

[0493] [ka] [In the formula, L 5A , L 5B and L 5C This represents monosaccharides such as GalNAc derivatives. It is particularly useful in conjunction with RNAi agents, such as RNAi agents used to inhibit the expression of target genes.

[0494] Suitable examples of divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above as formulas II, VII, XI, X, and XIII.

[0495] Representative U.S. patents teaching the preparation of RNA conjugates, each of which is incorporated herein by reference in its entirety, are U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; and 5,138,045. No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5, No. 608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,77 No. 9; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4 , 904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,1 No. 36; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292 , No. 873; No. 5,317,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; Same No. 5,451,463; Same No. 5,510,475; Same No. 5,512,667; Same No. 5,514,785; Same No. 5,56 This includes, but is not limited to, Nos. 5,552; Nos. 5,567,810; Nos. 5,574,142; Nos. 5,585,481; Nos. 5,587,371; Nos. 5,595,726; Nos. 5,597,696; Nos. 5,599,923; Nos. 5,599,928; Nos. 5,688,941; Nos. 6,294,664; Nos. 6,320,017; Nos. 6,576,752; Nos. 6,783,931; Nos. 6,900,297; Nos. 7,037,646; and Nos. 8,106,022.

[0496] It is not necessary for all positions within a given compound to be uniformly modified; in fact, more than one of the aforementioned modifications may be incorporated into a single compound, or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0497] In the context of the present invention, a “chimeric” iRNA compound or “chimera” is an iRNA compound, preferably a dsRNAi agent, that contains two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA has been modified to confer increased resistance to nuclease degradation, increased intracellular uptake, or increased binding affinity to a target nucleic acid. Further regions of the iRNA can be used as substrates for enzymes capable of cleaving RNA:DNA hybrids or RNA:RNA hybrids. For example, RNase H is an intracellular endonuclease that cleaves the RNA strand of an RNA:DNA double helix. Thus, activation of RNase H results in cleavage of an RNA target, thereby significantly enhancing the efficiency of gene expression inhibition by the iRNA. As a result, when chimeric dsRNAs are used, shorter iRNAs can often yield equivalent results compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can be routinely detected by gel electrophoresis and, if necessary, by nucleic acid hybridization methods known in the relevant art.

[0498] In certain cases, the RNA of iRNA can be modified with non-ligand groups. Numerous non-ligand molecules are conjugated to iRNA to enhance its activity, intracellular distribution, or uptake into cells, and procedures for performing such conjugations are available in the academic literature. Such non-ligand portions include lipid portions such as cholesterol [Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553], cholic acid [Manoharan et al., Biorg. Med. Chem. Lett., 1994, 4:1053], thioethers, for example, hexyl-S-tritylthiol [Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765], thiocholesterol [Oberhauser et al., Nucl. Acids Res., 1992, 20:533], aliphatic chains, e.g., dodecanediol or undecyl residues [Saison-Behmoaras et al., EMBO J, 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49], phospholipids, e.g., dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777], polyamine chains or polyethylene glycol chains [Manoharan et al.These included [Nucleosides & Nucleotides, 1995, 14:969], or adamantane acetate [Manoharan et al., Tetrahedron Lett., 1995, 36:3651], palmityl moiety [Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229], or octadecylamine moiety or hexylaminocarbonyloxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923]. Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA possessing an aminolinker at one or more positions in its sequence. The amino group is then reacted with the conjugated molecule using a suitable coupling or activating reagent. Conjugation reactions can be carried out with the RNA bound to a solid support, or after cleaving the RNA, in a solution phase. Purification of RNA conjugates by HPLC typically yields a pure conjugate.

[0499] IV. Delivery of the iRNAs of this Disclosure The delivery of the iRNAs of this disclosure to cells within a subject, such as a human subject (e.g., a subject susceptible to or diagnosed with PNPLA3-related disorders, e.g., NAFLD, or a subject requiring such delivery), can be achieved in a number of different ways. For example, delivery may be carried out in vitro or in vivo by contact of cells with the iRNAs of this disclosure. In vivo delivery may also be carried out directly by administering a composition containing the iRNA, e.g., dsRNA, to the subject. Alternatively, in vivo delivery may also be carried out indirectly by administering one or more vectors encoding the iRNA, resulting in direct expression of the iRNA. These alternative methods will be discussed further below.

[0500] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with iRNA as described herein [see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144; and WO94 / 02595, which are incorporated herein in their entirety by reference]. For in vivo delivery, factors to be considered for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in target tissue. RNA interference has also shown success through local delivery to the CNS via direct injection [Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602]. RNA modification or drug carriers can also enable the targeting of iRNAs to target tissues and avoid unwanted off-target effects. iRNA molecules can be modified by chemical conjugation with lipophilic groups such as cholesterol to enhance intracellular uptake and prevent degradation. For example, iRNA directed to ApoB conjugated with a lipophilic cholesterol moiety was systemically injected into mice, resulting in knockdown of apoB mRNA in both the liver and jejunum [Soutschek, J., et al (2004) Nature 432:173-178].

[0501] In alternative embodiments, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of negatively charged iRNA molecules and also enhance interactions with negatively charged cell membranes to enable efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may bind to iRNA and be induced to form vesicles or micelles that encapsulate the iRNA [see, for example, Kim SH, et al (2008) Journal of Controlled Release 129(2):107-116]. The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art [see, for example, Sorensen, DR, et al (2003) J. Mol. Biol 327:761-766; Verma, UN, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety].Some non-limiting examples of drug delivery systems useful for systemic iRNA delivery include DOTAP [Sorensen, DR., et al (2003), supra; Verma, UN, et al (2003), supra], "solid nucleic acid lipid particles" [Zimmermann, TS, et al (2006) Nature 441:111-114], cardiolipin [Chien, PY, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091], polyethyleneimine [Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659], and Arg-Gly-Asp(RGD) peptide [Liu, S. This includes [(2006) Mol. Pharm. 3:472-487] and polyamidoamines [Tomalia, DA, et al ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting intracellular PNPLA3 expression, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a complementary region to mRNA encoding patatin-like phospholipase domain 3 (PNPLA3), and the complementary region comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of Tables 2-11, 21, 24, 27, 30, 32, 33, 36, 37, 49, or 50.

2. It includes a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand is composed of nucleotides 677-721; 683-721; 773-817; 1185-1295; 1185-1241; 1202-1295; 1202-1241; 1255-1295; 1738-1792; 1901-1945; 1920-1945; 2108-2208; 2108-2166; 2108-2136; 2121-2166; 2121-22 Double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the nucleotide sequences 08;2169-2208;2176-2208; or 2239-2265, wherein the antisense strand comprises at least 15 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO:

2.

3. It includes a sense strand and an antisense strand forming a double-stranded region, the sense strand being the nucleotides of SEQ ID NO: 574-596; 677-699; 683-705; 699-721; 773-795; 795-817; 1185-1207; 1192-1214; 1202-1224; 1208-1230; 1209-1231; 1210-1232; 1211-1233; 1212-1234; 1213-1233; 1214-1234; 1214-1236; 1215-1237; 1216-1238; 1219-1237; 1219-1241; 125 5-1275; 1256-1276; 1257-1275; 1257-1277; 1258-1278; 1259-1279; 1260-1278; 1260-1280; 1261-1281; 1262-1282; 1263-1283; 1264-1282; 1264-12 84; 1265-1285; 1267-1285; 1267-1287; 1268-1290; 1269-1289; 1270-1290; 1271-1291; 1272-1292; 1273-1293; 1274-1294; 1275-1295; 1631-1653; 17 38-1760; 1739-1761; 1740-1760; 1740-1762; 1741-1763; 1744-1766; 1746-1766; 1750-1772; 1751-1773; 1752-1774; 1753-1775; 1754-1776; 1755-1 777; 1756-1778; 1757-1779; 1758-1780; 1759-1781; 1760-1782; 1761-1783; 1762-1782; 1762-1784; 1763-1785; 1764-1786; 1765-1787; 1766-1786; 1 766-1788; 1767-1787; 1768-1788; 1767-1789; 1769-1789; 1770-1788; 1770-1790; 1771-1791; 1772-1792; 1815-1837; 1901-1923; 1920-1942; 1923- 1945; 2112-2130; 2169-2191; 2171-2191; 2176-2198; 2177-2199; 2178-2200; 2179-2201; 2180-2202; 2181-2203; 2183-2205; 2184-2206; 2186-2208;A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising at least 15 consecutive nucleotides differing by three or fewer nucleotides from any one of the nucleotide sequences 2239-2261; 2241-2263; 2242-2264; or 2243-2265, wherein the antisense strand comprises at least 15 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO:

2.

4. The antisense chains are AD-517197.2; AD-517258.2; AD-516748.2; AD-516851.2; AD-519351.2; AD-519754.2; AD-519828.2; AD-520018.2; AD-520035.2; AD-520062. 2;AD-520064.2;AD-520065.2;AD-520067.2;AD-75289.2;AD-520069.2;AD -520099.2;AD-67575.7;AD-520101.2;AD-1193323.1;AD-1193344.1;AD-11 A dsRNA agent according to any one of claims 1 to 3, comprising one of the nucleotide sequences of the antisense strand of a double helix selected from the group consisting of 93350.1; AD-1193365.1; AD-1193379.1; AD-1193407.1; AD-1193421.1; AD-1193422.1; AD-1193429.1; AD-1193437.1; AD-1193443.1; AD-1193471.1; AD-1193481.1; and AD-67605.7, and at least 15 consecutive nucleotides that differ by 3 nucleotides or less.

5. The antisense chains are AD-519345.1, AD-519346.1, AD-519347.1, AD-67554.7, AD-519752.3, AD-1010731.1, AD-1010732.1, AD-519343.1, AD-519344.1, AD-519349.1, and AD-519350. 1, AD-519753.2, AD-519932.1, AD-519935.2, AD-520018.6, AD-517837.2, AD-80563 5.2, AD-519329.2, AD-520063.2, AD-519757.2, AD-805631.2, AD-516917.2, AD-516 A dsRNA agent according to any one of claims 1 to 3, comprising one of the nucleotide sequences of the antisense strand of a double helix selected from the group consisting of 828.2, AD-518983.2, AD-805636.2, AD-519754.7, AD-520062.2, AD-67575.9, AD-518923.3, AD-520053.4, AD-519667.2, AD-519773.2, AD-519354.2, AD-520060.4, AD-520061.4, AD-1010733.2, and AD-1010735.2, and at least 15 consecutive nucleotides that differ by 3 nucleotides or less.

6. The sensor includes a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand contains nucleotides 1200-1250, 1205-1250, 11210-1250, 1200-1245, 1200-1240, 1200-1235, 1200-1237, 1205-1237, 1210-1232, 1212-1237, 1212-1234, 1250-1300, 1255-1300, 1260-1300, 1250-1295, 1250-1390, 1250-1285, and 1250-1282 of SEQ ID NO:

1. Double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of intracellular patatin-like phospholipase domain-containing 3 (PNPLA3), comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences 1255-1282, 1260-1282, 1262-1300, 1262-1295, 1262-1390, and 1262-1282, wherein the antisense strand comprises at least 15 consecutive nucleotides derived from the corresponding nucleotide sequence of SEQ ID NO:

2.

7. A dsRNA agent according to any one of claims 1 to 6, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from one of the nucleotide sequences of the antisense strand of a double helix selected from the group consisting of AD-519351, AD-519345, AD-1193350, AD-1193365, AD-1193437; and AD-519347.

8. The dsRNA agent according to any one of claims 1 to 7, wherein the dsRNA agent comprises at least one modified nucleotide.

9. A dsRNA agent according to any one of claims 1 to 8, wherein substantially all of the nucleotides of the sense strand are modified; substantially all of the nucleotides of the antisense strand are modified; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified.

10. A dsRNA agent according to any one of claims 1 to 9, wherein all nucleotides of the sense strand contain modifications; all nucleotides of the antisense strand contain modifications; or all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

11. At least one of the modified nucleotides is a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a lock nucleotide, an unlock nucleotide, a conformation-fixed nucleotide, a restricted ethyl nucleotide, a debasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, or a morpholino nucleotide. A dsRNA agent according to any one of claims 8 to 10, selected from the group consisting of ocides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, thermally unstable nucleotides, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof.

12. A dsRNA agent according to any one of claims 8 to 10, wherein the nucleotide modification is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol; and combinations thereof.

13. The dsRNA according to any one of claims 8 to 10, wherein at least one of the modified nucleotides is selected from the group consisting of deoxyribonucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), and vinyl phosphonate nucleotides; and combinations thereof.

14. The dsRNA according to any one of claims 8 to 10, wherein at least one of the modifications on the nucleotide is a thermally unstable nucleotide modification.

15. The dsRNA according to claim 14, wherein the thermally unstable nucleotide modification is selected from the group consisting of debasic modification; mismatch with opposing nucleotide in the double helix; and unstable sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

16. A dsRNA agent according to any one of claims 1 to 15, wherein the double-stranded region is 19 to 30 nucleotide pairs long.

17. The dsRNA agent according to claim 16, wherein the double-stranded region has a length of 19 to 25 nucleotide pairs.

18. The dsRNA agent according to claim 16, wherein the double-stranded region has a length of 19 to 23 nucleotide pairs.

19. The dsRNA agent according to claim 16, wherein the double-stranded region has a length of 23 to 27 nucleotide pairs.

20. The dsRNA agent according to claim 16, wherein the double-stranded region has a length of 21 to 23 nucleotide pairs.

21. A dsRNA agent according to any one of claims 1 to 20, wherein each chain is independently 30 nucleotides or less in length.

22. A dsRNA agent according to any one of claims 1 to 21, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

23. The dsRNA agent according to any one of claims 1 to 22, wherein the complementary region is at least 17 nucleotides long.

24. A dsRNA agent according to any one of claims 1 to 23, wherein the complementary region has a length between 19 and 23 nucleotides.

25. A dsRNA agent according to any one of claims 1 to 24, wherein the complementary region is 19 nucleotides long.

26. A dsRNA agent according to any one of claims 1 to 25, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

27. A dsRNA agent according to any one of claims 1 to 25, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

28. A dsRNA agent according to any one of claims 1 to 27, further comprising a ligand.

29. The dsRNA agent according to claim 28, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

30. The dsRNA agent according to claim 28 or 29, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

31. The dsRNA agent according to any one of claims 28 to 30, wherein the ligand is one or more GalNAc derivatives conjugated via a monovalent, divalent, or trivalent branched linker.

32. The ligand is 【Chemistry 1】 The dsRNA agent according to claim 30 or 31.

33. The following diagram 【Chemistry 2】 The dsRNA agent according to claim 32, which is conjugated with a ligand shown in, where X is O or S.

34. The dsRNA agent according to claim 33, wherein X is O.

35. A dsRNA agent according to any one of claims 1 to 34, further comprising at least one phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage.

36. The dsRNA agent according to claim 35, wherein a phosphorothioate nucleotide linkage or a methylphosphonate nucleotide linkage is located at the 3' end of one strand.

37. The dsRNA agent according to claim 36, wherein the chain is an antisense chain.

38. The dsRNA agent according to claim 36, wherein the strand is a sense strand.

39. The dsRNA agent according to claim 35, wherein a phosphorothioate nucleotide linkage or a methylphosphonate nucleotide linkage is located at the 5' end of one strand.

40. The dsRNA agent according to claim 39, wherein the strand is an antisense strand.

41. The dsRNA agent according to claim 39, wherein the strand is a sense strand.

42. The dsRNA agent according to claim 35, wherein phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages are present at both the 5' and 3' ends of a single strand.

43. The dsRNA agent according to claim 42, wherein the strand is an antisense strand.

44. A dsRNA agent according to any one of claims 1 to 43, wherein the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

45. Cells containing the dsRNA agent according to any one of claims 1 to 44.

46. A pharmaceutical composition for inhibiting the expression of a gene encoding a patatin-like phospholipase domain 3 (PNPLA3), comprising a dsRNA agent according to any one of claims 1 to 44.

47. The pharmaceutical composition according to claim 46, wherein the dsRNA agent is in a non-buffer.

48. The pharmaceutical composition according to claim 47, wherein the non-buffer solution is physiological saline or water.

49. The pharmaceutical composition according to claim 46, wherein the dsRNA agent is in a buffer solution.

50. The pharmaceutical composition according to claim 49, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

51. The pharmaceutical composition according to claim 50, wherein the buffer solution is phosphate-buffered saline (PBS).

52. A method for inhibiting the expression of the intracellular patatin-like phospholipase domain-containing 3 (PNPLA3) gene, comprising contacting cells with a dsRNA agent according to any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 48 to 51, thereby inhibiting the expression of the intracellular PNPLA3 gene.

53. The method according to claim 52, wherein the cell is a cell inside the target.

54. The method according to claim 53, wherein the subject is a human.

55. The method according to claim 52, wherein the subject has PNPLA3-related disorders.

56. The method according to claim 55, wherein the PNPLA3-related disorder is selected from the group consisting of fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, and non-alcoholic fatty liver disease (NAFLD).

57. The method according to any one of claims 52 to 56, wherein contact of cells with a dsRNA agent inhibits PNPLA3 expression by at least 50%, 60%, 70%, 80%, 90%, or 95%.

58. The method according to any one of claims 52 to 57, wherein inhibiting the expression of PNPLA3 reduces the PNPLA3 protein level in the serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

59. A method for treating a subject having a disorder that would benefit from reduced expression of patatin-like phospholipase domain 3 (PNPLA3), comprising administering to the subject a therapeutically effective amount of the dsRNA agent according to any one of claims 1 to 44, or the pharmaceutical composition according to any one of claims 46 to 51, thereby treating the subject having a disorder that would benefit from reduced expression of PNPLA3.

60. A method for preventing at least one symptom in a subject having a disorder that benefits from reduced expression of patatin-like phospholipase domain 3 (PNPLA3), comprising administering to the subject a prophylactically effective amount of the dsRNA agent according to any one of claims 1 to 44, or the pharmaceutical composition according to any one of claims 46 to 51, thereby preventing at least one symptom in the subject having a disorder that benefits from reduced expression of PNPLA3.

61. The method according to claim 59 or 60, wherein the failure is a PNPLA3-related failure.

62. The method according to claim 61, wherein the PNPLA3-related disorder is selected from the group consisting of fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, and non-alcoholic fatty liver disease (NAFLD).

63. The method according to claim 61, wherein the PNPLA3-related disorder is NAFLD.

64. The method according to claim 61, wherein the subject is a human.

65. The method according to claim 59 or 60, wherein administration of the drug to a subject causes a reduction in PNPLA3 protein accumulation.

66. The method according to any one of claims 59 to 65, wherein the dsRNA agent is administered to the subject in a dose of approximately 0.01 mg / kg to approximately 50 mg / kg.

67. The method according to any one of claims 59 to 66, wherein the dsRNA agent is administered subcutaneously to the subject.

68. The method according to any one of claims 59 to 67, further comprising determining the PNPLA3 level in a sample(s) derived from the subject.

69. The method according to claim 68, wherein the PNPLA3 level in the target sample(s) is the PNPLA3 protein level in the blood sample(s) or serum sample(s).

70. The method according to any one of claims 59 to 69, further comprising administering to the subject an additional therapeutic agent for the treatment of PNPLA3-related disorder.

71. A kit comprising a dsRNA agent according to any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 46 to 51.

72. A vial comprising a dsRNA agent according to any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 46 to 51.

73. A syringe comprising a dsRNA agent according to any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 46 to 51.

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