iRNA Compositions and Methods for Silencing ANGPTL4

Abstract

The present invention relates to double-stranded ribonucleic acid (dsRNA) compositions that target the angiopoietin-like 4 (ANGPTL4) gene, and methods of using the dsRNA compositions to inhibit expression of ANGPTL4 and to treat subjects who would benefit from reduced expression of ANGPTL4, e.g., subjects having an ANGPTL4-associated disease, disorder, or condition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 245,693, filed September 17, 2021, and U.S. Provisional Patent Application No. 63 / 317,480, filed March 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in XML format, and is hereby incorporated by reference in its entirety. The XML copy created on September 13, 2022 is named A108868_1280WO_SL.XML and is 649,788 bytes in size.

[0003] The present disclosure relates to the specific inhibition of expression of ANGPTL4. [Background technology]

[0004] Angiopoietin-like 4 (ANGPTL4) is a member of the angiopoietin-like gene family and encodes a protein (ANGPTL4) involved in the regulation of lipid metabolism, glucose homeostasis and insulin sensitivity. In humans, the ANGPTL4 gene is located on the short (p) arm of chromosome 19 at position 13.2. The encoded protein, i.e., ANGPTL4, is an inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk and nutrient partitioning. ANGPTL4 is expressed in many tissues, including liver and adipose tissue. Consistent with its role in regulating lipid metabolism, ANGPTL4 has been identified as a gene involved in the development of nonalcoholic steatohepatitis (NASH).

[0005] Hepatocytes, which form the liver parenchyma, are responsible for mobilizing lipids for energy and storing excess lipids in the form of lipid droplets (LDs), making the liver the primary organ responsible for lipid homeostasis. Increased accumulation of LDs is associated with many metabolic diseases and chronic fibroinflammatory liver diseases, such as hepatic fibrosis, NASH, and NAFLD. Summary of the Invention [Problem to be solved by the invention]

[0006] At present, there is no treatment for chronic fibroinflammatory liver disease.Standard treatment for subjects with chronic fibroinflammatory liver disease includes lifestyle modification and management of related comorbidities, such as hypertension, dyslipidemia, diabetes, obesity, etc.Therefore, since the number of patients with chronic fibroinflammatory liver disease has been gradually increasing over the past decade and is expected to increase, there is a need in the art for alternative treatment methods for subjects with chronic fibroinflammatory liver disease. [Means for solving the problem]

[0007] The present invention provides an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of angiopoietin-like 4 (ANGPTL4) gene. ANGPTL4 can be in a cell, for example, in a cell in a subject, for example, a human. The present invention also provides a method of using the iRNA composition of the present invention to treat subjects who will benefit from the inhibition of expression of ANGPTL4 gene and / or the inhibition or reduction of expression of ANGPTL4 gene, for example, subjects who suffer from or are susceptible to ANGPTL4-related diseases, such as chronic fibroinflammatory liver disease, obesity, or metabolic disorders (for example, primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, diabetes, or insulin resistance).

[0008] Thus, in one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 4 (ANGPTL4) in cells. The dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 by no more than 1, 2 or 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 by no more than 1, 2 or 3 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8.

[0009] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of angiopoietin-like 4 (ANGPTL4) in cells. The dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises a region of complementarity to the mRNA that codes for ANGPTL4, comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in Tables 2 or 3 by no more than 1, 2 or 3 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises a region of complementarity to the mRNA that codes for ANGPTL4, comprising at least 15 consecutive nucleotides of any one of the antisense sequences listed in Tables 2 or 3.

[0010] In one embodiment, the region of complementarity is selected from nucleotides 26-48, 35-57, 42-64, 49-71, 56-78, 63-85, 76-98, 83-105, 122-144, 170-192, 179-201, 186-208, 276-298, 284-306, 294-316, 303-325, 310-332, 318-340, 486-508, 493-515, 503-525, 510-532, 519-541, 526-548, 533-555, 540-562, 548-570, 567-589, 576-598, 578-599, 580-599, 590-600, 592-601, 594-602, 596-603, 598-604, 598-605, 599-606, 599-607, 599-608, 601-602, 602-603, 603-604, 604-605, 605-606, 606-607, 607-608, 608-609, 610-611, 611-612, 612-613, 613-614, 614-615, 615-616, 616-617, 617-618, 618-61 89~611, 596~618, 606~628, 615~637, 623~645, 630~652, 639~661, 646~668, 662~684, 669~691, 678~700, 693~715, 702~724, 709~731, 716~738, 746~7 68, 755~777, 780~802, 787~809, 794~816, 818~840, 827~849, 836~858, 843~865, 850~872, 858~880, 865~887, 874~896, 881~903, 888~910, 895~917, 90 5~927, 948~970, 955~977, 962~984, 969~991, 1020~1042, 1032~1054, 1041~1063, 1050~1072, 1075~1097, 1083~1105, 1092~1114, 1155~1177, 1162~1 184, 1169-1191, 1176-1198, 1185-1207, 1193-1215, 1200-1222, 1212-1234, 1240-1262, 1249-1271, 1286-1308, 1293-1315, 1300-1322, 1307-1329, 1 315~1337, 1328~1350, 1337~1359, 1348~1370, 1355~1377, 1362~1384, 1373~1395, 1406~1428, 1418~1440, 1425~1447, 1435~1457, 1446~1468, 1453~ 1475, 1487~1509, 1494~1516, 1502~1524, 1510~1532, 1519~1541, 1527~1549, 1572~1594, 1580~1602, 1587~1609, 1597~1619, 1639~1661, 1648~1670,1661~1683, 1668~1690, 1677~1699, 1685~1707, 1708~1730, 1715~1737, 1722~1744, 1743~1765, 1766~1788, 1773~1795, 1780~1802, 1787~1809, 1817~1839, 1824~1846, 1836~1858, 1845~1867, 185 4 to 1876, 1880 to 1902, 1888 to 1910, 1895 to 1917, 1905 to 1927, 1912 to 1934, 1919 to 1941, or 1926 to 1948. The present invention includes at least 15 consecutive nucleotides that differ by 1, 2, or 3 nucleotides or less from the perfectly complementary nucleotides of any 15 consecutive nucleotides located within the following sequence:

[0011] In some embodiments, the region of complementarity is selected from nucleotides 26-48, 35-57, 42-64, 49-71, 56-78, 63-85, 76-98, 83-105, 122-144, 170-192, 179-201, 186-208, 276-298, 284-306, 294-316, 303-325, 310-332, 318-340, 486-508, 493-515, 503-525, 510-532, 519-541, 526-548, 533-555, 540-562, 548-570, 567-589, 576-598, 589~611, 596~618, 606~628, 615~637, 623~645, 630~652, 639~661, 646~668, 662~684, 669~691, 678~700, 693~715, 702~724, 709~731, 716~738, 746~ 768, 755~777, 780~802, 787~809, 794~816, 818~840, 827~849, 836~858, 843~865, 850~872, 858~880, 865~887, 874~896, 881~903, 888~910, 895~917, 9 05~927, 948~970, 955~977, 962~984, 969~991, 1020~1042, 1032~1054, 1041~1063, 1050~1072, 1075~1097, 1083~1105, 1092~1114, 1155~1177, 1162~ 1184, 1169~1191, 1176~1198, 1185~1207, 1193~1215, 1200~1222, 1212~1234, 1240~1262, 1249~1271, 1286~1308, 1293~1315, 1300~1322, 1307~1329, 1315~1337, 1328~1350, 1337~1359, 1348~1370, 1355~1377, 1362~1384, 1373~1395, 1406~1428, 1418~1440, 1425~1447, 1435~1457, 1446~1468, 1453~ 1475, 1487~1509, 1494~1516, 1502~1524, 1510~1532, 1519~1541, 1527~1549, 1572~1594, 1580~1602, 1587~1609, 1597~1619, 1639~1661, 1648~1670,1661~1683, 1668~1690, 1677~1699, 1685~1707, 1708~1730, 1715~1737, 1722~1744, 1743~1765, 1766~1788, 1773~1795, 1780~1802, 1787~1809, 1817~1839, 1824~1846, 1836~1858 , 1845-1867, 1854-1876, 1880-1902, 1888-1910, 1895-1917, 1905-1927, 1912-1934, 1919-1941, or 1926-1948.

[0012] In one embodiment, the sense strand is selected from nucleotides 28 to 48, 37 to 57, 44 to 64, 51 to 71, 58 to 78, 65 to 85, 78 to 98, 85 to 105, 124 to 144, 172 to 192, 181 to 201, 188 to 208, 278 to 298, 286 to 306, 296 to 316, 305 to 325, 312 to 332, 320 to 340, 488 to 508, 495 to 515, 505 to 525, 512 to 532, 521 to 541, 528 to 548, 535 to 555, 542 to 562, 550 to 570, 569 to 589, 578 to 598, 591 to 592, 593 to 594, 595 to 596, 597 to 598, 599 to 599, 591 to 599, 592 to 593, 594 to 595, 595 to 596, 597 to 598, 599 to 599, 598 to 599, 591 ... ~611, 598~618, 608~628, 617~637, 625~645, 632~652, 641~661, 648~668, 664~684, 671~691, 680~700, 695~715, 704~724, 711~731, 718~738, 748~768 , 757~777, 782~802, 789~809, 796~816, 820~840, 829~849, 838~858, 845~865, 852~872, 860~880, 867~887, 876~896, 883~903, 890~910, 897~917, 907 ~927, 950~970, 957~977, 964~984, 971~991, 1022~1042, 1034~1054, 1043~1063, 1052~1072, 1077~1097, 1085~1105, 1094~1114, 1157~1177, 1164~11 84, 1171-1191, 1178-1198, 1187-1207, 1195-1215, 1202-1222, 1214-1234, 1242-1262, 1251-1271, 1288-1308, 1295-1315, 1302-1322, 1309-1329, 1 317~1337, 1330~1350, 1339~1359, 1350~1370, 1357~1377, 1364~1384, 1375~1395, 1408~1428, 1420~1440, 1427~1447, 1437~1457, 1448~1468, 1455~ 1475, 1489~1509, 1496~1516, 1504~1524, 1512~1532, 1521~1541, 1529~1549, 1574~1594, 1582~1602, 1589~1609, 1599~1619, 1641~1661, 1650~1670,1663~1683, 1670~1690, 1679~1699, 1687~1707, 1710~1730, 1717~1737, 1724~1744, 1745~1765, 1768~1788, 1775~1795, 1782~1802, 1789~1809, 1819~1839, 1826~1846, 1838~1858, 1847~1867, 1856~1876, 1882~1902, 1890~1910 , 1897-1917, 1907-1927, 1914-1934, 1921-1941, or 1928-1948, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2 or 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 2, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2 or 3 nucleotides from the complementary nucleotide sequence of SEQ ID NO: 2.

[0013] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0014] In one embodiment, substantially all of the nucleotides in the sense strand contain a modification. In another embodiment, substantially all of the nucleotides in the antisense strand contain a modification. In yet another embodiment, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand contain a modification.

[0015] In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent is provided for inhibiting the expression of angiopoietin-like 4 (ANGPTL4) in cells.The dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 by 1, 2 or 3 nucleotides or less, the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 by 1, 2 or 3 nucleotides or less, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand which form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0016] In one embodiment, substantially all of the nucleotides in the sense strand contain a modification. In another embodiment, substantially all of the nucleotides in the antisense strand contain a modification. In yet another embodiment, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand contain a modification.

[0017] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide. modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5' phosphates, nucleotides containing 5' phosphate mimics, glycol modified nucleotides, and 2-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0018] In one embodiment, the nucleotide modifications are 2'-O-methyl and / or 2'-fluoro modifications.

[0019] The region of complementarity may be at least 17 nucleotides in length, 19-30 nucleotides in length, 19-25 nucleotides in length, or 21-23 nucleotides in length.

[0020] Each strand can be 30 nucleotides or less in length, for example, each strand is independently 19-30 nucleotides in length, each strand is independently 19-25 nucleotides in length, each strand is independently 21-23 nucleotides in length.

[0021] The dsRNA may comprise at least one strand comprising a 3' overhang of at least one nucleotide, or at least one strand comprising a 3' overhang of at least two nucleotides.

[0022] In some embodiments, the dsRNA agent further comprises a ligand.

[0023] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of a dsRNA agent.

[0024] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0025] In one embodiment, the ligand is

[0026] [ka] It is.

[0027] In one embodiment, the dsRNA agent has the following formula:

[0028] [ka] wherein X is O or S. The ligand is conjugated as shown in FIG.

[0029] In one embodiment, X is O.

[0030] In one embodiment, the region of complementarity comprises any one of the antisense sequences in Tables 2 or 3.

[0031] In one embodiment, the invention provides a duplex for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell, the dsRNA agent comprising a sense strand complementary to an anti-strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding ANGPTL4, each strand being about 14 to about 30 nucleotides in length, the dsRNA agent comprising a sequence represented by formula (Ii): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense:3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(Ii) [In the formula, i, j, k and l each independently represent 0 or 1; p, p', q and q' each independently represents 0 to 6; each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence comprising 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; Each n may or may not be present p , n p ',n q and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N b The modification on Y is different from the modification on N. b The modification on Y' is different from the modification on Y', and the sense strand is conjugated to at least one ligand.

[0032] In one embodiment, i is 0, j is 0, i is 1, j is 1, i and j are both 0, or i and j are both 1. In another embodiment, k is 0, l is 0, k is 1, l is 1, k and l are both 0, or k and l are both 1.

[0033] In one embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0034] In one embodiment, the YYY motif is present at or near the cleavage site on the sense strand, eg, a Y'Y'Y' motif is present at positions 11, 12, and 13 on the antisense strand from the 5' end.

[0035] In one embodiment, formula (Ii) may be represented by formula (Ij): Sense:5'n p -N a -YYY-N a -n q 3' Antisense:3'n p -N a -Y'Y'Y'-N a’ -n q’ 5'(Ij) It is represented by:

[0036] In another embodiment, formula (Ii) has formula (Ik): Sense: 5' n p -N a -YYY-Nb -ZZZ-N a -n q 3' Antisense: 3' n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(Ik) [In the formula, each N b and N b ' independently represents an oligonucleotide containing 1 to 5 modified nucleotides. It is represented by:

[0037] In another embodiment, formula (Ii) has formula (Il): Sense:5'n p -N a -XXX-N b -YYY-N a -n q 3' Antisense:3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5'(Il) [In the formula, each N b and N b ' independently represents an oligonucleotide containing 1 to 5 modified nucleotides. It is represented by:

[0038] In another embodiment, formula (Ii) has formula (Im): Sense:5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense:3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(Im) [In the formula, each N b and N b ' independently represents an oligonucleotide containing 1 to 5 modified nucleotides, a and N a ' independently represents an oligonucleotide containing 2 to 10 modified nucleotides. It is represented by:

[0039] The region of complementarity may be at least 17 nucleotides in length, 19-30 nucleotides in length, 19-25 nucleotides in length, or 21-23 nucleotides in length.

[0040] Each strand can be 30 nucleotides or less in length, for example, each strand is independently 19-30 nucleotides in length.

[0041] In one embodiment, the modification on the nucleotide is selected from the group consisting of LNA, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-fluoro, 2'-O-methyl, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

[0042] In one embodiment, the modification on the nucleotide is a 2'-O-methyl or a 2'-fluoro modification.

[0043] In one embodiment, Y' is a 2'-O-methyl or 2'-fluoro modified nucleotide.

[0044] In one embodiment, at least one strand of the dsRNA agent can include a 3' overhang of at least one nucleotide, or a 3' overhang of at least two nucleotides.

[0045] In one embodiment, the dsRNA agent may further include at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0046] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0047] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0048] In one embodiment, the strand is the antisense strand, hi another embodiment, the strand is the sense strand.

[0049] In one embodiment, phosphorothioate or methylphosphonate internucleotide linkages are present at both the 5' and 3' ends of one strand.

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

[0051] In one embodiment, p'>0. In another embodiment, p'=2. In one embodiment, q'=0, p=0, q=0 and p' overhanging nucleotides are complementary to the target mRNA. In another embodiment, q'=0, p=0, q=0 and p' overhanging nucleotides are non-complementary to the target mRNA.

[0052] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0053] In one embodiment, at least one n p In another embodiment, every n' is linked to an adjacent nucleotide via a phosphorothioate linkage. p ' is linked to the adjacent nucleotide via a phosphorothioate linkage.

[0054] In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

[0055] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of a dsRNA agent.

[0056] In one embodiment, the Ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached via a monovalent, divalent or trivalent branched linker.

[0057] In one embodiment, the ligand is

[0058] [ka] It is.

[0059] In one embodiment, the dsRNA agent has the following formula:

[0060] [ka] wherein X is O or S. The ligand is conjugated as shown in FIG.

[0061] In one embodiment, X is O.

[0062] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 2 and shown below.

[0063] [ka]

[0064] In some embodiments, the RNAi agent is, for example, uL96, as shown below:

[0065] [ka] The nucleic acid may contain a 3'-terminal L96 modified nucleotide such as (2'-O-methyluridine-3'-phosphate ((2S,4R)-1-[29-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-14,14-bis[[3-[[3-[[5-[[2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-1-oxopentyl]amino]propyl]amino]-3-oxopropoxy]methyl]-1,12,19,25-tetraoxo-16-oxa-13,20,24-triazanonacos-1-yl]-4-hydroxy-2-pyrrolidinyl)methyl ester).

[0066] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell. The dsRNA agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding ANGPTL4, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent has the formula (Ii): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense:3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(II) [In the formula, i, j, k and l each independently represent 0 or 1; p, p', q and q' each independently represents 0 to 6; each N aand N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence comprising 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; Each n may or may not be present p , n p ',n q and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N. b The modification on Y' is different from the modification on Y', and the sense strand is conjugated to at least one ligand.

[0067] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell. The dsRNA agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding ANGPTL4, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent has the formula (Ii): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense:3'n p '-N a'-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(II) [In the formula, i, j, k and l each independently represent 0 or 1; Each n may or may not be present p , n q and n q ' independently represents an overhanging nucleotide; p, q and q' each independently represents 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence comprising 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N. b The modification on Y' is different from the modification on Y', and the sense strand is conjugated to at least one ligand.

[0068] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell. The dsRNA agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding ANGPTL4, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent has the formula (Ii): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense:3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(II) [In the formula, i, j, k and l each independently represent 0 or 1; Each n may or may not be present p , n q and n q ' independently represents an overhanging nucleotide; p, q and q' each independently represents 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b' represents an oligonucleotide sequence comprising 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N. b ' is different from the modification on Y'], and the sense strand is conjugated to at least one ligand, which is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0069] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell. The dsRNA agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding ANGPTL4, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent has the formula (Ii): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense:3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(Ii) [In the formula, i, j, k and l each independently represent 0 or 1; Each n may or may not be present p , nq and n q ' independently represents an overhanging nucleotide; p, q and q' each independently represents 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence comprising 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N. b ' is different from the modification on Y'], the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to at least one ligand, which is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0070] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell. The dsRNA agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding ANGPTL4, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent has the formula (Ii): Sense:5'n p -Na -YYY-N a -n q 3' Antisense:3'n p '-N a '-Y'Y'Y'-N a '-n q '5'(Ij) [In the formula, Each n may or may not be present p , n q and n q ' independently represents an overhanging nucleotide; p, q and q' each independently represents 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl and / or 2'-fluoro modifications, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to at least one ligand, which is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0071] In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell is provided. The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, 3, 5 or 7, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, 4, 6 or 8, substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' terminus and substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' terminus and two phosphorothioate internucleotide linkages at the 3' terminus, and the sense strand is conjugated at the 3' terminus to one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8, substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end, and substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand is conjugated at the 3' end to one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0072] In one embodiment, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0073] In one embodiment, the region of complementarity comprises any one of the antisense sequences listed in Tables 2 or 3.

[0074] In one embodiment, the drug is AD-1663650、AD-1663659、AD-1663666、AD-1663673、AD-1663680、AD-1663687、AD-1663700、AD-1663707、AD-1663725、AD-1663726、 AD-1663735、AD-1663742、AD-1663789、AD-1663797、AD-1663807、AD-1663816、AD-1663823、AD-1663831、AD-1663898、AD-1663905、AD-1663915、AD- 1663922、AD-1663931、AD-1663938、AD-1663945、AD-1663952、AD-1663960、AD-1663973、AD-1663982、AD-1663995、AD-1664002、AD-1664012、AD-166 4021、AD-1664029、AD-1664036、AD-1664045、AD-1664052、AD-1664067、AD-1664074、AD-1664083、AD-1664096、AD-1664105、AD-1664112、AD-166411 9、AD-1664137、AD-1664146、AD-1664151、AD-1664158、AD-1664165、AD-1664169、AD-1664178、AD-1664187、AD-1664194、AD-1664201、AD-1664209 D-1664216、AD-1664225、AD-1664232、AD-1664239、AD-1664246、AD-1664256、AD-1664266、AD-1664273、AD-1664280、AD-1664287、AD-1664302、AD-1 664314、AD-1664323、AD-1664332、AD-1664354、AD-1664362、AD-1664371、AD-1664395、AD-1664402、AD-1664409、AD-1664416、AD-1664425、AD-1664 433、AD-1664440、AD-1664452、AD-1664460、AD-1664469、AD-1664479、AD-1664486、AD-1664493、AD-1664500、AD-1664508、AD-1664521、AD-1664530、AD-1664541, AD-1664548, AD-1664555, AD-1664566, AD-1664579, AD-1664589 , AD-1664596, AD-1664606, AD-1664617, AD-1664624, AD-1664646, AD-166465 3, AD-1664661, AD-1664669, AD-1664678, AD-1664686, AD-1664696, AD-16647 04, AD-1664711, AD-1664721, AD-1664724, AD-1664733, AD-1664746, AD-1664 753, AD-1664762, AD-1664770, AD-1664773, AD-1664780, AD-1664787, AD-1664788, AD-1664791, AD-1664798, AD-1664805, AD-1664812, AD-1664822, AD-1664829, AD-1664841, AD-1664850, AD-1664859, AD-1664860, AD-1664868, AD-1664875, AD-1664885, AD-1664892, AD-1664899, or AD-1664906.

[0075] In one embodiment, the sense and antisense strands comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of any one of the agents listed in Tables 2 or 3.

[0076] In various embodiments of the dsRNA agent, the dsRNA agent targets a hotspot region of the mRNA encoding ANGPTL4.

[0077] In another embodiment, the invention provides dsRNA agents targeting hotspot regions of myosin regulatory light chain-interacting protein (ANGPTL4) mRNA.

[0078] The present invention also provides cells, vectors and pharmaceutical compositions that contain any of the dsRNA agents of the present invention.The dsRNA agent can be formulated in unbuffered solution, such as saline or water, or in buffer solution, such as acetate, citrate, prolamin, carbonate or phosphate, or a combination thereof.In one embodiment, the buffer solution is phosphate buffered saline (PBS).

[0079] In one embodiment, the present invention provides a method for inhibiting the expression of Angiopoietin-like 4 (ANGPTL4) in a cell. The method comprises contacting the cell with a dsRNA agent or pharmaceutical composition of the present invention, thereby inhibiting the expression of ANGPTL4 in the cell.

[0080] The cell may be in a subject, such as a human subject.

[0081] In one embodiment, ANGPTL4 expression is inhibited by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or below the level of detection of ANGPTL4 expression relative to control levels.

[0082] In one embodiment, the human subject suffers from ANGPTL4-related disease, disorder and condition.In one embodiment, the ANGPTL4-related disease, disorder and condition is chronic fibroinflammatory liver disease.In one embodiment, the chronic fibroinflammatory liver disease is selected from the group consisting of liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related liver cirrhosis, drug-induced liver injury and hepatocyte necrosis.

[0083] In one embodiment, ANGPTL4-related disease, disorder or condition is obesity.In one embodiment, ANGPTL4-related disease, disorder or condition is metabolic disorder.In certain embodiments, metabolic disorder is primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, type 1 diabetes, type 2 diabetes, prediabetes or insulin resistance.

[0084] In one embodiment, the present invention provides a method for inhibiting the expression of ANGPTL4 in a subject.The method comprises administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby inhibiting the expression of ANGPTL4 in the subject.

[0085] In one embodiment, the present invention provides the method for inhibiting the accumulation of lipid droplets in the liver of the subject suffering from ANGPTL4-related disease, disorder or condition.The method includes administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby inhibiting the accumulation of lipid droplets in the liver of the subject suffering from ANGPTL4-related disease, disorder or condition.

[0086] In another embodiment, the present invention provides the method for treating the subject suffering from ANGPTL4-related disease, disorder or condition.The method comprises administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby treating the subject suffering from ANGPTL4-related disease, disorder or condition.

[0087] In another embodiment, the present invention provides the method for preventing at least one symptom in the subject with disease, disorder or condition that will benefit from the reduction of the expression of ANGPTL4 gene.The method comprises administering to the subject a preventive effective amount of the agent of the dsRNA agent or pharmaceutical composition of the present invention, thereby preventing at least one symptom in the subject with disease, disorder or condition that will benefit from the reduction of the expression of ANGPTL4 gene.

[0088] In another aspect, the present invention provides the method for reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in subject.The method comprises administering to subject the preventively effective amount or therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in subject.

[0089] In another aspect, the present invention provides the method for reducing the risk of developing chronic liver disease in subjects with steatosis.The method comprises administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby reducing the risk of developing chronic liver disease in subjects with steatosis.

[0090] In yet another embodiment, the present invention provides a method for inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis.The method comprises administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

[0091] In one embodiment, the present invention provides the method for inhibiting the accumulation of lipid droplets in the liver of the subject suffering from ANGPTL4-related disease, disorder or condition.The method includes administering to the subject a therapeutically effective amount of the present dsRNA agent or pharmaceutical composition and the dsRNA agent that targets HSD17B13 gene, or the pharmaceutical composition that comprises the dsRNA agent that targets HSD17B13 gene, thereby inhibiting the accumulation of lipid droplets in the liver of the subject suffering from ANGPTL4-related disease, disorder or condition.

[0092] In another embodiment, the present invention provides the method for treating the subject suffering from ANGPTL4-related disease, disorder or condition.The method includes administering to the subject a therapeutically effective amount of the present invention dsRNA agent or pharmaceutical composition and the dsRNA agent that targets HSD17B13 gene or the pharmaceutical composition that comprises the dsRNA agent that targets HSD17B13 gene, thereby treating the subject suffering from ANGPTL4-related disease, disorder or condition.

[0093] In another embodiment, the present invention provides the method for preventing at least one symptom in the subject with disease, disorder or condition that will benefit from the reduction of the expression of ANGPTL4 gene.The method comprises administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention and the dsRNA agent that targets HSD17B13 gene, or the pharmaceutical composition that comprises the dsRNA agent that targets HSD17B13 gene, thereby preventing at least one symptom in the subject with disease, disorder or condition that will benefit from the reduction of the expression of ANGPTL4 gene.

[0094] In another embodiment, the present invention provides the method for reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in subject.The method includes administering to subject a therapeutically effective amount of the present dsRNA agent or pharmaceutical composition and the dsRNA agent that targets HSD17B13 gene or the pharmaceutical composition that comprises the dsRNA agent that targets HSD17B13 gene, thereby reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in subject.

[0095] In another aspect, the present invention provides the method for reducing the risk of developing chronic liver disease in subjects with steatosis.The method includes administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention and the dsRNA agent that targets HSD17B13 gene or the pharmaceutical composition that comprises the dsRNA agent that targets HSD17B13 gene, thereby reducing the risk of developing chronic liver disease in subjects with steatosis.

[0096] In another aspect, the present invention provides the method for inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis.The method comprises administering to the subject a therapeutically effective amount of the present invention dsRNA agent or pharmaceutical composition and the dsRNA agent that targets HSD17B13 gene or the pharmaceutical composition that comprises the dsRNA agent that targets HSD17B13 gene, thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

[0097] In one embodiment, administering a dsRNA agent or pharmaceutical composition to a subject results in decreased ANGPTL4 enzyme activity, decreased ANGPTL4 protein accumulation, decreased HSD17B13 enzyme activity, decreased HSD17B13 protein accumulation, and / or decreased fat accumulation and / or spread of lipid droplets in the subject's liver.

[0098] In one embodiment, the ANGPTL4-related disease, disorder or condition is a chronic fibroinflammatory liver disease.

[0099] In one embodiment, the chronic fibroinflammatory liver disease is selected from the group consisting of fat accumulation in the liver, liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, and hepatocellular necrosis.

[0100] In one embodiment, the chronic fibroinflammatory liver disease is non-alcoholic steatohepatitis (NASH).

[0101] In one embodiment, the subject is obese.

[0102] In one embodiment, ANGPTL4-related disease, disorder or condition is obesity.In one embodiment, ANGPTL4-related disease, disorder or condition is metabolic disorder.In certain embodiments, metabolic disorder is primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, type 1 diabetes, type 2 diabetes, prediabetes or insulin resistance.

[0103] In one embodiment, the methods and uses of the invention further comprise administering to the subject an additional therapeutic agent.

[0104] In certain embodiments, the subject may be administered a therapeutic amount of dsRNA, such as about 0.01 mg / kg to about 200 mg / kg. In other embodiments, the subject may be administered a therapeutic amount of dsRNA, such as about 0.01 mg / kg to about 500 mg / kg. In yet other embodiments, the subject may be administered a therapeutic amount of dsRNA of about 500 mg / kg or more.

[0105] In one embodiment, the dsRNA agent is administered to a subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

[0106] The agent is administered to the subject intravenously, intramuscularly or subcutaneously, hi one embodiment, the agent is administered to the subject subcutaneously.

[0107] In one embodiment, the methods and uses of the invention further comprise determining the level of ANGPTL4 in the subject.

[0108] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand which form a double-stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the agents in Table 2 or 3, and the antisense strand comprises the nucleotide sequence of any one of the agents in Table 2 or 3, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the dsRNA agent is conjugated to a ligand. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] The present invention provides an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of ANGPTL4 gene.ANGPTL4 gene can be in a cell, for example, in a subject, such as a human.The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of ANGPTL4 gene and to treat subjects who will benefit from inhibiting or reducing the expression of ANGPTL4 gene, for example, subjects who will benefit from reducing inflammation in the liver, for example, subjects who suffer from or are susceptible to ANGPTL4-related disease, disorder or condition, for example, subjects who suffer from or are susceptible to liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), liver cirrhosis, HCV-related liver cirrhosis, drug-induced liver injury and liver cell necrosis.

[0110] The iRNA of the present invention targeting ANGPTL4 is about 30 nucleotides in length or less, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 1 The ANGPTL4 gene may include an RNA strand (antisense strand) having a region that is 9 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 and that is substantially complementary to at least a portion of an mRNA transcript of the ANGPTL4 gene.

[0111] In some embodiments, one or both strands of a double-stranded RNAi agent of the invention are 66 nucleotides or less in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, including a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the ANGPTL4 gene. In some embodiments, the iRNA agent having a longer antisense strand may include a second RNA strand (sense strand) that is 20-60 nucleotides in length, such that the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0112] The use of the iRNA agents described herein allows for the targeted degradation of the mRNA of the ANGPTL4 gene in a mammal.

[0113] In particular, very low doses of iRNA can specifically and efficiently mediate RNA interference (RNAi) to significantly inhibit the expression of ANGPTL4 gene.Therefore, the method and composition comprising these iRNAs are useful for treating subjects who will benefit from inhibiting or reducing the expression of ANGPTL4 gene, such as subjects who suffer from or are prone to ANGPTL4-related diseases, disorders or conditions, such as subjects who suffer from or are prone to liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), liver cirrhosis, HCV-related liver cirrhosis, drug-induced liver injury and liver cell necrosis.

[0114] The following detailed description discloses how to make and use compositions containing iRNA to inhibit expression of the ANGPTL4 gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibition and / or reduction of expression of this gene.

[0115] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. In addition, whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0116] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0117] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0118] The term "about" is used herein to mean within the typical tolerance range in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is present before a series of numbers or ranges, it will be understood that "about" can modify each of the numbers or ranges in the series.

[0119] The term "at least" before a number or a series of numbers is understood to include the number next to the term "at least" and all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20 or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0120] As used herein, "no more than" or "or less" is understood as the value next to the phrase and the logical lower value or integer, up to zero, as is logical from the context. For example, a duplex with an overhang of "2 or less nucleotides" has 2, 1 or 0 nucleotide overhangs. When "or less" is present before a series of numbers or a range, it is understood that "or less" can modify each of the numbers in the series or range. As used herein, a range includes both upper and lower limits.

[0121] As used herein, the term "at least about" refers to measurable values ​​such as parameters and amounts, and includes + / -20% variation from a particular value, such as + / -10%, + / -5% or + / -1%, as long as such variation is appropriate for carrying out the disclosed invention.For example, "at least about 25%" inhibition of the expression of ANGPTL4 gene means that the inhibition of the expression of ANGPTL4 gene can be measured to be any value of + / -20% of the particular 25%, i.e., 20%, 30%, or any intermediate value between 20% and 30%.

[0122] As used herein, "control level" refers to the expression level of a gene, or the expression level of an RNA molecule, or the expression level of one or more proteins or protein subunits in a non-modulated cell, tissue or system identical to the cell, tissue or system in which the RNAi agent described herein is expressed. The cell, tissue or system in which the RNAi agent is expressed has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, or 5-fold or more expression of the gene, RNA and / or protein described above than the expression observed in the absence of the RNAi agent. The % and / or fold difference can be expressed relative to the control level, for example,

[0123]

number

[0124] As used herein, a method of detection can include determining that the amount of analyte present is below the level of detection of the method.

[0125] In the event of a conflict between a depicted target site and the nucleotide sequence for the sense or antisense strand, the depicted sequence controls.

[0126] In the event of a conflict between a chemical structure and a chemical name, the chemical structure takes precedence.

[0127] Unless otherwise specified, the term "ANGPTL4," also known as "angiopoietin-like 4," "angiopoietin-like protein 4," "ARP4," "HARP," "HFARP," "PGAR," "FIAF," "ARP4," "Pp1158," "NL2," "UNQ171," "TGQTL," "peroxisome proliferator-activated receptor (PPAR)," "gamma-induced angiopoietin-related protein," "hepatic angiopoietin-related protein," and "starvation-induced adipose factor," refers to the well-known gene encoding angiopoietin-like 4 protein from any vertebrate or mammalian source, including, without limitation, human, bovine, chicken, rodent, mouse, rat, pig, sheep, primate, monkey, and guinea pig.

[0128] The term also refers to fragments and variants of native ANGPTL4 that maintain at least one in vivo or in vitro activity of native ANGPTL4. The term encompasses full-length unprocessed precursor forms of ANGPTL4, as well as mature forms that result from post-translational cleavage of the signal peptide and forms that result from proteolytic processing.

[0129] The human ANGPTL4 gene has seven exons. Four transcript variants of the human ANGPTL4 gene have already been identified. The nucleotide and amino acid sequence of the human ANGPTL4 transcript variant X1 can be found, for example, in GenBank reference sequence XM_005272484.3 (SEQ ID NO: 1, reverse complement SEQ ID NO: 2). The nucleotide and amino acid sequence of the human ANGPTL4 transcript variant X2 can be found, for example, in GenBank reference sequence XM_005272485.3 (SEQ ID NO: 3, reverse complement SEQ ID NO: 4). The nucleotide and amino acid sequence of the human ANGPTL4 transcript variant 3 can be found, for example, in GenBank reference sequence NM_001039667.3 (SEQ ID NO: 5, reverse complement SEQ ID NO: 6). The nucleotide and amino acid sequence of the human ANGPTL4 transcript variant 1 can be found, for example, in GenBank reference sequence NM_139314.3 (SEQ ID NO: 7, reverse complement SEQ ID NO: 8).

[0130] ANGPTL4 gene is located in chromosomal region 19p13.2. The nucleotide sequence of the genomic region of human chromosome containing ANGPTL4 gene can be found, for example, in Genome Reference Consortium Human Build 38 (also called human genome build38 or GRCh38), available at GenBank. The nucleotide sequence of the genomic region of human chromosome 19 containing ANGPTL4 gene can also be found, for example, in GenBank accession number NC_000019.10, which corresponds to nucleotides 8364129-8374373 of human chromosome 19.

[0131] There is one known transcript variant of the mouse ANGPTL4 gene. The nucleotide and amino acid sequence of mouse ANGPTL4 transcript variant 1 can be found, for example, in GenBank reference sequence NM_020581.2 (SEQ ID NO: 9, reverse complement SEQ ID NO: 10).

[0132] There is one known transcript variant of the rat ANGPTL4 gene. The nucleotide and amino acid sequence of rat ANGPTL4 transcript variant 1 can be found, for example, in GenBank reference sequence NM_199115.2 (SEQ ID NO: 11, reverse complement SEQ ID NO: 12).

[0133] There are two known transcript variants of the rhesus monkey ANGPTL4 gene.The nucleotide and amino acid sequence of the rhesus monkey ANGPTL4 transcript variant X1 can be found, for example, in GenBank reference sequence XM_028839189.1 (SEQ ID NO: 13, reverse complement SEQ ID NO: 14).The nucleotide and amino acid sequence of the rhesus monkey ANGPTL4 transcript variant X2 can be found, for example, in GenBank reference sequence XM_015122727.2 (SEQ ID NO: 15, reverse complement SEQ ID NO: 16).

[0134] There is one known transcript variant of the cynomolgus monkey ANGPTL4 gene. The nucleotide and amino acid sequence of the rhesus monkey ANGPTL4 transcript variant X1 can be found, for example, in GenBank reference sequence XM_005587832.2 (SEQ ID NO: 17, reverse complement SEQ ID NO: 18).

[0135] Further examples of ANGPTL4 mRNA sequences can be easily obtained using publicly available databases, such as GenBank, UniProt and OMIM.Further information on ANGPTL4 can be found, for example, at https: / / www.ncbi.nlm.nih.gov / gene / 51129.The term ANGPTL4 as used herein also refers to the variation of ANGPTL4 gene presented in clinical variant databases, such as http: / / www.ncbi.nlm.nih.gov / clinvar / ?term=ANGPTL4[gene].

[0136] The term "ANGPTL4" as used herein also refers to the specific polypeptide expressed in cells due to the naturally occurring DNA sequence variation of ANGPTL4 gene, for example, the single nucleotide polymorphism in ANGPTL4 gene.Many SNPs in ANGPTL4 gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0137] Angiopoietin-like 4 (ANGPTL4), also known as ARP4, HARP, HFARP, PGAR, FIAF, ARP4, Pp1158, NL2, UNQ171 or TGQTL, is a member of the angiopoietin-like gene family and encodes a protein (ANGPTL4) involved in the regulation of lipid metabolism, glucose homeostasis and insulin sensitivity. The ANGPTL4 gene is located on the short (p) arm of chromosome 19 at position 13.2. The encoded protein, i.e., ANGPTL4, is an inhibitor of lipoprotein lipase that modulates coronary atherosclerosis risk and nutrient partitioning. ANGPTL4 is expressed in many tissues, including liver and adipose tissue (Kersten, et al. (2000). Journal of Biological Chemistry, 275(37), 28488-28493).

[0138] ANGPTL4 contains an N-terminal coiled-coil domain and a C-terminal fibrinogen (FBN)-like domain (Kim et al. (2000) Biochem. 346: 603-610). The coiled-coil region of ANGPTL4 inhibits lipoprotein lipase-mediated triglyceride clearance. Lipoprotein lipase has a central role in lipoprotein metabolism, including maintaining lipoprotein levels in the blood. Consistent with its role in regulating lipid metabolism, ANGPTL4 was identified as a gene involved in the development of NASH. (Liu, et al (2021). International journal of clinical and experimental pathology, 14(5), 567). ANGPTL4 loss-of-function mutations (e.g., found in human subjects) and antibody inhibition of ANGPTL4 (e.g., found in mice and cynomolgus monkeys) have been associated with reduced levels of plasma triglycerides (Dewey, et al. (2016). New England Journal of Medicine, 374(12), 1123-1133). Furthermore, genetic inactivation of ANGPTL4 has been associated with improved glucose homeostasis, reduced risk of diabetes, and reduced atherosclerosis (Gusarova, et al. (2018). ] Nature communications, 9(1), 1-11; Singh, et al. (2020). bioRxiv.; Aryal, et al. (2018). JCI insight, 3(6)).

[0139] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of ANGPTL4 gene, such as the mRNA that is the product of RNA processing of the primary transcription product.In one embodiment, the target portion of the sequence will be at least sufficiently long to serve as a substrate for iRNA-dependent cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed during transcription of ANGPTL4 gene.

[0140] The target sequence of the ANGPTL4 gene may be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence may be about 15 to 30 nucleotides in length, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 1 It may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 nucleotides in length. Ranges and lengths intermediate to the above listed ranges and lengths are also contemplated as part of the invention.

[0141] As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a strand of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0142] "G", "C", "A", "T" and "U" each generally represent a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or substitute replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moieties. For example, but not limited to, a nucleotide that contains inosine as its base can base pair with a nucleotide that contains adenine, cytosine or uracil. Thus, a nucleotide that contains uracil, guanine or adenine can be replaced with a nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA featured in the present invention. In another example, adenine and cytosine in any of the oligonucleotides can be replaced with guanine and uracil, respectively, to form G-U Wobble base pairs with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the present invention.

[0143] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent", used interchangeably herein, refer to an agent that contains RNA as the term is defined herein and mediates targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, e.g. inhibits, the expression of ANGPTL4 gene in cells, e.g. in cells in a subject, e.g. in a mammalian subject.

[0144] In one embodiment, the RNAi agent of the present invention comprises a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL4 target mRNA sequence, to direct cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into cells is degraded into siRNAs 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 19-23 base pair small interfering RNAs with characteristic two base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. Then, siRNA is introduced into RNA-induced silencing complex (RISC), where one or more helicases unwind siRNA duplex and allow complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one aspect, the present invention relates to single-stranded RNA (sssiRNA) that is generated in cells and promotes the formation of RISC complex, thereby silencing target gene, i.e., ANGPTL4 gene. Thus, the term "siRNA" is used herein to mean RNAi as described above.

[0145] In another embodiment, the RNAi agent can be a single-stranded RNAi agent introduced into a cell or organism to inhibit target mRNA. Single-stranded RNAi agents (ssRNAi) bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAi agents is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs described herein or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0146] In another embodiment, the "iRNA" for use in the compositions and methods of the present invention is double-stranded RNA, and is referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent", or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel substantially complementary nucleic acid strands, said to have "sense" or "antisense" orientation with respect to the target RNA, i.e., the ANGPTL4 gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0147] Generally, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but each or both strands can also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides, as detailed herein.In addition, as used herein, "RNAi agent" can include ribonucleotides with chemical modifications; RNAi agent can include substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase.Thus, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional groups or atoms, to the internucleoside linkage, sugar moiety, or nucleobase.Modifications suitable for use in the agent of the present invention encompass all types of modifications disclosed herein or known in the art.Any such modifications used in siRNA type molecules are encompassed by "RNAi agent" for the purposes of this specification and claims.

[0148] The duplex region may be of any length that allows for specific degradation of the desired target RNA through the RISC pathway, ranging from about 9-36 base pairs in length, e.g., about 15-30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, , 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19~ It may be 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the ranges and lengths listed above are also contemplated as part of the invention.

[0149] The two strands forming the duplex structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of a larger molecule and may be connected by a continuous chain of nucleotides between the 3' end of one strand forming the duplex structure and the 5' end of the other strand, respectively, the connecting RNA is called a "hairpin loop". A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 7, at least 8, at least 9, at least 10, at least 20, or at least 23 or more unpaired nucleotides, or nucleotides not directed to the target site of the dsRNA. 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 4-10 unpaired nucleotides. In some embodiments, a hairpin loop may be 4-8 nucleotides.

[0150] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, which can, but do not necessarily, be covalently linked. When the two strands are covalently linked by means other than an uninterrupted strand of nucleotides between the 3' end of one strand and the 5' end of each other strand that forms a duplex structure, the connecting structure is called a "linker". The RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus all overhangs present in the duplex. In addition to the duplex structure, the RNAi can include one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, for example, 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 comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises 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 other embodiments, the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0151] In one embodiment, the RNAi agent of the present invention is a dsRNA, each of which contains less than 30 nucleotides, for example, 17-27, 19-27, 17-25, 19-25, or 19-23 nucleotides, that interacts with a target RNA sequence, for example, an ANGPTL4 target mRNA sequence, to induce cleavage of the target RNA. In another embodiment, the RNAi agent of the present invention is a dsRNA, each of which contains 19-23 nucleotides, that interacts with a target RNA sequence, for example, an ANGPTL4 target mRNA sequence, to induce cleavage of the target RNA. In one embodiment, the sense strand is 21 nucleotides long. In another embodiment, the antisense strand is 23 nucleotides long.

[0152] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of iRNA, such as dsRNA. For example, when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa, a nucleotide overhang exists. A dsRNA can include at least one nucleotide overhang; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. A nucleotide overhang can include or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. An overhang can be on the sense strand, antisense strand, or any combination thereof. Moreover, the nucleotide of the overhang can be present on the 5'-end, 3'-end, or both of the antisense strand or sense strand of a dsRNA.

[0153] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' and / or 5' end, for example, an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' and / or 5' end, for example, an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0154] In certain embodiments, the overhang on the sense strand or the antisense strand or both strands may comprise an extended length of more than 10 nucleotides, for example, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, the extended overhang is in the sense strand of the duplex. In certain embodiments, the extended overhang is at the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is at the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is at the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 3' end of the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 5' end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang are replaced with a nucleoside thiophosphate.

[0155] The term "blunt" or "blunt ended" as used herein with respect to dsRNA means that there is no unpaired nucleotide or nucleotide analog at a given end of the dsRNA, i.e., there is no nucleotide overhang. One or both ends of the dsRNA can be blunt. If both ends of the dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there is no nucleotide overhang at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its length.

[0156] The terms "antisense strand" or "guide strand" refer to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., ANGPTL4 mRNA.

[0157] As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an ANGPTL4 nucleotide sequence, as defined herein. If the region of complementarity is not completely complementary to the target sequence, the mismatch may be in an internal region or a terminal region of the molecule. In general, the most tolerated mismatch is in the terminal region, e.g., in the 5' and / or 3' end of the iRNA. In some embodiments, the double-stranded RNA agent of the present invention comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises no more than 4 mismatches with the target mRNA, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises no more than 4 mismatches with the sense strand, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention comprises a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the antisense strand, for example, the sense strand comprises 4, 3, 2, 1 or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3' end of the iRNA agent. In another embodiment, the nucleotide mismatch is, for example, at the 3' end nucleotide of the iRNA agent. In some embodiments, the mismatch is not present in the seed region.

[0158] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0159] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases that are directly adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases that are directly adjacent to either end of the cleavage site. In some embodiments, specifically, the cleavage site is located at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0160] As used herein, unless otherwise specified, the term "complementary" when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, as understood by those skilled in the art, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions. Such conditions can be, for example, "stringent conditions" including, but not limited to, 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). As used herein, "stringent conditions" or "stringent hybridization conditions" refer to conditions under which an antisense compound hybridizes to its target sequence but minimizes the number of other sequences with which it hybridizes. Stringent conditions are sequence-dependent and vary in different situations, and the "stringent conditions" that antisense compounds hybridize to target sequences are determined by the nature and composition of antisense compounds and the assays that they are examined in.Other conditions can also be applied, such as physiologically relevant conditions that may be encountered inside an organism.Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the final application of hybridized nucleotide.

[0161] The complementary sequence in iRNA, for example, in dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to the oligonucleotide or polynucleotide comprising a second nucleotide sequence throughout the length of one or both nucleotide sequences.Such sequences can be referred to herein as "fully complementary" with respect to each other.However, when the first sequence is referred to herein as "substantially complementary" with respect to the second sequence, the two sequences can be fully complementary, or can form one or more mismatched base pairs, generally no more than 5, 4, 3 or 2, during hybridization for up to 30 double strands. In some embodiments, the "substantially complementary" sequence disclosed herein comprises a continuous nucleotide sequence that is at least about 80% complementary to the corresponding region of target ANGPTL4 sequence throughout its entire length, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary.However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered as mismatches in terms of determining complementarity.For example, a dsRNA that comprises one oligonucleotide that is 21 nucleotides long and another oligonucleotide that is 23 nucleotides long, where the longer oligonucleotide comprises a 21 nucleotide sequence that is completely complementary to the shorter oligonucleotide, can still be considered as "completely complementary" for the purposes described herein.

[0162] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs, and / or base pairs formed from non-natural modified nucleotides, so long 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 Wobble or Hoogsteen base pairing.

[0163] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein in reference to base matching between two oligonucleotides or polynucleotides, e.g., between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as can be understood in the context of their usage.

[0164] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding ANGPTL4). For example, a polynucleotide is complementary to at least a portion of an ANGPTL4 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding ANGPTL4.

[0165] Thus, in some embodiments, the antisense strand polynucleotide disclosed herein is fully complementary to target ANGPTL4 sequence (e.g., human ANGPTL4 sequence).In other embodiments, the antisense strand polynucleotide disclosed herein is substantially complementary to target ANGPTL4 sequence (e.g., human ANGPTL4 sequence), and comprises the contiguous nucleotide sequence that is at least about 80% complementary to the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 or the equivalent region of the fragment of SEQ ID NO: 1, 3, 5 or 7 throughout its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary.

[0166] In one embodiment, the RNAi agent of the present invention is substantially complementary to an antisense polynucleotide that is further complementary to a target ANGPTL4 sequence (e.g., a human ANGPTL4 sequence), and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary to the nucleotide sequence of SEQ ID NO:2, 4, 6 or 8, or an equivalent region of a fragment of any one of SEQ ID NOs:2, 4, 6 or 8, throughout its length.

[0167] In some embodiments, the antisense strand polynucleotide disclosed herein is fully complementary to target mouse ANGPTL4 sequence.In other embodiments, the antisense strand polynucleotide disclosed herein is substantially complementary to mouse ANGPTL4, and comprises the continuous nucleotide sequence that is at least about 80% complementary to the nucleotide sequence of SEQ ID NO:9 or the equivalent region of any one of the fragments of SEQ ID NO:9 throughout its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary.

[0168] In one embodiment, the RNAi agent of the present invention is substantially complementary to an antisense polynucleotide that is further complementary to a mouse ANGPTL4 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary to the nucleotide sequence of SEQ ID NO:10 or the equivalent region of any one of the fragments of SEQ ID NO:10 throughout its length.

[0169] In some embodiments, the iRNA of the invention comprises an antisense strand that is substantially complementary to a target ANGPTL4 sequence and comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary, to the nucleotide sequence of any one of the sense strands in Tables 2 or 3, or the equivalent region of a fragment of any one of the sense strands in Tables 2, 3, 4, 5, 6, 7, 8 or 9, throughout its length.

[0170] The term "inhibiting," as used herein, is used synonymously with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0171] The phrase "inhibiting expression of the ANGPTL4 gene," as used herein, includes inhibition of expression of any ANGPTL4 gene (e.g., mouse ANGPTL4 gene, rat ANGPTL4 gene, monkey ANGPTL4 gene or human ANGPTL4 gene, etc.), as well as variants or mutants of the ANGPTL4 gene that encode the ANGPTL4 gene.

[0172] "Inhibiting expression of the ANGPTL4 gene" includes any level of inhibition of the ANGPTL4 gene, for example, at least partial suppression of the ANGPTL4 gene, for example, at least about 20% inhibition. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% relative to control levels.

[0173] The expression of ANGPTL4 gene may be evaluated based on the level of any variable associated with the expression of ANGPTL4 gene, for example, ANGPTL4 mRNA level or ANGPTL4 protein level.The expression of ANGPTL4 gene may be evaluated indirectly based on, for example, the enzyme activity of ANGPTL4 in tissue samples (for example, GTPase activating protein (GAP) activity), or the level of ANGPTL4-mediated signal transduction in tissue samples, such as liver samples.Inhibition can be evaluated by the absolute or relative level reduction of one or more of these variables compared to control level.The control level can be any kind of control level used in the art, for example, pre-administration baseline level or level determined from similar subjects, cells or samples that are untreated or treated with a control (for example, buffer only control or inactive drug control, etc.).

[0174] In one embodiment, at least partial suppression of expression of the ANGPTL4 gene is assessed by a reduction in the amount of ANGPTL4 mRNA, which can be isolated from or detected in a first cell or group of cells in which the ANGPTL4 gene is transcribed and which has been treated or has been treated so that expression of the ANGPTL4 gene is inhibited, and compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has been or has not been so treated.

[0175] The degree of inhibition may be expressed by the following formula:

number

[0176] The phrase "contacting a cell with an RNAi agent" such as dsRNA, as used herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo.Contacting can be performed directly or indirectly.Thus, for example, an RNAi agent can be physically contacted with a cell by carrying out a method separately, or the RNAi agent can be placed in a situation that can allow or cause it to contact a cell later.

[0177] Contacting cells in vitro can be done, for example, by incubating cells with RNAi agents.Contacting cells in vivo can be done, for example, by injecting RNAi agents into or near the tissue where the cells are located, or by injecting into another area, for example, into the bloodstream (i.e., intravenously) or subcutaneous space, so that the agent then reaches the tissue where the cells to be contacted are located.For example, the RNAi agent can contain and / or be coupled to a ligand, for example, GalNAc3, that directs the RNAi agent to a specific site, for example, the liver.Combination of in vitro and in vivo methods for contacting is also possible.For example, the cell can be contacted with the RNAi agent in vitro and then transferred to a subject.

[0178] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering an iRNA into a cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below and / or known in the art.

[0179] The term "lipophilic moiety" or "lipophilic substance" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is the octanol / water partition coefficient, log K ow This is due to K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol / water partition coefficient is a property of a substance measured on an experimental scale. However, it may be predicted by using coefficients ascribed to the chemical of its structural components, calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of its logK owis greater than 0, it is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol is ow For example, the log K of cholesteryl N-(hexan-6-ol) carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0180] The lipophilicity of a molecule can be altered in relation to the functional groups it carries. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety increases the partition coefficient (e.g., logK ow ) value can be increased or decreased.

[0181] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated to one or more lipophilic moieties can be measured by its protein binding properties.For example, in certain embodiments, the unbound fraction in the plasma protein binding assay of double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, which can then be positively correlated with the silencing activity of double-stranded RNAi agent.

[0182] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift analysis (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, in PCT / US2019 / 031170. Briefly, the duplexes were incubated with human serum albumin and the unbound fraction was determined. An exemplary assay protocol includes duplexes at a stock concentration of 10 μM diluted to a final concentration of 0.5 μM (total volume of 20 μL) in 1×PBS with 0, 20 or 90% serum. Samples can be mixed and centrifuged for 30 seconds, followed by incubation at room temperature for 10 minutes. Once the incubation step is complete, 4 μL of 6×EMSA gel loading solution can be added to each sample, centrifuged for 30 seconds, and 12 μL of each sample can be loaded onto a 26-well BioRad 10% PAGE (polyacrylamide gel electrophoresis). Gels can be run at 100 volts for 1 hour. After the run is complete, the gel is removed from the casing and washed with 50 mL of 10% TBE (Tris base, boric acid and EDTA). Once washing is complete, 5 μL of SYBR Gold can be added to the gel, which is then incubated at room temperature for 10 minutes, and the gel can be washed again with 50 mL of 10% TBE. In this exemplary assay, the gel can be read using the Gel Doc XR+ gel documentation system with the following parameters: Imaging application set to SYBR Gold, size set to Bio-Rad reference gel, exposure set to automatic for intense bands, pixel saturation display set to one, and color set to gray. Detection, molecular weight analysis, and output can all be disabled. Once a clean picture of the gel is obtained, the image can be processed using Image Lab5.2. Lanes and bands can be manually set and band intensities measured. Band intensities for each sample can be normalized to PBS to obtain the fraction of unbound siRNA. This measurement allows the relative hydrophobicity to be determined.The hydrophobicity of the double-stranded RNAi agent, as measured by the fraction of unbound siRNA in a binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 to enhance in vivo delivery of the siRNA.

[0183] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides increased hydrophobicity to enhance in vivo delivery of the siRNA.

[0184] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharma- ceutically active molecule, such as a nucleic acid molecule, such as an iRNA or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0185] As used herein, a "subject" is an animal, e.g., a mammal, including a primate (e.g., a human, a non-human primate, such as, for example, monkeys and chimpanzees), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose).

[0186] In embodiments, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition that is expected to benefit from reduced ANGPTL4 expression; a human being at risk for a disease, disorder, or condition that is expected to benefit from reduced ANGPTL4 expression; a human having a disease, disorder, or condition that is expected to benefit from reduced ANGPTL4 expression; and / or a human being treated for a disease, disorder, or condition that is expected to benefit from reduced ANGPTL4 expression as described herein.

[0187] In another embodiment, the subject is homozygous for the ANGPTL4 gene. Each allele of the gene may code for a functional ANGPTL4 protein. In yet another embodiment, the subject is heterozygous for the ANGPTL4 gene. The subject may have an allele that codes for a functional ANGPTL4 protein and an allele that codes for a loss-of-function mutant of ANGPTL4. In one embodiment, the subject is homozygous for the HSD17B13 gene. Each allele of the gene may code for a functional HSD17B13 protein. In another embodiment, the subject is heterozygous for the HSD17B13 gene. The subject may have an allele that codes for a functional HSD17B13 protein and an allele that codes for a loss-of-function mutant of HSD17B13. In another embodiment, the subject is not a carrier of the HSD17B13 rs72613567 variant, for example, HSD17B13 rs72613567:TA.

[0188] In some embodiments, the subject is heterozygous for the gene encoding patatin-like phospholipase domain-containing protein 3 (PNPLA3). In one embodiment, one of the alleles encodes an I148M variation (e.g., encoded by rs738409C>G). In one embodiment, one of the alleles encodes an I144M variation. In some embodiments, the subject is homozygous for PNPLA3. In one embodiment, each allele of the gene encodes an I148M variation. In one embodiment, each allele of the gene encodes a PNPLA3 I144M variation. In one embodiment, each allele of the gene encodes a functional PNPLA3 protein.

[0189] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, including, without limitation, alleviation or amelioration of one or more symptoms associated with ANGPTL4 gene expression and / or ANGPTL4 protein production. In some embodiments, a condition associated with ANGPTL4 gene expression and / or ANGPTL4 protein production can be a symptom of a disease or disorder whose pathology or cause is unrelated to ANGPTL4 expression and / or ANGPTL4 protein production, but which can nevertheless be corrected, treated or abrogated by inhibiting ANGPTL4 gene expression and / or ANGPTL4 protein production, e.g., an ANGPTL4-related disease, e.g., obesity, metabolic disorders (e.g., type 1 diabetes, type 2 diabetes, prediabetes or insulin resistance, or chronic fibroinflammatory liver disease, e.g., liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocellular necrosis and / or hepatocellular carcinoma. "Treatment" can also mean prolonging survival as compared to expected survival if no treatment is performed.

[0190] The term "lower" in the context of the level of ANGPTL4 gene expression or ANGPTL4 protein production in a subject, or disease marker or symptom, refers to a statistically significant decrease in said level.Decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or less than the level of detection in the relevant cell or tissue, such as liver cell, or other subject sample, such as blood or serum, urine derived therefrom, according to the detection method.In certain embodiments, decrease is at least 20%.

[0191] As used herein, "prevention" or "preventing" when used in relation to a disease, disorder or condition that would benefit from reducing the expression of ANGPTL4 gene refers to the reduction in the probability that a subject will develop symptoms associated with the disease, disorder or condition, such as symptoms of ANGPTL4 gene expression, such as liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocellular necrosis and / or hepatocellular carcinoma.Not developing a disease, disorder or condition, or reducing the onset of symptoms associated with such disease, disorder or condition (e.g., at least about 10% reduction in the clinically acceptable magnitude for the disease or disorder), or delaying the presentation of delayed symptoms (e.g., reducing lipid accumulation in the liver and / or spreading of lipid droplets in the liver) (e.g., delaying by days, weeks, months, or years) is considered to be effective prevention.

[0192] As used herein, the term "ANGPTL4-associated disease" is a disease or disorder caused by or associated with ANGPTL4 gene expression or ANGPTL4 protein production. The term "ANGPTL4-associated disease" includes diseases, disorders, or conditions that would benefit from reduced ANGPTL4 gene expression or protein activity. For example, "ANGPTL4-associated disease" includes diseases or disorders that do not result from ANGPTL4 gene expression and / or ANGPTL4 protein production, but in which reduced ANGPTL4 gene expression and / or ANGPTL4 protein production can nevertheless alleviate the symptoms of the disease or disorder or abrogate or correct its deleterious physiological effects. Subjects having or at risk for an ANGPTL4-associated disease or disorder include those that exhibit wild-type ANGPTL4 gene and / or instead exhibit normal / healthy levels of ANGPTL4 gene expression and ANGPTL4 protein production. ANGPTL4-associated diseases further include diseases in which subjects carry missense mutations and / or deletions in the ANGPTL4 gene, or diseases in subjects with reduced expression of ANGPTL4 that would otherwise benefit from further reduction in ANGPTL4 expression.

[0193] In one embodiment, "ANGPTL4-related disease" is chronic fibroinflammatory liver disease. "Chronic fibroinflammatory liver disease" is any disease, disorder or condition related to chronic liver inflammation and / or fibrosis. Non-limiting examples of chronic fibroinflammatory liver disease include, for example, liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related liver cirrhosis, drug-induced liver injury, hepatocellular necrosis and / or hepatocellular carcinoma.

[0194] "ANGPTL4-associated diseases" include, but are not limited to, those involving lipid metabolism, such as in primary dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperlipoproteinemia, or atherogenic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (such as obesity, metabolic syndrome, diabetes mellitus), lipodystrophy, subcutaneous lipoatrophy, and dyslipidemias, including, for example, decreased LPL activity and / or LPL deficiency, decreased LDL receptor activity and / or LDL receptor deficiency, alterations in ApoC2, ApoE deficiency, increased ApoB, increased production and / or decreased shedding of very low density lipoprotein (VLDL), certain drug treatments (e.g., glucocorticoid treatment-induced dyslipidemia), any genetic predisposition, dietary or lifestyle-related causes.

[0195] Other ANGPTL4-related diseases or disorders associated with or caused by hyperlipidemia, hyperlipoproteinemia and / or dyslipidemia include, but are not limited to, cardiovascular diseases or disorders, such as atherosclerosis, aneurysm, hypertension, angina, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction or peripheral vascular disease.

[0196] In one embodiment, the "ANGPTL4-associated disease" is a metabolic disorder such as type 1 diabetes, type 2 diabetes, prediabetes, metabolic syndrome or insulin resistance. In one embodiment, the ANGPTL4-associated disease is obesity.

[0197] "Therapeutically effective amount" as used herein is intended to include the amount of RNAi agent that is sufficient to effectively treat the disease (e.g., by attenuating, improving or maintaining the existing disease or one or more symptoms of the disease) when administered to a subject with an ANGPTL4-related disease, disorder or condition. "Therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0198] "Prophylactically effective amount" as used herein is intended to include an amount of iRNA that is sufficient to prevent or ameliorate the disease, or one or more symptoms of the disease, when administered to a subject with an ANGPTL4-related disease, disorder, or condition. Ameliorating the disease includes slowing the course of the disease or reducing the severity of a later-onset disease. A "prophylactically effective amount" may vary depending on the iRNA, how the agent is administered, the degree of risk of the disease, as well as the medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0199] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNAs used in the methods of the invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0200] The phrase "pharmacologically acceptable" is used herein to mean compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human and animal subjects, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0201] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in the transport or transportation of a compound of interest from one organ, or part of the body, to another organ, e.g., part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, as well as not deleterious to the subject being treated. Some examples of materials which may function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, peppermint oil, and oleaginous oils. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic affinity substances used in pharmaceutical formulations.

[0202] The term "sample" as used herein includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be obtained from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., the entire liver, or a certain part of the liver, or a certain type of cell in the liver, such as hepatocytes). In some embodiments, a "sample from a subject" refers to blood or plasma obtained from a subject.

[0203] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specific substituent, including, without limitation, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, halo, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

[0204] The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains, which may be linear or branched, containing the indicated number of carbon atoms, optionally interrupted by N, O, or S, including, without limitation, propyl, allyl, or propargyl. For example, "(C1-C6) alkyl" refers to a radical having 1 to 6 carbon atoms in a linear or branched arrangement. "(C1-C6) alkyl" includes, for example, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, pentyl, and hexyl. In certain embodiments, the lipophilic moieties of the present disclosure may include C6-C18 alkyl hydrocarbon chains.

[0205] The term "alkylene" refers to an optionally substituted saturated aliphatic branched or straight-chain divalent hydrocarbon radical having the specified number of carbon atoms. For example, "(C1-C6)alkylene" refers to a divalent saturated aliphatic radical having 1 to 6 carbon atoms in a linear arrangement, e.g., [(CH 2 ) n ], where n is an integer from 1 to 6. "(C1-C6)alkylene includes methylene, ethylene, propylene, butylene, pentylene, and hexylene. Alternatively, "(C1-C6)alkylene" refers to a divalent saturated radical having 1 to 6 carbon atoms in a branched arrangement, such as [(CH 2 CH 2 CH 2 CH 2 CH(CH 3 )], [(CH 2 CH 2 CH 2 CH 2 C(CH 3 ) 2 ] and [(CH 2 C(CH 3 ) 2 CH(CH 3 ))], etc. The term "alkylenedioxo" refers to a divalent species of the structure -ORO-, where R represents alkylene.

[0206] The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0207] The term "halo" refers to any radical of fluorine, chlorine, bromine, or iodine. "Halogen" and "halo" are used interchangeably herein.

[0208] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms, unless otherwise specified. For example, "(C3-C10)cycloalkyl" refers to a hydrocarbon radical of a (3-10) membered saturated aliphatic cyclic hydrocarbon ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may contain multiple spiro or fused rings. Cycloalkyl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valence.

[0209] As used herein, the term "alkenyl" refers to a non-aromatic hydrocarbon radical that is straight-chained or branched, contains at least one carbon-carbon double bond, and has 2 to 10 carbon atoms unless otherwise specified. There may be up to five carbon-carbon double bonds in the group. For example, a "C2-C6" alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight-chain, branched, or cyclic portions of the alkenyl group may contain double bonds and may be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valence. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

[0210] As used herein, the term "alkynyl" refers to a hydrocarbon radical, straight or branched, having 2 to 10 carbon atoms unless otherwise specified, and containing at least one carbon-carbon triple bond. Up to five carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an alkynyl radical having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight or branched portions of the alkynyl group may contain triple bonds as permitted by normal valence, and may be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valence.

[0211] As used herein, "alkoxyl" or "alkoxy" refers to an alkyl group as defined above with the specified number of carbon atoms attached through an oxygen bridge. For example, "(C1-C3)alkoxy" includes methoxy, ethoxy, and propoxy. For example, "(C1-C6)alkoxy" is intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. For example, "(C1-C8)alkoxy" is intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy groups. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy. "Alkylthio" refers to an alkyl radical attached through a sulfur linking atom. The term "alkylamino" or "aminoalkyl" refers to an alkyl radical attached through an NH linkage. "Dialkylamino" refers to two alkyl radicals attached through a nitrogen linking atom. The amino group may be unsubstituted, mono-substituted or di-substituted. In some embodiments, the two alkyl radicals are the same (e.g., N,N-dimethylamino). In some embodiments, the two alkyl radicals are different (e.g., N-ethyl-N-methylamino).

[0212] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbocyclic ring having up to seven atoms in each ring, with at least one ring being aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, attachment is understood to be via the aromatic ring. The aryl group may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valence. The term "arylalkyl" or "aralkyl" refers to an alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0213] "Hetero" refers to the replacement of at least one carbon atom in a ring system with at least one heteroatom selected from N, S and O. "Hetero" also refers to the replacement of at least one carbon atom in an acyclic system. A heterocyclic or heteroacyclic system may, for example, have 1, 2 or 3 carbon atoms replaced with a heteroatom.

[0214] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic ring having up to seven atoms in each ring, at least one ring being aromatic, and containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazoyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolinyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. "Heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. When a heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, attachment is understood to be via the aromatic ring or via the heteroatom-containing ring. Heteroaryl groups may be mono-, di-, tri-, tetra- or penta-substituted at any position as permitted by normal valence.

[0215] As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" refers to a 3-14 membered aromatic or non-aromatic heterocycle containing 1-4 heteroatoms selected from the group consisting of O, N and S, including polycyclic groups. As used herein, the term "heterocyclic" is also considered synonymous with the terms "heterocycle" and "heterocyclyl" and is understood to have the same definition as provided herein. "Heterocyclyl" includes the heteroaryls described above, as well as dihydro and tetrahydro analogs thereof.Examples of heterocyclyl groups include azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, and oxooxazolidine. aryl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, Thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, Heterocyclyl groups include, but are not limited to, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of the heterocyclyl substituent can be via a carbon atom or via a heteroatom. Heterocyclyl groups can be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valence.

[0216] "Heterocycloalkyl" refers to a cycloalkyl residue in which one to four of the carbons are replaced by a heteroatom such as oxygen, nitrogen, or sulfur. Examples of heterocycles in which the radical is a heterocyclyl group include tetrahydropyran, morpholine, pyrrolidine, piperidine, thiazolidine, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran, and the like.

[0217] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic or 1-9 heteroatoms if tricyclic, selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 N, O or S heteroatoms if monocyclic, bicyclic or tricyclic, respectively), optionally substituted on 0, 1, 2, 3 or 4 atoms of each ring. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl and thiazolyl. The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0218] The term "cycloalkyl" as used herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, and e.g., 3 to 6 carbons, where the cycloalkyl group may be optionally further substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0219] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted with substituents.

[0220] As used herein, "keto" refers to an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl group as defined herein attached through a carbonyl bridge.

[0221] Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethinoyl, propinoyl, butynoyl, pentinoyl, hexinoyl), aryloyl (e.g., benzoyl), heteroaryloyl (e.g., pyrroloyl, imidazoloyl, quinolinoyl, pyridinoyl).

[0222] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above attached through a carbonyl bridge (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0223] As used herein, "aryloxycarbonyl" refers to any aryl group, as defined herein, attached through an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0224] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group, as defined herein, attached through an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0225] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0226] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Thus, as used herein, the structures disclosed herein contemplate that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to include the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment, as those skilled in the art will readily understand.

[0227] II. iRNA of the Invention The iRNA described herein inhibits the expression of target gene.In one embodiment, the iRNA inhibits the expression of ANGPTL4 gene.In one embodiment, the iRNA agent comprises double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of ANGPTL4 gene in cells, such as hepatocytes, for example, hepatocytes in subjects, such as mammals, for example, humans with obesity, metabolic disorder, or chronic fibroinflammatory liver disease, disorder or condition, such as lipid droplet accumulation and / or expansion in liver, and / or liver fibrosis.

[0228] The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed in the expression of the ANGPTL4 gene. The region of complementarity is about 30 nucleotides or less (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When the iRNA contacts a cell that expresses the target gene, it inhibits the expression of the target gene (e.g., human, primate, non-primate, or rodent target gene) by at least about 10% compared to a similar cell that is not contacted with the RNAi agent or an RNAi agent that is not complementary to the ANGPTL4 gene. The expression of the gene may be assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, for example, by immunofluorescence analysis using Western blotting or flow cell technology. In one embodiment, the level of knockdown is assayed in human A549 cells. In some embodiments, the level of knockdown is assayed in primary mouse hepatocytes.

[0229] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a region of complementarity that is substantially complementary or completely complementary to the target sequence. The target sequence can be derived from the sequence of the mRNA formed during the expression of ANGPTL4 gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that the two strands hybridize to form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be comprised as a self-complementary region of a single nucleic acid molecule, so that it is opposite on separate oligonucleotides.

[0230] Generally, the duplex structure is 15-30 base pairs long, 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, 19-2 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 length. Ranges and lengths intermediate to the above listed ranges and lengths are also contemplated as part of the invention.

[0231] Similarly, the region of complementarity to the target sequence may be from 15 to 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, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above listed ranges and lengths are also contemplated as part of the invention.

[0232] In some embodiments, the sense and antisense strands of the dsRNA are each independently about 15 to about 30 nucleotides in length, or about 25 to about 30 nucleotides in length, e.g., each strand is independently 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, 18-26, 18-28, 18-24, 18-26 ... 23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the dsRNA is about 15 to about 23 nucleotides in length, about 25 to about 30 nucleotides in length. Generally, dsRNA is long enough to serve as a substrate for Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can function as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage is most often a part of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "part" of an mRNA target is a continuous sequence of the mRNA target that is long enough to allow it to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0233] Those skilled in the art will appreciate that the double-stranded region of dsRNA, the main functional portion, e.g., about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-35, 34, 9~33, 10~33, 11~33, 12~33, 13~33, 14~33, 15~33, 9~32, 10~32, 11~32, 12~32, 13~32, 14~32, 15~32, 9~31, 10~31, 11~31, 12~31, 13~32, 14~31, 15~31, 15~30, 15~29, 15~28, 15~27, 15~26, 15~25 , 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-2 It will also be appreciated that the duplex region of 3, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, an RNA molecule or a complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, so long as it is processed into a functional duplex, for example, 15-30 base pairs, that targets the desired RNA for cleavage. Thus, one skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting ANGPTL4 expression is not generated in a target cell by cleavage of a larger dsRNA.

[0234] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.The dsRNA with at least one nucleotide overhang can have unexpectedly superior inhibitory properties compared to their blunt-end counterparts.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.The overhang can be on the sense strand, on the antisense strand, or any combination thereof.Moreover, the nucleotide of the overhang can be on the 5'-end, 3'-end, or both of the antisense strand or the sense strand of the dsRNA.

[0235] dsRNA can be synthesized by standard methods known in the art, as further described below, for example, by using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0236] The iRNA compounds of the present invention can be prepared using a two-step approach. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the strands of the components are annealed. The individual strands of the dsRNA compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands containing non-natural nucleotides or modified nucleotides can be easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0237] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences presented in Table 2 or 3, and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences in Table 2 or 3. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of the mRNA generated upon expression of the ANGPTL4 gene. Therefore, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide being described as the sense strand (passenger strand) in Table 2 or 3, and the second oligonucleotide being described as the corresponding antisense strand (guide strand) to the sense strand in Table 2 or 3. In one embodiment, the substantially complementary sequence to the dsRNA is comprised in a separate oligonucleotide. In another embodiment, the substantially complementary sequence to the dsRNA is comprised in a single oligonucleotide.

[0238] Although the sequences in Tables 2 or 3 are described as modified, unmodified, unconjugated, and / or conjugated sequences, the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, may comprise any one of the sequences set forth in Tables 2 or 3 that is unmodified, unconjugated, and / or modified and / or conjugated differently than described.

[0239] Those skilled in the art are well aware that dsRNAs with duplex structures of about 20-23 base pairs, e.g., 21 base pairs, are recognized to be particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the above-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein may include at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes, minus a few nucleotides at one or both ends, may be similarly effective compared to the dsRNAs described above. Thus, it is contemplated that dsRNAs having a sequence of at least 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides derived from one of the sequences presented herein and whose ability to inhibit expression of the ANGPTL4 gene compared to control levels differs from a dsRNA containing the complete sequence by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% inhibition.

[0240] In addition, the RNA agent described in Table 2 or 3 identifies the site in ANGPTL4 mRNA transcript that is susceptible to RISC-mediated cleavage.Therefore, the present invention further features an iRNA that targets within this site.As used herein, an iRNA is said to "target within" a specific site of an mRNA transcript if it promotes the cleavage of the mRNA transcript at any of the specific sites.Such an iRNA will generally comprise at least about 15 consecutive nucleotides from one of the sequences provided herein coupled with additional nucleotide sequences taken from the region adjacent to the selected sequence in the gene.

[0241] Target sequences are generally about 15-30 nucleotides in length, although there is great variation in the suitability of particular sequences in this range for inducing cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for the identification of optimal targets for any given gene target, one can also take an empirical approach in which a "window" or "mask" of a given size (21 nucleotides as a non-limiting example) is literally or figuratively (including, for example, in silico) placed over the RNA sequence to identify sequences in a size range that can serve as target sequences. By progressively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence location, the next potential target sequence can be identified until a complete set of possible sequences is identified for any given target size selected. This process, when coupled with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art) to identify the sequences that work best, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted by an iRNA agent. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency can be achieved by incrementally moving the window one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0242] Further optimization can be achieved for any sequence identified herein by systematically removing or adding nucleotides to generate longer or shorter sequences, and testing the generated sequences by moving windows of longer or shorter size from the points up or down the target RNA. This approach of generating new candidate targets can also be coupled with testing the effectiveness of iRNAs based on those target sequences in inhibition assays known in the art and / or described herein, resulting in further improvements in the efficiency of inhibition. Furthermore, the optimized sequence can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., improving serum stabilization or circulating half-life, improving thermostability, enhancing transmembrane delivery, targeting to specific locations or cell types, enhancing interaction with silencing pathway enzymes, increasing release from endosomes).

[0243] The iRNA agent described herein may contain one or more mismatches to the target sequence. In one embodiment, the iRNA described herein contains no more than three mismatches (i.e., 3, 2, 1 or 0 mismatches). In one embodiment, the RNAi agent described herein contains no more than two mismatches. In one embodiment, the RNAi agent described herein contains no more than one mismatch. In one embodiment, the RNAi agent described herein contains 0 mismatches. In certain embodiments, when the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch can be appropriately limited to within the last 5 nucleotides from the 5' or 3' end of the region of complementarity. For example, in such embodiments, in a 23-nucleotide RNAi agent, the strand complementary to a region of the ANGPTL4 gene generally does not contain a mismatch within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting expression of the ANGPTL4 gene. For example, Jackson et al. (Nat. Biotechnol. 2003; 21: 635-637) described that the expression of a small set of genes with sequence identity to MAPK14 siRNA at only 12-18 nt of the sense strand was downregulated by MAPK14 with similar kinetics. Similarly, Lin et al. (Nucleic Acids Res. 2005; 33(14): 4527-4535) using qPCR and reporter assays showed that 7 nt complementarity between siRNA and target was sufficient to trigger target mRNA degradation. It is important to consider the efficacy of iRNAs with mismatches in inhibiting expression of the ANGPTL4 gene, especially when the region of specific complementarity in the ANGPTL4 gene is known to have polymorphic sequence variation within the population.

[0244] An RNA target may have a region or span of the target RNA nucleotide sequence that is relatively more susceptible or suitable for mediated cleavage of the RNA target via RNA interference induced by binding of an RNAi agent to the region than other regions of the RNA target. Increased susceptibility to RNA interference within the "hotspot region" (or simply "hotspot") means that an iRNA agent targeting the region is likely to be more effective at inducing iRNA interference than an iRNA agent targeting other regions of the target RNA. For example, without being bound by theory, the accessibility of a target region of a target RNA may affect the effectiveness of an iRNA agent targeting that region, and some hotspot regions may be more accessible. For example, secondary structures that form an RNA target (e.g., within or near a hotspot region) may affect the ability of an iRNA agent to bind to the target region and induce RNA interference.

[0245] According to certain embodiments of the invention, an iRNA agent may be designed to target a hotspot region of any of the target RNAs described herein, including any identified portion of the target RNA (e.g., a particular exon). As used herein, a hotspot region may refer to an approximately 19-200, 19-150, 19-100, 19-75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, or 100-150 nucleotide region of a target RNA sequence where targeting with an RNAi agent results in a significantly higher probability of effective silencing compared to targeting other regions of the same target RNA. According to certain aspects of the present invention, hotspot region may comprise a limited region of target RNA, in some cases, a substantially limited region of targeting, for example, comprising less than half the length of target RNA, for example, about 5%, 10%, 15%, 20%, 25% or 30% of the length of target RNA.On the contrary, other regions to which hotspot is compared may cumulatively comprise at least half or more of the length of target RNA.For example, other regions may cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of target RNA.

[0246] The compared regions of the target RNA may be experimentally evaluated to identify hotspots using efficacy data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting various regions spanning the target RNA may be compared for the frequency of effective iRNA agents binding to each region (e.g., the amount of target gene expression inhibited, as measured by mRNA expression or protein expression, etc.). In general, hotspots can be recognized by observing the clustering of a large number of effective RNAi agents binding to a limited region of the RNA target. Hotspots may be fully characterized in themselves by observing the efficacy of iRNA agents that cumulatively span at least about 60% of the target region identified as a hotspot, e.g., about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e., at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides in the region, including the nucleotides at each end of the region, are targeted by the iRNA agent). According to some embodiments of the invention, an iRNA agent that exhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% inhibition to a region (e.g., about 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less or 5% or less of mRNA remaining) may be identified as efficacious.

[0247] Quantitative comparison of inhibition measurements across different regions of defined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nt) may be used to assess the suitability of RNA regions for targeting. For example, an average level of inhibition may be determined for each region, and the averages of each region may be compared. The average level of inhibition within a hotspot region may be substantially higher than the average of the averages for all evaluated regions. According to some embodiments, the average level of inhibition in a hotspot region may be at least about 10%, 20%, 30%, 40% or 50% higher than the average of the averages. According to some embodiments, the average level of inhibition in a hotspot region may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 standard deviations above the average of the averages. The average level of inhibition may be higher by a statistically significant (e.g., p<0.05) amount. According to some embodiments, each inhibition measurement within a hotspot region may exceed a threshold amount (e.g., at or below a threshold amount of mRNA remaining). According to some embodiments, each inhibition measurement within a region may be substantially higher than the average of all inhibition measurements across all measured regions. For example, each inhibition measurement in a hotspot region may be at least about 10%, 20%, 30%, 40% or 50% higher than the average of all measurements. According to some embodiments, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 standard deviations above the average of all inhibition measurements. Each inhibition measurement may be higher than all inhibition measurements by a statistically significant (e.g., p<0.05) amount. Criteria for assessing hotspots may include various combinations of the above criteria where compatible (e.g., an average level of inhibition with no inhibition measurements below a threshold level of a second amount at least about the same as the first amount and less than the first amount).

[0248] Therefore, it is expressly contemplated that any iRNA agent, including certain exemplary iRNA agents described herein, that targets a hotspot region of a target RNA may be preferably selected to induce RNA interference of target mRNA, since targeting the hotspot region is more likely to show a stronger inhibitory response than targeting a region that is not a hotspot region. An RNAi agent that targets a target sequence that substantially overlaps (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95% of the length of the target sequence) or preferably exists entirely within a hotspot region may be considered to target a hotspot region. A hotspot region of an RNA target of the present invention may include any region that the data disclosed herein shows is more frequently targeted by an effective RNAi agent, including any of the standards described elsewhere herein, regardless of whether the scope of the hotspot region is clearly specified.

[0249] In various embodiments, a dsRNA agent of the invention targets a hotspot region of the mRNA encoding ANGPTL4.

[0250] III. Modified iRNAs of the Invention In one embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain modified nucleotides, e.g., chemical modifications and / or conjugations known in the art and described herein. In another embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to improve stability or other beneficial properties. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all of the nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention in which "substantially all of the nucleotides are modified" is generally, but not completely, modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0251] In some aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent comprises 10 or fewer nucleotides that contain 2'-fluoro modifications (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). For example, in some embodiments, the sense strand comprises 4 or fewer nucleotides that contain 2'-fluoro modifications (e.g., 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other embodiments, the antisense strand comprises 6 or fewer nucleotides that contain 2'-fluoro modifications (e.g., 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

[0252] In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides that contain 2'-fluoro modifications (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

[0253] In one embodiment, the double-stranded RNAi agent of the present invention further comprises 5'-phosphate or 5'-phosphate mimic at the 5' nucleotide of antisense strand.In another embodiment, the double-stranded RNAi agent further comprises 5'-phosphate mimic at the 5' nucleotide of antisense strand.In a specific embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0254] Nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.), base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire partner, removal of a base (abasic nucleotide) or a conjugated base, sugar modifications (e.g., at the 2' or 4' position) or replacement of a sugar, and / or backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones and do not contain natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have phosphorus atom in backbones are included.For the purpose of this specification, as sometimes referred to in the art, the modified RNAs that do not have phosphorus atom in their internucleoside backbones can also be considered as oligonucleosides.In some embodiments, modified iRNAs have phosphorus atom in their internucleoside backbones.

[0255] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof and those with reversed polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms. 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, the dsRNA agent of the present invention is in sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as counterion to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists as counterion to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0256] Representative United States patents which teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and 5,312,715. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 , No. 925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,5 87,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639 Nos. 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029 and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0257] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (some formed from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and mixed N, O, S and CH 2 Others having component parts are included.

[0258] Representative United States patents which teach the preparation of the above oligonucleosides include, but are not limited to, 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, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0259] In other embodiments, suitable RNA mimetics are contemplated for use in iRNA, in which both the sugar and the internucleoside linkage, i.e., backbone, of nucleotide units are replaced with alternative groups. Nucleobase units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic that has been found to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. Nucleobases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331 and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0260] Some embodiments featured in the present invention include RNAs having phosphorothioate backbones, as well as heteroatom backbones, particularly the --CH 2 --NH--CH 2 -, --CH 2 --N(CH 3 )--O--CH 2 -- [also known as the methylene (methylimino) or NMI backbone], --CH 2 --O--N(CH 3 )--CH 2 --, --CH 2 --N(CH 3 )--N(CH 3 )--CH 2 -- and --N(CH 3 )--CH 2 --CH 2--, and oligonucleotides having the amide backbone of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone is -OP(O)(OH)-OCH 2 - can be expressed as

[0261] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein may contain one of the following at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n O.N.H. 2 and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are from 1 to about 10. In another embodiment, the dsRNA is 1 ~C 10 Alkyl, substituted alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 At the 2' position, the modification comprises one of the following: heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, an acceptor group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and a substituent having similar properties. In some embodiments, the modification is 2'-methoxyethoxy (2'-O--CH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE). 2 CH 2 OCH 3 ) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification includes O(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described herein below in the Examples. 2 ) 2 ON(CH 3 ) 2 groups and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH 2 --O--CH 2 --N(CH 3 ) 2 Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both the R and S isomers in these three families), 2'-alkoxyalkyls and 2'-NMA (N-methylacetamide).

[0262] Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in a 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. An iRNA can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,56 Nos. 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, certain of which are co-owned with this application, the entire contents of each of which are incorporated herein by reference.

[0263] The iRNAs of the present invention may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil, cytosine, thymine, ... These include anal (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 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 of these modified 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 and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitution has been shown to increase nucleic acid duplex 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 an exemplary base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modification.

[0264] Representative U.S. patents which teach the preparation of certain of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, and 5,587,469. Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672 and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0265] The iRNA of the present invention can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability 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].

[0266] The iRNA of the present invention can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring structure. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agent of the present invention can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-endo structural conformation. The addition of locked nucleic acid to siRNA has been shown to increase siRNA stability 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 the polynucleotide of the present invention include, but are not limited to, nucleosides that contain a bridge between 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides that contain a bridge from 4' to 2'.Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,233,631). No. 278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. 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. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134) and 4'-CH2-C(-CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426), the entire contents of each of which are incorporated herein by reference.

[0267] Additional representative United States patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, Nos. 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618 and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0268] For example, any of the above bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0269] The iRNA of the invention can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0 to 2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0270] The iRNA of the present invention may also contain one or more "conformation-restricting nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation and increase hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0271] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0272] In some embodiments, the iRNA of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any of the sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the linkage between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' linkage of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference].

[0273] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227 and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0274] RNAi agents of the present disclosure may also include one or more "cyclohexene nucleic acids" or ("CeNAs) which are nucleotide analogs that have a cyclohexene ring replacing the furanose moiety of DNA. The inclusion of a cyclohexenyl nucleoside in the DNA strand increases the stability of DNA / RNA hybrids. CeNA is stable to degradation in serum, and CeNA / RNA hybrids can activate E. coli ribonuclease H resulting in cleavage of the RNA strand (see Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595-8602, incorporated herein by reference).

[0275] Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0276] Other modifications of the iRNA of the present invention include 5' phosphate or 5' phosphate mimic, for example, 5' terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent.Suitable phosphate mimic is disclosed, for example, in US Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0277] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In a specific embodiment, the 5'-phosphate mimic is a 5'-vinyl phosphonate (5'-VP). In one embodiment, the phosphate mimic is a 5'-cyclopropyl phosphonate (VP). In some embodiments, the 5' end of the antisense strand of the double-stranded iRNA agent does not comprise a 5'-vinyl phosphonate (VP).

[0278] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, nucleotides with 2' phosphates, such as G2p, C2p, A2p, or U2p, and vinyl-phosphonate nucleotides; and combinations thereof. In other embodiments, each of the duplexes in Tables 3, 5, 7, and 9 may be individually modified to provide another double-stranded iRNA agent of the present disclosure. In one embodiment, the 3' end of each sense duplex may be modified by removing the 3' terminal L96 ligand and replacing the two phosphodiester internucleotide linkages between the three 3' terminal nucleotides with phosphorothioate internucleotide linkages. That is, the formula: 5'-N 1 -...-N n-2 N n-1 N n L963' The three 3'-terminal nucleotides (N) of the sense sequence of 1 -...-N n-2 sN n-1 sN n 3' may be substituted.

[0279] That is, for example, the antisense sequence is left unchanged, AD-1663650, the sense sequence: gsasagccGfaGfCfUfgagcggaucuL96 (SEQ ID NO: 293), gsasagccGfaGfCfUfgagcggauscsu (SEQ ID NO: 698) may be substituted to provide another double-stranded iRNA agent of the disclosure.

[0280] In certain embodiments, the RNAi agent of the present invention is an agent that inhibits the expression of the ANGPTL4 gene selected from the group of agents described in Tables 2 or 3. Any of these agents may further comprise a ligand.

[0281] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the invention, the double-stranded RNAi agents of the invention include agents having chemical modifications disclosed, for example, in International Publication WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.

[0282] Thus, the present invention provides a double-stranded RNAi agent capable of inhibiting expression of a target gene (i.e., the ANGPTL4 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may range from 12 to 30 nucleotides in length. For example, each strand may be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In one embodiment, the sense strand is 21 nucleotides in length. In one embodiment, the antisense strand is 23 nucleotides in length.

[0283] The sense and antisense strands typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of the RNAi agent can be 12-30 nucleotide pairs in length. For example, the duplex region can be 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0284] In one embodiment, the RNAi agent may contain one or more overhang regions and / or capping groups at the 3', 5' or both ends of one or both strands. The overhangs may be 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhangs may be the result of one strand being longer than the other or of two strands of the same length being staggered. The overhangs may form a mismatch with the target mRNA, or may be complementary to the gene sequence being targeted, or may be another sequence. The first and second strands may also be joined by additional bases, e.g., forming a hairpin, or by other non-basic linkers.

[0285] In one embodiment, the nucleotides in the overhang region of the RNAi agent can be modified or unmodified nucleotides, including but not limited to 2'-sugar modified, such as 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.For example, TT can be the overhang sequence at either end on either strand.The overhang can form a mismatch with the target mRNA, or can be complementary to the gene sequence to be targeted, or can be another sequence.

[0286] The 5'- or 3'-overhang of the sense strand, antisense strand or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides with phosphorothioate between them, and the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0287] RNAi agent may contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, single-stranded overhang may be located at the 3' end of sense strand or at the 3' end of antisense strand. RNAi may also have a blunt end located at the 5' end of antisense strand (i.e., the 3' end of sense strand), or vice versa. In general, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and the 5' end is blunt. Without wishing to be bound by theory, the blunt end at the 5' end of asymmetric antisense strand and the 3' end overhang of antisense strand are favorable for guide strand loading into RISC process.

[0288] In one embodiment, the RNAi agent is 19 nucleotides long and double blunt-ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0289] In another embodiment, the RNAi agent is 20 nucleotides long and double blunt-ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from 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 from the 5' end.

[0290] In yet another embodiment, the RNAi agent is 21 nucleotides long and double blunt-ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from 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 from the 5' end.

[0291] In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, one end of the RNAi agent is blunt and the other end includes a two nucleotide overhang. The two nucleotide overhang may be present at the 3' end of the antisense strand.

[0292] When a two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three 3' nucleotides at the end of the antisense strand, two of which are overhanging nucleotides, and the third nucleotide is the paired nucleotide next to the overhanging nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages between the three nucleotides at the end at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, every nucleotide in the sense and antisense strands of the RNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 2'-fluoro in an alternating motif. The RNAi agent may be modified with a ligand (e.g., GalNAc 3 ) may further be included.

[0293] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the sense strand is 25-30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), positions 1-23 of the first strand comprise at least 8 ribonucleotides, the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions that pair with positions 1-23 of the sense strand to form a duplex, at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides, and the 5' end of the antisense strand comprises 10-30 consecutive ribonucleotides that are not paired with the sense strand. The nucleic acid sequence of the present invention comprises a nucleotide sequence of at least one nucleotide of the sense strand that is complementary to a nucleotide sequence of the antisense strand, the nucleotide sequence of the antisense strand being at least 19 ribonucleotides long ...

[0294] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the RNAi agent comprises a first strand having a length that is at least 25 and at most 29 nucleotides, and a second strand having a length that is at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from 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 is 1-4 nucleotides longer at its 3' end than the first strand, the duplex region is a region that is at least 25 nucleotides long, the second strand is sufficiently complementary to a target mRNA along a length of the second strand of at least 19 nucleotides, the RNAi agent reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially results in an siRNA that includes the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent may further comprise a ligand.

[0295] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the cleavage site in the sense strand.

[0296] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs being at or near the cleavage site in the antisense strand.

[0297] For RNAi agents having a duplex region 17-23 nucleotides long, the cleavage sites in the antisense strand are usually approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs may be present in the antisense strand at positions 9, 10, and 11, positions 10, 11, and 12, positions 11, 12, and 13, positions 12, 13, and 14, or positions 13, 14, and 15, with the numbers starting from the first nucleotide from the 5' end of the antisense strand, or the numbers starting from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the RNAi from the 5' end.

[0298] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the break site of the strand, and the antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the break site of the strand.When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be arranged 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, i.e., at least one of the three nucleotides of the motif in the sense strand base pairs with at least one of the three nucleotides of the motif in the antisense strand.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0299] In one embodiment, the sense strand of the RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may be at or near the cleavage site of the strand, and the other motif may be a wing modification. As used herein, the term "wing modification" refers to a motif that is present in another part of the strand away from the motif at or near the cleavage site of the same strand. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemistry of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemistry may be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each wing modification may be at one end to the first motif at or near the cleavage site, or on either side of the lead motif.

[0300] Similar to the sense strand, the antisense strand of the RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs being present at or near the site of strand breakage.The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present on the sense strand.

[0301] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0302] In another embodiment, the wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0303] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can fall at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0304] When the sense or antisense strand of an RNAi agent each contains at least two wing modifications, the sense and antisense strands can be arranged such that the two modifications from one strand each reside at one end of the duplex region and have an overlap of 1, 2 or 3 nucleotides, the two modifications from one strand each reside at the other end of the duplex region and have an overlap of 1, 2 or 3 nucleotides, and the two modifications one strand reside on each side of the lead motif and have an overlap of 1, 2 or 3 nucleotides in the duplex region.

[0305] In one embodiment, all nucleotides in the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or both of the non-linked phosphate oxygens and / or one or more of the linking phosphate oxygens, changes to components of the ribose sugar, such as the 2' hydroxyl on the ribose sugar, wholesale replacement of the phosphate moiety with a "dephospho" linker, modification or replacement of naturally occurring bases, and replacement or modification of the ribose-phosphate backbone.

[0306] Nucleic acids are polymers of subunits, so many of the modifications are present at positions that are repeated within the nucleic acid, such as modifications of the base or phosphate moiety or non-linked O of the phosphate moiety. In some cases, modifications are present at all of the target positions in the nucleic acid, but in many cases they do not occur. For example, modifications may only be present at the 3' or 5' terminal positions, and may only be present in the terminal region, such as at the position on the terminal nucleotide of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications may be present in double-stranded regions, single-stranded regions, or both. Modifications may only be present in the double-stranded regions of the RNA, or only in the single-stranded regions of the RNA. For example, phosphorothioate modifications at non-linked O positions may only be present at one or both ends, and may only be present in the terminal region, such as at the position on the terminal nucleotide of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides, or may be present in double-stranded and single-stranded regions, especially at the terminals. The 5' end or both ends may be phosphorylated.

[0307] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, with modifications known in the art, for example, the use of modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, in place of the ribosugar of the nucleobase, and modifications at the phosphate group, for example, phosphorothioate modifications. The overhang does not have to be homologous to the target sequence.

[0308] In one embodiment, each residue of the sense strand and the antisense strand is independently modified with locked nucleic acid (LNA), non-locked nucleic acid (UNA), conformationally restricted nucleotide (CRN), constrained ethyl nucleotide (cET), HNA, cyclohexene nucleic acid (CeNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro. A strand may contain two or more modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0309] At least two different modifications are usually present on the sense and antisense strands. The two modifications can be 2'-O-methyl or 2'-fluoro modifications, or other modifications.

[0310] In one embodiment, N a and / or N b includes an alternating pattern of modifications. The term "alternating motif" as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a strand. Alternating nucleotides may refer to one every other nucleotide or one every third nucleotide or similar patterns. 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...", "AABBBAAABBB..." or "ABCABCABCABC...", etc.

[0311] The types of modifications contained in the alternating motifs can be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleotide, the alternating turns, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0312] In one embodiment, the RNAi agent of the present invention comprises a modification pattern of an alternating motif on the sense strand that is shifted relative to the modification pattern of the alternating motif on the antisense strand. The shift can be such that the modified group of the nucleotide of the sense strand corresponds to the differently modified group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from the 5'-3' of the strand, and the alternating motif in the antisense strand may start with "BABABA" from the 5'-3' of the strand in the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" from the 5'-3' of the strand, and the alternating motif in the antisense strand may start with "BBAABBAA" from the 5'-3' of the strand in the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0313] In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl modification, and the 2'-F modification on the sense strand has a pattern of alternating motifs of 2'-O-methyl modification initially and a pattern of alternating motifs of 2'-F modification initially on the antisense strand, i.e., the 2'-O-methyl modified nucleotide on the sense strand base pairs with the 2'-F modified nucleotide on the antisense strand, and vice versa.The 1st position of the sense strand may start with the 2'-F modification, and the 1st position of the antisense strand may start with the 2'-O-methyl modification.

[0314] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand interrupts the initial modification pattern present in sense strand and / or antisense strand.The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand introduces the modification pattern of sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand, and unexpectedly enhances the gene silencing activity against target gene.

[0315] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is designated "N a YYYN b " [wherein "Y" represents a motif of three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification to the nucleotide next to the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications. Alternatively, if wing modifications are present, N a and / or N b may or may not be present.

[0316] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present on any nucleotide of the sense strand or the antisense strand or both strands at any position of the strand. For example, the internucleotide linkage modification may be present on every nucleotide on the sense strand and / or the antisense strand, each nucleotide linkage modification may be present in an alternating pattern on the sense strand and / or the antisense strand, or the sense strand or the antisense strand may comprise both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkages on the sense strand may be the same or different from the antisense strand, and the alternating pattern of internucleotide linkages on the sense strand may have a shift relative to the alternating pattern of internucleotide linkages on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at the 5' end or the 3' end.

[0317] In one embodiment, the RNAi comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region can comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be an additional phosphorothioate or methylphosphonate internucleotide linkage that links the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides may be at the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, and / or at the 5' of the antisense strand.

[0318] In one embodiment, the 2-nucleotide overhang is at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the paired nucleotide next to the overhanging nucleotide.Optionally, the RNAi agent can further have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0319] In one embodiment, the RNAi agent contains a mismatch(es) or combinations thereof in the duplex with the target. The mismatch may be in the overhang region or in the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular pairing, the most straightforward approach is to look at pairs on an individual pairing basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings that include universal bases are preferred over canonical pairings.

[0320] In one embodiment, the RNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs in the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairs or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0321] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0322] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxy-thymidine nucleotides, e.g., two dT nucleotides at the 3' end of the sense and / or antisense strands.

[0323] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide, Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In one embodiment, YYY are all 2'-F modified nucleotides.

[0324] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0325] In one embodiment, the YYY motif is present at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can be present at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), the numbers starting from the first nucleotide from the 5' end, or, optionally, the numbers starting from the first paired nucleotide in the duplex region from the 5' end.

[0326] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 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 expressed as:

[0327] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0328] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a may independently represent an oligonucleotide sequence that contains from 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0329] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0330] Each of X, Y and Z may be the same as or different from each other.

[0331] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' n p -N a -YYY-N a -n q 3' (Ia) It can be expressed as:

[0332] When the sense strand is represented by formula (Ia), each N a can independently comprise an oligonucleotide sequence containing from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0333] In one embodiment, the antisense strand sequence of the RNAi has the formula (Ie): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b'-(X'X'X') l -N' a -n p '3' (Ie) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represents 0 to 6; each N a ' independently represent an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b 'representing an oligonucleotide sequence comprising, independently, 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; N b ' and Y' do not have the same modification, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It can be expressed as:

[0334] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0335] The Y'Y'Y' motif is present at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 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, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. In certain embodiments, the Y'Y'Y' motif is present at positions 11, 12, 13.

[0336] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0337] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0338] Thus, the antisense strand has the formula: 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (If), 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (Ig), or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (Ih) It can be expressed as:

[0339] When the antisense strand is represented by formula (If), N b ’ represents an oligonucleotide sequence 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.

[0340] When the sense strand is represented as formula (Ic), Nb 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 Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0341] When the antisense strand is represented by formula (Ih), each N b Each N' independently 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. a In certain embodiments, N' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0342] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia) It can be expressed as:

[0343] When the antisense strand is represented by formula (Ie), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0344] Each of X', Y' and Z' may be the same as or different from each other.

[0345] Each nucleotide of sense strand and antisense strand 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 of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' may specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0346] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif present at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally the numbers starting from the first paired nucleotide in the duplex region from the 5' end, where Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0347] In one embodiment, the antisense strand may contain a Y'Y'Y' motif present at positions 11, 12, 13 of the strand, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0348] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (Ie), (If), (Ig) and (Ih), respectively.

[0349] Thus, an RNAi agent for use in the methods of the invention can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex can have the formula (Ii): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (II) [In the formula, i, j, k and l each independently represent 0 or 1; p, p', q and q' each independently represents 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ',n p , n q ' and n q independently represent overhanging nucleotides, each of which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It is represented by:

[0350] 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 i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0 and k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0351] An exemplary combination of sense and antisense strands that form an RNAi duplex has 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' (Ij) 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' (Ik) 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' (IIl) 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' (Im) Includes.

[0352] When the RNAi agent is represented by formula (Ij), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0353] When the RNAi agent is represented by formula (Ik), each N b Each N independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0354] When an RNAi agent is represented as formula (II), each Nb, Nb' independently 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 Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0355] When an RNAi agent is represented as formula (Im), each Nb, Nb' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0356] Each of X, Y and Z in formulae (Ii), (Ij), (Ik), (Il) and (Im) may be the same or different.

[0357] When the RNAi agent is represented by formula (Ii), (Ij), (Ik), (Il) and (Im), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired with a corresponding Y' nucleotide, or all three of the Y nucleotides are base-paired with a corresponding Y' nucleotide.

[0358] When the RNAi agent is represented by formula (Ik) or (Im), at least one of the Z nucleotides may be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide, or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0359] When an RNAi agent is represented by formula (Il) or (Im), at least one of the X nucleotides can be base-paired with one of the X' nucleotides. Alternatively, at least two of the X nucleotides are base-paired with the corresponding X' nucleotide, or all three of the X nucleotides are base-paired with the corresponding X' nucleotide.

[0360] In one embodiment, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0361] In one embodiment, when the RNAi agent is represented by formula (Im), N aThe modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (Im), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p np' is linked to adjacent nucleotides via phosphorothioate linkages. In yet another embodiment, when the RNAi agent is represented by formula (Im), the Na modification is 2'-O-methyl or 2'-fluoro modified, np'>0, and at least one np' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (below). In another embodiment, when the RNAi agent is represented by formula (Im), the Na modification is 2'-O-methyl or 2'-fluoro modified, np'>0, and at least one np' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (below).

[0362] In one embodiment, when the RNAi agent is represented by formula (Ij), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a divalent or trivalent branched linker.

[0363] In one embodiment, the RNAi agent is a multimer that contains at least two duplexes represented by formula (Ii), (Ij), (Ik), (Il) and (Im), and the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further include a ligand. Each of the duplexes may target the same gene, or may target two different genes, or each of the duplexes may target the same gene at two different target sites.

[0364] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (Ii), (Ij), (Ik), (Il) and (Im), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further include a ligand. Each of the double strands may target the same gene, or may target two different genes, or each of the double strands may target the same gene at two different target sites.

[0365] In one embodiment, two RNAi agents represented by formula (Ii), (Ij), (Ik), (Il) and (Im) are linked to each other at the 5' end, and one or both of the 3' ends can be conjugated to a ligand.Each agent can target the same gene, or can target two different genes, or each agent can target the same gene at two different target sites.

[0366] In certain embodiments, the RNAi agent of the present invention may comprise a small number of nucleotides that comprise 2'-fluoro modification, for example, 10 or less nucleotides that have 2'-fluoro modification. For example, the RNAi agent may comprise 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides that have 2'-fluoro modification. In a specific embodiment, the RNAi agent of the present invention comprises 10 nucleotides that have 2'-fluoro modification, for example, 4 nucleotides that have 2'-fluoro modification in the sense strand and 6 nucleotides that have 2'-fluoro modification in the antisense strand. In another specific embodiment, the RNAi agent of the present invention comprises 6 nucleotides that have 2'-fluoro modification, for example, 4 nucleotides that have 2'-fluoro modification in the sense strand and 2 nucleotides that have 2'-fluoro modification in the antisense strand.

[0367] In other embodiments, the iRNA agent of the present invention may comprise a very small number of nucleotides that comprise 2'-fluoro modification, for example, 2 or less nucleotides that comprise 2'-fluoro modification.For example, the RNAi agent may comprise 2, 1 or 0 nucleotides that have 2'-fluoro modification.In a specific embodiment, the RNAi agent may comprise 2 nucleotides that have 2'-fluoro modification, for example, 0 nucleotides that have 2'-fluoro modification in the sense strand and 2 nucleotides that have 2'-fluoro modification in the antisense strand.

[0368] Various publications describe the multimeric RNAi agent that can be used in the method of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of which is incorporated herein by reference in its entirety.

[0369] As described in more detail below, an RNAi agent that includes one or more carbohydrate moieties conjugated to the RNAi agent can improve one or more properties of the RNAi agent. In many cases, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (e.g., cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). A cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, e.g., nitrogen, oxygen, sulfur. A heterocyclic carrier can be a monocyclic ring system or can include two or more double bonds, e.g., fused rings. A cyclic carrier can be a fully saturated ring system or can include one or more double bonds.

[0370] Ligands can be attached to polynucleotides via carriers. Carriers include (i) at least one "backbone attachment point", e.g., two "backbone attachment points", and (ii) at least one "tethering attachment point". "Backbone attachment point", as used herein, refers to a functional group, e.g., a hydroxyl group, or a bond that is generally available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., a sulfur-containing backbone, of a ribonucleic acid. "Tethering attachment point" (TAP) refers in some embodiments to a constituent ring atom, e.g., a carbon atom or a heteroatom (apart from the atom that provides the backbone attachment point), of a cyclic carrier that connects a selected moiety. The moiety can be, for example, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers will often include a functional group, e.g., an amino group, or generally provide a bond that is suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the constituent ring.

[0371] RNAi agent may be conjugated to ligand through carrier, and carrier can be cyclic or acyclic group.Cyclic group can be selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin.Acyclic group can be selected from serinol backbone or diethanolamine backbone.

[0372] In another embodiment of the invention, the iRNA agent includes a sense strand and an antisense strand, each strand having between 14 and 40 nucleotides. The RNAi agent has the formula (L):

[0373] [ka] It may be represented by:

[0374] In formula (L), B1, B2, B3, B1', B2', B3' and B4' are each independently a nucleotide 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 certain embodiments, B1, B2, B3, B1', B2', B3' and B4' each contain a 2'-OMe or 2'-F modification. In certain embodiments, at least one of B1, B2, B3, B1', B2', B3' and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0375] C1 is a heat destabilizing nucleotide located at a site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is at a position in the sense strand that pairs with the nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is at position 15 from the 5' end of the sense strand. The C1 nucleotide has a heat destabilizing modification that can include an abasic modification, a mismatch with the opposing nucleotide in the duplex, and a sugar modification, e.g., a 2'-deoxy modification or an acyclic nucleotide, e.g., an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In certain embodiments, C1 is i) a mismatch with the opposing nucleotide in the antisense strand, ii)

[0376] [ka] and iii) an abasic modification selected from the group consisting of

[0377] [ka] [wherein B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR 3 or alkyl, R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In certain embodiments, the thermolabile modification in C1 is a mismatch 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, and optionally at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermolabile modification in C1 is GNA, or

[0378] [ka] It is.

[0379] T1, T1', T2' and T3' each independently represent a nucleotide that includes a modification that provides the nucleotide with a steric volume that is less than or equal to the steric volume of the 2'-OMe modification. Steric volume refers to the sum of the steric effects of the modifications. Methods for determining the steric effect of a modification of a nucleotide are known to those skilled in the art. The modification can be at the 2' position of the ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, an acyclic nucleotide, or the backbone of the nucleotide that is similar or equivalent to the 2' position of the ribose sugar, providing the nucleotide with a steric volume that is less than or equal to the steric volume of the 2'-OMe modification. For example, T1, T1', T2' and T3' each independently are selected from DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl. In certain embodiments, T1 is DNA. In certain embodiments, T1' is DNA, RNA or LNA. In certain embodiments, T1' is DNA, RNA or LNA. In certain embodiments, T2' is DNA or RNA. In certain embodiments, T3' is DNA or RNA.

[0380] n 1 , n 3 and q 1 are independently 4 to 15 nucleotides in length.

[0381] n 5 , q 3 and q 7 are independently 1 to 6 nucleotides in length.

[0382] n 4 , q 2 and q 6 are independently 1 to 3 nucleotides in length, or 4 is 0.

[0383] q 5 are independently 0 to 10 nucleotides in length.

[0384] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0385] Or, n 4 is 0 to 3 nucleotides in length.

[0386] In certain embodiments, n 4 can be 0. In one example, n 4 is 0, and q 2 and q 6 is 1. In another example, n 4 is 0, and q 2 and q 6 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0387] In certain embodiments, n 4 , q 2 and q 6 are 1, respectively.

[0388] In certain embodiments, n 2 , n 4 , q 2 , q 4 and q 6 are 1, respectively.

[0389] In certain embodiments, C1 is a nucleotide sequence in which the sense strand is 19 to 22 nucleotides in length, 4 When C1 is 1, C1 is at position 14-17 of the 5' end of the sense strand. In certain embodiments, C1 is at position 15 of the 5' end of the sense strand.

[0390] In certain embodiments, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand and 6 is equal to 1.

[0391] In certain embodiments, T1' begins at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand and q 2 is equal to 1.

[0392] In one exemplary embodiment, T3' starts at position 2 from the 5' end of the antisense strand and T1' starts at position 14 from the 5' end of the antisense strand. In one example, T3' starts at position 2 from the 5' end of the antisense strand and q 6 is equal to 1, T1' starts at position 14 from the 5' end of the antisense strand, and q 2 is equal to 1.

[0393] In certain embodiments, T1' and T3' are 11 nucleotides apart in length (ie, not counting T1' and T3').

[0394] In certain embodiments, T1' is at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand and q 2 is equal to 1 and is at the 2' position or at a non-ribose, acyclic or backbone position that provides less steric bulk than 2'-OMe ribose.

[0395] T3' is at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand and q 6 is equal to 1 and is at the 2' position or at a non-ribose, acyclic or backbone position that provides less steric bulk than 2'-OMe ribose.

[0396] In certain embodiments, T1 is at the cleavage site of the sense strand. In one example, the sense strand is 19-22 nucleotides in length, 2 When n is 1, T1 is at position 11 from the 5' end of the sense strand. In one exemplary embodiment, the sense strand is 19-22 nucleotides in length and n 2 When T1 is 1, T1 is at the cleavage site of the sense strand at position 11 from the 5' end of the sense strand.

[0397] In certain embodiments, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at positions 6-10 from the 5' end of the antisense strand, and 4 is 1.

[0398] In one exemplary embodiment, the sense strand is 19-22 nucleotides in length, 2 is 1, T1 is at the cleavage site of the sense strand, e.g., position 11 from the 5' end of the sense strand, T1' is at position 14 from the 5' end of the antisense strand, and q 2 is equal to 1, the modification to T1' is at the 2' position on the ribose sugar or at a non-ribose, acyclic or backbone position that provides less steric bulk than 2'-OMe ribose. T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1, T3' is at position 2 from the 5' end of the antisense strand, and q 6 is equal to 1 and the modification to T3' is at the 2' position on the ribose sugar or at a non-ribose, acyclic or backbone position that offers less steric bulk than 2'-OMe ribose.

[0399] In certain embodiments, T2' starts at position 8 from the 5' end of the antisense strand. In one example, T2' starts at position 8 from the 5' end of the antisense strand and q 4 is 2.

[0400] In certain embodiments, T2' begins at position 9 from the 5' end of the antisense strand. In one example, T2' is at position 9 from the 5' end of the antisense strand and 4 is 1.

[0401] In certain embodiments, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0402] In certain embodiments, n 4 is 0, B3 is 2'-OMe, 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 7is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0403] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0404] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0405] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0406] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0407] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1.

[0408] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0409] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 there may be at least two additional TTs at the 3' end of the antisense strand.

[0410] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 there may be at least two additional TTs at the 3' end of the antisense strand, including two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), as well as two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0411] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1.

[0412] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0413] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q2 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'-F, and q 7 is 1.

[0414] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0415] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, 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.

[0416] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0417] The RNAi agent may contain a phosphorus-containing group at the 5' end of the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS 2 ), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl (

[0418] [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., a trans-vinyl phosphonate).

[0419] [ka] ), 5'-Z-VP isomers (i.e., cis-vinyl phosphonates

[0420] [ka] ), or a mixture of these.

[0421] In certain embodiments, an RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand.In certain embodiments, an RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0422] In certain embodiments, the RNAi agent comprises a 5'-P. In certain embodiments, the RNAi agent comprises a 5'-P in the antisense strand.

[0423] In certain embodiments, the RNAi agent comprises a 5'-PS. In certain embodiments, the RNAi agent comprises a 5'-PS on the antisense strand.

[0424] In certain embodiments, the RNAi agent comprises a 5'-VP. In certain embodiments, the RNAi agent comprises a 5'-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5'-E-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5'-Z-VP in the antisense strand.

[0425] In certain embodiments, the RNAi agent is a 5'-PS 2 In certain embodiments, the RNAi agent comprises a 5'-PS 2 Includes.

[0426] In certain embodiments, the RNAi agent is a 5'-PS 2 In certain embodiments, the RNAi agent comprises 5'-deoxy-5'-C-malonyl in the antisense strand.

[0427] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. The RNAi agent also contains a 5'-PS.

[0428] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. The RNAi agent also contains a 5'-P.

[0429] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0430] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. The RNAi agent is a 5'-PS 2 Also includes.

[0431] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0432] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0433] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0434] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0435] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 Also includes.

[0436] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0437] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1. The RNAi agent also contains a 5'-P.

[0438] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0439] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0440] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1. The RNAi agent is a 5'-PS 2 Also includes.

[0441] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0442] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-P.

[0443] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS.

[0444] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0445] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1 and includes two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). 2 Also includes.

[0446] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl.

[0447] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0448] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0449] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5is 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'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0450] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1. dsRNA RNAi agents are 5'-PS 2 Also includes.

[0451] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n5 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'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0452] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0453] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0454] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0455] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 Also includes.

[0456] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0457] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0458] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. The RNAi agent also contains a 5'-PS.

[0459] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0460] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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. The RNAi agent is a 5'-PS 2 Also includes.

[0461] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0462] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0463] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0464] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0465] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 Also includes.

[0466] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0467] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In certain embodiments, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0468] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, including two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0469] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0470] In certain embodiments, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0471] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 and a targeting agent. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0472] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting agent. In certain embodiments, the 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.

[0473] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-P and a targeting agent. In certain embodiments, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0474] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS and a targeting agent. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0475] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In certain embodiments, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0476] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1 and includes two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). 2 and a targeting agent. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0477] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 is 1, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl and a targeting agent. In certain embodiments, the 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.

[0478] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In certain embodiments, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0479] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, 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'-F, and q 7 is 1, including two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0480] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7is 1, including two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In certain embodiments, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0481] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 and a targeting agent. 2is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0482] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting agent. In certain embodiments, the 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.

[0483] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5is 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In certain embodiments, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0484] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 7is 1, including two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0485] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, including two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In certain embodiments, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0486] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 and includes two phosphorothioate interoligonucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate interoligonucleotide linkage modifications within positions 1 and 2 of the antisense strand and two phosphorothioate interoligonucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 and a targeting agent. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0487] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q2 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, including two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (as counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (as counted from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting agent. In certain embodiments, the 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.

[0488] In certain embodiments, the RNAi agent of the invention comprises (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) a sense strand having 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); (b)(i) 23 nucleotides in length; (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) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end); The dsRNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0489] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) a sense strand having 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 (as counted from the 5' end); and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (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) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0490] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached 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 a deoxy-nucleotide (e.g., dT) at position 11 (as counted from the 5' end); and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 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) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0491] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15, and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (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, 14, 16, 18, and 20 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0492] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (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) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0493] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F at positions 1, 3, 5, 7, 9-11, and 13, and 2'-OMe at positions 2, 4, 6, 8, 12, and 14-21, and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (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) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0494] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, and 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18 (counting from the 5' end); and (iii) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a 4 nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0495] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11, and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (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) an antisense strand having phosphorothioate internucleotide linkages 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); The RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0496] In another particular embodiment, the RNAi agent of the invention comprises: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached at its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11, and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); (b)(i) 23 nucleotides in length; (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) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide...

Claims

1. 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding ANGPTL4 comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences set forth in Table 2 or 3.

2. (a) the dsRNA agent comprises at least one modified nucleotide; (b) substantially all of the nucleotides of the sense strand comprise a modification; (c) substantially all of the nucleotides of the antisense strand comprise a modification; (d) substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification; (e) all of the nucleotides of the sense strand contain a modification; (f) all of the nucleotides of the antisense strand contain a modification; (g) all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification; (h) the dsRNA agent comprises at least one modified nucleotide, wherein the at least one modified nucleotide is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic 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'-methoxy and / or selected from the group consisting of diethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5' phosphates, nucleotides containing 5' phosphate mimics, glycol-modified nucleotides, and 2-O-(N-methylacetamido)-modified nucleotides, and combinations thereof; and / or (i) the dsRNA agent comprises at least one modified nucleotide, wherein the at least one modified nucleotide is 2'-O-methyl and / or 2'-fluoro modified; The dsRNA agent of claim 1.

3. (a) the region of complementarity is at least 17 nucleotides in length; (b) the region of complementarity is 19-30, 19-25, or 21-23 nucleotides in length; (c) the sense strand and the antisense strand are each 30 nucleotides or less in length; (d) each of the sense strand and the antisense strand is independently 19-30, 19-25, or 21-23 nucleotides in length; (e) at least one of the sense strand or antisense strand comprises a 3' overhang of at least one nucleotide; (f) at least one of the sense strand or antisense strand comprises a 3' overhang of at least two nucleotides; (g) the region of complementarity comprises any of the antisense sequences listed in Table 2 or 3; (h) the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of any of the agents listed in Table 2 or 3; and / or (i) the dsRNA agent targets a hotspot region of the mRNA encoding ANGPTL4; The dsRNA agent of claim 1.

4. (a) the dsRNA agent comprises a ligand; (b) the dsRNA agent includes a ligand conjugated to the 3' end of the sense strand of the dsRNA agent; (c) the dsRNA agent comprises a ligand, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative; (d) the dsRNA agent comprises a ligand, wherein the ligand is 【Chemistry 1】 (Formula II); and / or (e) the dsRNA agent has the following formula: 【Chemistry 2】 wherein X is O or S. The dsRNA agent of claim 1.

5. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 4 (ANGPTL4) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any of the sense sequences listed in Table 2 or 3, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any of the antisense sequences listed in Table 2 or 3; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand contains two phosphorothioate internucleotide linkages at the 5' end; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus; A dsRNA agent wherein the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached through a monovalent, divalent, or trivalent branched linker.

6. (a) all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides; (b) the antisense sequence comprises any of the antisense sequences listed in Table 2 or 3; (c) the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of an agent listed in any of Tables 2 or 3; and / or (d) the dsRNA agent targets a hotspot region of the mRNA encoding ANGPTL4; The dsRNA agent of claim 5.

7. A cell comprising the dsRNA agent of any one of claims 1-6.

8. A vector encoding at least one strand of the dsRNA agent of any one of claims 1-6.

9. A pharmaceutical composition for inhibiting expression of the Angiopoietin-like 4 (ANGPTL4) gene, comprising the dsRNA agent of any one of claims 1-6.

10. (a) the dsRNA agent is formulated in an unbuffered solution; (b) the dsRNA agent is formulated in an unbuffered solution, wherein the unbuffered solution is saline or water; (c) the dsRNA agent is formulated in a buffer solution; (d) the dsRNA agent is formulated with a buffer solution, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof; and / or (e) the dsRNA agent is formulated in a buffer solution, wherein the buffer solution is phosphate buffered saline (PBS); The pharmaceutical composition of claim 9.

11. 10. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-6 for use in inhibiting angiopoietin-like 4 (ANGPTL4) expression in a cell, wherein inhibiting comprises introducing the dsRNA agent into the cell and inhibiting expression of ANGPTL4 in the cell.

12. (a) the cell is in a subject; (b) the cell is in a human subject; (c) the ANGPTL4 expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or is inhibited to below the level of detection of ANGPTL4 expression; (d) the cell is in a human subject suffering from an ANGPTL4-associated disease, disorder, or condition; (e) the cell is in a human subject suffering from a chronic fibroinflammatory liver disease; (f) the cell is in a human subject suffering from a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is associated with the accumulation and / or spread of lipid droplets in the liver; (g) the cell is in a human subject suffering from a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is selected from the group consisting of liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, and hepatocellular necrosis; (h) the cell is in a human subject suffering from an ANGPTL4-associated disease, disorder, or condition, wherein the ANGPTL4-associated disease, disorder, or condition is obesity; (i) the cell is in a human subject suffering from an ANGPTL4-associated disease, disorder, or condition, wherein the ANGPTL4-associated disease, disorder, or condition is a metabolic disorder; and / or (j) the cell is in a human subject suffering from an ANGPTL4-associated disease, disorder, or condition, wherein the ANGPTL4-associated disease, disorder, or condition is a metabolic disorder, wherein the metabolic disorder is primary dyslipidemia, hypertriglyceridemia, metabolic syndrome, type 1 diabetes, type 2 diabetes, prediabetes, or insulin resistance; The pharmaceutical composition of claim 11.

13. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-6, (a) inhibiting expression of ANGPTL4 in a subject, comprising administering to said subject a therapeutically effective amount of said dsRNA agent, thereby inhibiting expression of ANGPTL4 in said subject; (b) preventing at least one symptom in a subject having a disease, disorder, or condition that would benefit from reduced expression of the ANGPTL4 gene, comprising administering to the subject a prophylactically effective amount of the dsRNA agent of claim 2, thereby preventing at least one symptom in a subject having a disease, disorder, or condition that would benefit from reduced expression of the ANGPTL4 gene; (c) reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in a subject, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 2, thereby reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in the subject; (d) preventing at least one symptom in a subject having a disease, disorder, or condition that would benefit from reduced expression of the ANGPTL4 gene, comprising administering to the subject a prophylactically effective amount of the dsRNA agent of claim 2 and a dsRNA agent targeting the HSD17B13 gene or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby preventing at least one symptom in a subject having a disease, disorder, or condition that would benefit from reduced expression of the ANGPTL4 gene; and / or (e) reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in a subject, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 2 and a dsRNA agent targeting the HSD17B13 gene or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby reducing the risk of developing hepatic steatosis or worsening hepatic steatosis in the subject; A pharmaceutical composition for use in

14. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-6, (a) treating a subject suffering from an ANGPTL4-related disease, disorder, or condition, comprising administering to said subject a therapeutically effective amount of said dsRNA agent, thereby treating the subject suffering from an ANGPTL4-related disease, disorder, or condition; (b) inhibiting lipid droplet accumulation in the liver of a subject suffering from an ANGPTL4-related disease, disorder, or condition, comprising administering to the subject a therapeutically effective amount of the dsRNA agent, and a dsRNA agent targeting the HSD17B13 gene, or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby inhibiting fat accumulation in the liver of a subject suffering from an ANGPTL4-related disease, disorder, or condition; (c) inhibiting lipid droplet accumulation in the liver of a subject suffering from an ANGPTL4-related disease, disorder, or condition, comprising administering to the subject a therapeutically effective amount of the dsRNA agent, and a dsRNA agent targeting the HSD17B13 gene, or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby inhibiting fat accumulation in the liver of a subject suffering from an ANGPTL4-related disease, disorder, or condition; and / or (d) treating a subject suffering from an ANGPTL4-related disease, disorder, or condition, comprising administering to the subject a therapeutically effective amount of the dsRNA agent, and a dsRNA agent targeting the HSD17B13 gene, or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby treating the subject suffering from an ANGPTL4-related disease, disorder, or condition; A pharmaceutical composition for use in

15. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-6, (a) reducing the risk of developing chronic liver disease in a subject with steatosis, comprising administering to said subject a therapeutically effective amount of said dsRNA agent, thereby reducing the risk of developing chronic liver disease in the subject with steatosis; (b) inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis, comprising administering to said subject a therapeutically effective amount of said dsRNA agent, thereby inhibiting the progression of steatosis to steatohepatitis in the subject; (c) reducing the risk of developing chronic liver disease in a subject with steatosis, comprising administering to the subject a therapeutically effective amount of the dsRNA agent, and a dsRNA agent targeting the HSD17B13 gene, or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby reducing the risk of developing chronic liver disease in the subject with steatosis; and / or (d) inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis, comprising administering to the subject a therapeutically effective amount of the dsRNA agent, and a dsRNA agent targeting the HSD17B13 gene, or a pharmaceutical composition comprising a dsRNA agent targeting the HSD17B13 gene, thereby inhibiting the progression of steatosis to steatohepatitis in the subject; A pharmaceutical composition for use in

16. (a) administration of the dsRNA agent to a subject results in decreased ANGPTL4 enzyme activity, decreased ANGPTL4 protein accumulation, decreased HSD17B13 enzyme activity, decreased HSD17B13 protein accumulation, and / or decreased fat accumulation and / or lipid droplet prevalence in the liver of the subject; (b) the ANGPTL4-related disease, disorder, or condition is chronic fibroinflammatory liver disease; (c) the ANGPTL4-related disease, disorder, or condition is a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is associated with the accumulation and / or spread of lipid droplets in the liver; (d) the ANGPTL4-related disease, disorder, or condition is a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is selected from the group consisting of fat accumulation in the liver, liver inflammation, liver fibrosis, fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, and hepatocellular necrosis; (e) the ANGPTL4-related disease, disorder, or condition is a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is nonalcoholic steatohepatitis (NASH); (f) the subject is obese; (g) the method includes administering to the subject an additional therapeutic agent; (h) the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg; and / or (i) the method includes determining the level of ANGPTL4 in the subject; The pharmaceutical composition of claim 14.

17. (a) administration of the dsRNA agent or pharmaceutical composition to a subject results in decreased ANGPTL4 enzyme activity, decreased ANGPTL4 protein accumulation, decreased HSD17B13 enzyme activity, decreased HSD17B13 protein accumulation, and / or decreased fat accumulation and / or lipid droplet prevalence in the liver of the subject; (b) the subject is obese; (c) the use includes administering to the subject an additional therapeutic agent; (d) the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg; and / or (e) The use includes determining the level of ANGPTL4 in the subject. The pharmaceutical composition of claim 12.

18. (a) administration of the dsRNA agent or pharmaceutical composition to a subject results in decreased ANGPTL4 enzyme activity, decreased ANGPTL4 protein accumulation, decreased HSD17B13 enzyme activity, decreased HSD17B13 protein accumulation, and / or decreased fat accumulation and / or lipid droplet prevalence in the liver of the subject; (b) the subject is suffering from an ANGPTL4-related disease, disorder, or condition; (c) the ANGPTL4-related disease, disorder, or condition is chronic fibroinflammatory liver disease; (d) the ANGPTL4-related disease, disorder, or condition is a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is associated with the accumulation and / or spread of lipid droplets in the liver; (e) the ANGPTL4-related disease, disorder, or condition is a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is selected from the group consisting of fat accumulation in the liver, liver inflammation, liver fibrosis, fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, and hepatocellular necrosis; (f) the ANGPTL4-related disease, disorder, or condition is a chronic fibroinflammatory liver disease, wherein the chronic fibroinflammatory liver disease is nonalcoholic steatohepatitis (NASH); (g) the subject is obese; (h) the use includes administering to the subject an additional therapeutic agent; (i) the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg; and / or (j) the use comprises determining the level of ANGPTL4 in the subject; The pharmaceutical composition of claim 13.

19. (a) administration of the dsRNA agent to a subject results in decreased ANGPTL4 enzyme activity, decreased ANGPTL4 protein accumulation, decreased HSD17B13 enzyme activity, decreased HSD17B13 protein accumulation, and / or decreased fat accumulation and / or lipid droplet prevalence in the liver of the subject; (b) the subject is obese; (c) the use includes administering to the subject an additional therapeutic agent; (d) the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg; and / or (e) the use comprises determining the level of ANGPTL4 in the subject; 16. The pharmaceutical composition of claim 15.

Images