iRNA Compositions and Methods for Silencing MYLIP

Abstract

The present invention relates to double-stranded ribonucleic acid (dsRNA) compositions that target the myosin regulatory light chain-interacting protein (MYLIP) gene, and methods of using the dsRNA compositions to inhibit expression of MYLIP and to treat subjects who would benefit from reduced expression of MYLIP, e.g., subjects having a MYLIP-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 / 286,891, filed December 7, 2021, and U.S. Provisional Patent Application No. 63 / 341,885, filed May 13, 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 December 6, 2022 is named A108868_1560WO_SL.XML and is 644,814 bytes in size.

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

[0004] Myosin regulatory light chain-interacting protein (MYLIP) is a cytoplasmic protein that promotes the ubiquitination and subsequent degradation of the low-density lipoprotein (LDL) receptor (LDLR) and other cell surface receptors that recognize lipoproteins. LDLR is primarily present in the liver and helps maintain plasma levels of LDL and cholesterol by mediating the endocytosis of cholesterol-rich LDL. LDL is normally rapidly recycled to the cell surface after internalization. Modulation of LDLR expression is controlled by multiple pathways, and dysregulation is associated with atherosclerosis and cardiovascular disease due to increased accumulation of circulating LDL-cholesterol. Hepatocytes, which form the liver parenchyma, receive lipids from the systemic circulation and are responsible for mobilizing lipids for energy and storing excess lipids in the form of lipid droplets (LDs). This makes the liver the primary organ responsible for lipid homeostasis. Summary of the Invention [Problem to be solved by the invention]

[0005] The current standard treatment for the subject with lipid imbalance, especially high LDL cholesterol, is mainly statin treatment, and can also include dietary or lifestyle modification and management of comorbidities such as hypertension, diabetes, obesity, etc. However, statin is associated with many side effects, including increased risk of diabetes, liver dysfunction / injury, hemorrhagic stroke, muscle damage, neuropathy, pancreatic dysfunction and sexual dysfunction.In addition, many subjects cannot reach target cholesterol level with statin alone, and it has been shown that statin increases the level of PCSK9, which promotes the degradation of LDLR.Therefore, there is a need in the art for alternative treatment methods for the subject with lipid imbalance and related conditions. [Means for solving the problem]

[0006] The present invention provides an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the myosin regulatory light chain-interacting protein (MYLIP) gene. The MYLIP gene may be present in a cell, for example, in a cell of 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 would benefit from inhibiting and / or inhibiting or reducing the expression of the MYLIP gene, for example, subjects suffering from or susceptible to MYLIP-related diseases, such as lipid imbalances (e.g., hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, etc.) or pathological conditions associated therewith (e.g., atherosclerosis, coronary heart disease, other cardiovascular disorders, etc.).

[0007] Thus, in one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell. The dsRNA agent comprises a sense strand and an antisense strand, wherein 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, 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. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, and comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2.

[0008] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell. The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding MYLIP comprising at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from any one of the antisense sequences listed in Tables 3 or 4. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding MYLIP comprising at least 15 contiguous nucleotides of any one of the antisense sequences listed in Tables 3 or 4.

[0009] In one embodiment, the region of complementarity is selected from nucleotides 113-133, 161-181, 190-210, 235-255, 265-285, 286-306, 312-332, 329-349, 343-363, 359-379, 380-400, 397-417, 424-444, 454-474, 468-488, 487-507, 506-526, 520-540, 537-557, 553-573, 580-600, 613-633, 640-660, 667-687, 681-701, 697-717, 711-731, 746 of SEQ ID NO: 1. ~766, 760~780, 774~794, 789~809, 811~831, 826~846, 857~877, 874~894, 888~908, 904~924, 934~954, 954~974, 972~992, 991~1011, 1028~1048, 1052~ 1072, 1066~1086, 1085~1105, 1105~1125, 1129~1149, 1156~1176, 1190~1210, 1223~1243, 1247~1267, 1268~1288, 1282~1302, 1296~1316, 1357~1377, 1371~1391, 1389~1409, 1416~1436, 1432~1452, 1446~1466, 1475~1495, 1490~1510, 1507~1527, 1522~1542, 1536~1556, 1550~1570, 1570~1590, 1584~ 1604, 1598~1618, 1614~1634, 1632~1652, 1647~1667, 1661~1681, 1675~1695, 1714~1734, 1749~1769, 1765~1785, 1802~1822, 1832~1852, 1855~1875, 1872~1892, 1905~1925, 1921~1941, 1940~1960, 1965~1985, 1996~2016, 2021~2041, 2040~2060, 2059~2079, 2073~2093, 2088~2108, 2110~2130, 2125~ 2145, 2157~2177, 2172~2192, 2192~2212, 2216~2236, 2256~2276, 2277~2297, 2293~2313, 2335~2355, 2349~2369, 2369~2389, 2395~2415, 2417~2437,2442~2462, 2462~2482, 2482~2502, 2496~2516, 2513~2533, 2529~2549, 2545~2565, 2587~2607, 2601~2621, 2666~2686, 2682~2702, 2696~2716, 2720~2740, 2736~2756, 2750~2770, 2777~2797, 2793~2813, 2808~2828, 2823~2843, 283 7 to 2857, 2854 to 2874, 2868 to 2888, 2891 to 2911, 2918 to 2938, 2938 to 2958, 2958 to 2978, 2999 to 3019, 3013 to 3033, 3030 to 3050, or 3045 to 3065. It contains at least 15 consecutive nucleotides that differ by 1, 2, or 3 nucleotides or less from nucleotides that are perfectly complementary to any 15 consecutive nucleotides located within 2857 to 2857, 2854 to 2874, 2868 to 2888, 2891 to 2911, 2918 to 2938, 2938 to 2958, 2958 to 2978, 2999 to 3019, 3013 to 3033, 3030 to 3050, or 3045 to 3065.

[0010] In some embodiments, the region of complementarity is selected from nucleotides 113-133, 161-181, 190-210, 235-255, 265-285, 286-306, 312-332, 329-349, 343-363, 359-379, 380-400, 397-417, 424-444, 454-474, 468-488, 487-507, 506-526, 520-540, 537-557, 553-573, 580-600, 613-633, 640-660, 667-687, 681-701, 697-717, 711-731, 74 6~766, 760~780, 774~794, 789~809, 811~831, 826~846, 857~877, 874~894, 888~908, 904~924, 934~954, 954~974, 972~992, 991~1011, 1028~1048, 1052 ~1072, 1066~1086, 1085~1105, 1105~1125, 1129~1149, 1156~1176, 1190~1210, 1223~1243, 1247~1267, 1268~1288, 1282~1302, 1296~1316, 1357~1377 , 1371~1391, 1389~1409, 1416~1436, 1432~1452, 1446~1466, 1475~1495, 1490~1510, 1507~1527, 1522~1542, 1536~1556, 1550~1570, 1570~1590, 1584 ~1604, 1598~1618, 1614~1634, 1632~1652, 1647~1667, 1661~1681, 1675~1695, 1714~1734, 1749~1769, 1765~1785, 1802~1822, 1832~1852, 1855~1875 , 1872~1892, 1905~1925, 1921~1941, 1940~1960, 1965~1985, 1996~2016, 2021~2041, 2040~2060, 2059~2079, 2073~2093, 2088~2108, 2110~2130, 2125 ~2145, 2157~2177, 2172~2192, 2192~2212, 2216~2236, 2256~2276, 2277~2297, 2293~2313, 2335~2355, 2349~2369, 2369~2389, 2395~2415, 2417~2437,2442~2462, 2462~2482, 2482~2502, 2496~2516, 2513~2533, 2529~2549, 2545~2565, 2587~2607, 2601~2621, 2666~2686, 2682~2702, 2696~2716, 2720~2740, 2736~2756, 2750~2770, 2777~2797, 2793~2813, 2808~2828 , 2823 to 2843, 2837 to 2857, 2854 to 2874, 2868 to 2888, 2891 to 2911, 2918 to 2938, 2938 to 2958, 2958 to 2978, 2999 to 3019, 3013 to 3033, 3030 to 3050, or 3045 to 3065.

[0011] In one embodiment, the region of complementarity is selected from nucleotides 111-133, 159-181, 188-210, 233-255, 263-285, 284-306, 310-332, 327-349, 341-363, 357-379, 378-400, 395-417, 422-444, 452-474, 466-488, 485-507, 504-526, 518-540, 535-557, 551-573, 578-600, 611-633, 638-660, 665-687, 679-701, 695-717, 709-731, 744 of SEQ ID NO: 1. ~766, 758~780, 772~794, 787~809, 809~831, 824~846, 855~877, 872~894, 886~908, 902~924, 932~954, 952~974, 970~992, 989~1011, 1026~1048, 1050~ 1072, 1064~1086, 1083~1105, 1103~1125, 1127~1149, 1154~1176, 1188~1210, 1221~1243, 1245~1267, 1266~1288, 1280~1302, 1294~1316, 1355~1377, 1369~1391, 1387~1409, 1414~1436, 1430~1452, 1444~1466, 1473~1495, 1488~1510, 1505~1527, 1520~1542, 1534~1556, 1548~1570, 1568~1590, 1582~ 1604, 1596~1618, 1612~1634, 1630~1652, 1645~1667, 1659~1681, 1673~1695, 1712~1734, 1747~1769, 1763~1785, 1800~1822, 1830~1852, 1853~1875, 1870~1892, 1903~1925, 1919~1941, 1938~1960, 1963~1985, 1994~2016, 2019~2041, 2038~2060, 2057~2079, 2071~2093, 2086~2108, 2108~2130, 2123~ 2145, 2155~2177, 2170~2192, 2190~2212, 2214~2236, 2254~2276, 2275~2297, 2291~2313, 2333~2355, 2347~2369, 2367~2389, 2393~2415, 2415~2437,2440~2462, 2460~2482, 2480~2502, 2494~2516, 2511~2533, 2527~2549, 2543~2565, 2585~2607, 2599~2621, 2664~2686, 2680~2702, 2694~2716, 2718~2740, 2734~2756, 2748~2770, 2775~2797, 2791~2813, 2806~2828, 2821~2843, 283 2852 to 2874, 2866 to 2888, 2889 to 2911, 2916 to 2938, 2936 to 2958, 2956 to 2978, 2997 to 3019, 3011 to 3033, 3028 to 3050, or 3043 to 3065.

[0012] In some embodiments, the region of complementarity is selected from nucleotides 111-133, 159-181, 188-210, 233-255, 263-285, 284-306, 310-332, 327-349, 341-363, 357-379, 378-400, 395-417, 422-444, 452-474, 466-488, 485-507, 504-526, 518-540, 535-557, 551-573, 578-600, 611-633, 638-660, 665-687, 679-701, 695-717, 709-731, 74 4~766, 758~780, 772~794, 787~809, 809~831, 824~846, 855~877, 872~894, 886~908, 902~924, 932~954, 952~974, 970~992, 989~1011, 1026~1048, 1050 ~1072, 1064~1086, 1083~1105, 1103~1125, 1127~1149, 1154~1176, 1188~1210, 1221~1243, 1245~1267, 1266~1288, 1280~1302, 1294~1316, 1355~1377 , 1369~1391, 1387~1409, 1414~1436, 1430~1452, 1444~1466, 1473~1495, 1488~1510, 1505~1527, 1520~1542, 1534~1556, 1548~1570, 1568~1590, 1582 ~1604, 1596~1618, 1612~1634, 1630~1652, 1645~1667, 1659~1681, 1673~1695, 1712~1734, 1747~1769, 1763~1785, 1800~1822, 1830~1852, 1853~1875 , 1870~1892, 1903~1925, 1919~1941, 1938~1960, 1963~1985, 1994~2016, 2019~2041, 2038~2060, 2057~2079, 2071~2093, 2086~2108, 2108~2130, 2123 ~2145, 2155~2177, 2170~2192, 2190~2212, 2214~2236, 2254~2276, 2275~2297, 2291~2313, 2333~2355, 2347~2369, 2367~2389, 2393~2415, 2415~2437,2440~2462, 2460~2482, 2480~2502, 2494~2516, 2511~2533, 2527~2549, 2543~2565, 2585~2607, 2599~2621, 2664~2686, 2680~2702, 2694~2716, 2718~2740, 2734~2756, 2748~2770, 2775~2797, 2791~2813, 2806~2828 , 2821 to 2843, 2835 to 2857, 2852 to 2874, 2866 to 2888, 2889 to 2911, 2916 to 2938, 2936 to 2958, 2956 to 2978, 2997 to 3019, 3011-3033, 3028 to 3050, or 3043 to 3065.

[0013] In one embodiment, the sense strand comprises nucleotides 113 to 133, 161 to 181, 190 to 210, 235 to 255, 265 to 285, 286 to 306, 312 to 332, 329 to 349, 343 to 363, 359 to 379, 380 to 400, 397 to 417, 424 to 444, 454 to 474, 468 to 488, 487 to 507, 506 to 526, 520 to 540, 537 to 557, 553 to 573, 580 to 600, 613 to 633, 640 to 660, 667 to 687, 681 to 701, 697 to 717, 711 to 731, 746 to 750, 751 to 760, 761 to 770, 771 to 780, 781 to 790, 791 to 800, 801 to 810, 811 to 820, 821 to 830, 822 to 830, 831 to 840, 832 to 842, 833 to 850, 834 to 852, 835 to 856, 837 to 860, 841 to 850, 842 to 854, 843 to 865, 844 to 856, 845 to 858, 846 to 869, 850 to 850, 66, 760-780, 774-794, 789-809, 811-831, 826-846, 857-877, 874-894, 888-908, 904-924, 934-954, 954-974, 972-992, 991-1011, 1028-1048, 1052-1060 072, 1066~1086, 1085~1105, 1105~1125, 1129~1149, 1156~1176, 1190~1210, 1223~1243, 1247~1267, 1268~1288, 1282~1302, 1296~1316, 1357~1377, 1 371~1391, 1389~1409, 1416~1436, 1432~1452, 1446~1466, 1475~1495, 1490~1510, 1507~1527, 1522~1542, 1536~1556, 1550~1570, 1570~1590, 1584~ 1604, 1598~1618, 1614~1634, 1632~1652, 1647~1667, 1661~1681, 1675~1695, 1714~1734, 1749~1769, 1765~1785, 1802~1822, 1832~1852, 1855~1875, 1872~1892, 1905~1925, 1921~1941, 1940~1960, 1965~1985, 1996~2016, 2021~2041, 2040~2060, 2059~2079, 2073~2093, 2088~2108, 2110~2130, 2125~ 2145, 2157~2177, 2172~2192, 2192~2212, 2216~2236, 2256~2276, 2277~2297, 2293~2313, 2335~2355, 2349~2369, 2369~2389, 2395~2415, 2417~2437,2442~2462, 2462~2482, 2482~2502, 2496~2516, 2513~2533, 2529~2549, 2545~2565, 2587~2607, 2601~2621, 2666~2686, 2682~2702, 2696~2716, 2720~2740, 2736~2756, 2750~2770, 2777~2797, 2793~2813, 2808~2828, 2823~2843, 2837~2857, 2854~2874, 2868~288 The antisense strand comprises at least 15 consecutive nucleotides that differ by 1, 2, or no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 8, 2891-2911, 2918-2938, 2938-2958, 2958-2978, 2999-3019, 3013-3033, 3030-3050, and 3045-3065, and the antisense strand comprises at least 15 consecutive nucleotides that differ by 1, 2, or no more than 3 nucleotides from the complementary nucleotide sequence of SEQ ID NO: 2.

[0014] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0015] 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.

[0016] In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell is provided. The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 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 by no more than 1, 2, or 3 nucleotides, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in 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 that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached to its 3' end.

[0017] 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.

[0018] 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 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.

[0019] In one embodiment, the nucleotide modifications are 2'-O-methyl and / or 2'-fluoro modifications.

[0020] 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.

[0021] 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, and each strand is independently 21-23 nucleotides in length.

[0022] The dsRNA may comprise at least one strand comprising a 3' overhang of at least 1 nucleotide, or at least one strand comprising a 3' overhang of at least 2 nucleotides.

[0023] In some embodiments, the dsRNA agent further comprises a ligand.

[0024] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0025] In one embodiment, the ligand is an N-acetylgalactosamine (Ga1NAc) derivative.

[0026] In one embodiment, the ligand is

[0027] [ka] is.

[0028] In one embodiment, the dsRNA agent has the following structure:

[0029] [ka] wherein X is O or S. The compound is conjugated to a ligand as shown in

[0030] In one embodiment, X is O.

[0031] In one embodiment, the region of complementarity comprises any one of the antisense sequences in Tables 3 or 4.

[0032] In one embodiment, the invention provides a duplex for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, wherein 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 MYLIP, 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 represent 0 to 6; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides that are independently modified or unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; Each n may or may not exist 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 differs 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.

[0033] 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.

[0034] 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'.

[0035] In one embodiment, the YYY motif is present at or near the cleavage site on the sense strand, eg, the Y'Y'Y' motif is present at positions 11, 12, and 13 on the antisense strand from the 5' end.

[0036] 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) is expressed by

[0037] In another embodiment, Formula (Ii) may be represented by 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] is expressed by

[0038] 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] is expressed by

[0039] 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, and each N a and N a ' independently represents an oligonucleotide containing 2 to 10 modified nucleotides] is expressed by

[0040] 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.

[0041] Each strand can be 30 nucleotides or less in length, for example, each strand is independently 19-30 nucleotides in length.

[0042] 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.

[0043] In one embodiment, the modification on the nucleotide is a 2'-O-methyl or a 2'-fluoro modification.

[0044] In one embodiment, Y' is a 2'-O-methyl or 2'-fluoro modified nucleotide.

[0045] 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.

[0046] In one embodiment, the dsRNA agent may further include at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0047] 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.

[0048] 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.

[0049] In one embodiment, the strand is the antisense strand, hi another embodiment, the strand is the sense strand.

[0050] In one embodiment, phosphorothioate or methylphosphonate internucleotide linkages are present at both the 5' and 3' ends of one strand.

[0051] 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.

[0052] In one embodiment, p'>0. In another embodiment, p'=2.

[0053] 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.

[0054] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0055] 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.

[0056] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0057] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0058] In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0059] In one embodiment, the ligand is

[0060] [ka] is.

[0061] In one embodiment, the dsRNA agent has the following structure:

[0062] [ka] wherein X is O or S. The compound is conjugated to a ligand as shown in

[0063] In one embodiment, X is O.

[0064] In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 2 and shown below.

[0065] [ka]

[0066] In some embodiments, the RNAi agent is, for example, uL96, shown below:

[0067] [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).

[0068] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding MYLIP, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having a structure 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 represent 0 to 6; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides that are independently modified or unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which are independently modified or unmodified, or a combination thereof; Each n may or may not exist 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 differs 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.

[0069] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding MYLIP, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having a structure 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; Each n may or may not exist 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 to 25 nucleotides that are independently modified or unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 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 differs 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.

[0070] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding MYLIP, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having a structure 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; Each n may or may not exist 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 to 25 nucleotides that are independently modified or unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and Nb ' represents an oligonucleotide sequence containing 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 differs 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, which is one or more GalNAc derivatives attached through a monovalent, divalent, or trivalent branched linker.

[0071] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding MYLIP, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having a structure 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; Each n may or may not existp , 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 to 25 nucleotides that are independently modified or unmodified, or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 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 differs from the modification on N b ' the modification on Y' is different from the modification on Y', wherein 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.

[0072] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding MYLIP, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having a structure represented by formula (Ii): Sense:5'np -N a -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 exist 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 to 25 nucleotides that are independently modified or unmodified, or a combination thereof, and each sequence contains 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, the sense strand comprising at least one phosphorothioate linkage, and the sense strand conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0073] In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell is provided. The dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein 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, 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, wherein substantially all of the nucleotides of the sense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end and substantially all of the nucleotides of the antisense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and wherein the sense strand is conjugated at the 3' end 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 that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2, wherein substantially all of the nucleotides of the sense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end, and wherein substantially all of the nucleotides of the antisense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and wherein the sense strand is conjugated at the 3' end to one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0074] In one embodiment, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

[0075] In one embodiment, the region of complementarity comprises any one of the antisense sequences listed in Tables 3 or 4.

[0076] In one embodiment, the drug is AD-1753226、AD-1753250、AD-1753259、AD-1753324、AD-1753346、AD-1753413、AD-1753439、AD-1753456、AD-1753520、AD-1753536、 AD-1753557、AD-1753624、AD-1753651、AD-1753661、AD-1753725、AD-1753744、AD-1753813、AD-1753827、AD-1753844、AD-1753860、AD-1753917、AD- 1753949、AD-1754026、AD-1754053、AD-1754117、AD-1754133、AD-1754147、AD-1754162、AD-1754226、AD-1754240、AD-1754255、AD-1754327、AD-175 4342、AD-1754353、AD-1754420、AD-1754434、AD-1754450、AD-1754530、AD-1754550、AD-1754618、AD-1754637、AD-1754661、AD-1754735、AD-175474 9、AD-1754818、AD-1754838、AD-1754862、AD-1754939、AD-1755013、AD-1755046、AD-1755120、AD-1755141、AD-1755155、AD-1755219、AD-1755326、A D-1755340、AD-1755358、AD-1755435、AD-1755451、AD-1755515、AD-1755539、AD-1755554、AD-1755621、AD-1755636、AD-1755650、AD-1755714、AD-1 755734、AD-1755747、AD-1755761、AD-1755827、AD-1755830、AD-1755845、AD-1755859、AD-1755923、AD-1755943、AD-1756028、AD-1756044、AD-1756 131、AD-1756146、AD-1756219、AD-1756236、AD-1756319、AD-1756335、AD-1756354、AD-1756429、AD-1756439、AD-1756514、AD-1756533、AD-1756552、AD-1756616, AD-1756631, AD-1756653, AD-1756718, AD-1756749, AD-1756814 , AD-1756834, AD-1756858, AD-1756915, AD-1756932, AD-1756947, AD-175701 9, AD-1757032, AD-1757052, AD-1757124, AD-1757146, AD-1757171, AD-17571 91, AD-1757211, AD-1757225, AD-1757242, AD-1757258, AD-1757274, AD-1757 278, AD-1757292, AD-1757308, AD-1757323, AD-1757337, AD-1757361, AD-1757377, AD-1757391, AD-1757418, AD-1757434, AD-1757449, AD-1757464, AD-1757478, AD-1757495, AD-1757509, AD-1757532, AD-1757544, AD-1757564, AD-1757583, AD-1757603, AD-1757617, AD-1757634 and AD-1757649.

[0077] 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 3 or 4.

[0078] In another embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a duplex region. The sense strand comprises the nucleotide sequence of any one of the agents in Table 3 or 4, and the antisense strand comprises the nucleotide sequence of any one of the agents in Table 3 or 4. Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the dsRNA agent is conjugated to a ligand.

[0079] In various embodiments of the dsRNA agent, the dsRNA agent targets a hotspot region of the mRNA encoding MYLIP. In one embodiment, the hotspot region comprises nucleotides 341-417 of SEQ ID NO: 1. The dsRNA agent may be selected from the group consisting of AD-1753624, AD-1753557, AD-1753536, and AD-1753520.

[0080] In another embodiment, the invention provides dsRNA agents that target hotspot regions of myosin regulatory light chain-interacting protein (MYLIP) mRNA.

[0081] 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).

[0082] In one aspect, the present invention provides a method for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, comprising contacting the cell with a dsRNA agent or pharmaceutical composition of the present invention, thereby inhibiting expression of MYLIP in the cell.

[0083] The cell may be in a subject, such as a human subject.

[0084] In one embodiment, MYLIP 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% relative to control levels, or to below the level of detection of MYLIP expression.

[0085] In one embodiment, the human subject is suffering from a MYLIP-related disease, disorder, or condition. In one embodiment, the MYLIP-related disease, disorder, or condition is a lipid imbalance (e.g., hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, etc.) or a pathological condition associated therewith (e.g., atherosclerosis, coronary heart disease, other cardiovascular disorders, etc.). The hypercholesterolemia can be hyper-LDL cholesterolemia.

[0086] In one aspect, the invention provides a method for inhibiting expression of MYLIP in a subject, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, thereby inhibiting expression of MYLIP in the subject.

[0087] In another aspect, the present invention provides a method for treating a subject suffering from a MYLIP-related disease, disorder or condition.The method comprises administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject suffering from a MYLIP-related disease, disorder or condition.The MYLIP-related disease, disorder or condition can be any one of the diseases, disorders or conditions described above.

[0088] In another aspect, the present invention provides a method for preventing at least one symptom in a subject with a disease, disorder or condition that would benefit from reducing the expression of the MYLIP gene.The method comprises administering to the subject a preventively effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby preventing at least one symptom in a subject with a disease, disorder or condition that would benefit from reducing the expression of the MYLIP gene.

[0089] In another aspect, the present invention provides the method for reducing the risk of developing cardiovascular disease (such as atherosclerosis) or the risk of aggravating cardiovascular disease in subject.The method comprises administering to subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby reducing the risk of developing cardiovascular disease or the risk of aggravating cardiovascular disease in subject.

[0090] In yet another aspect, the present invention provides a method for reducing the plasma cholesterol level in a subject with elevated plasma low-density lipoprotein (LDL) cholesterol.The method comprises administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention to the subject, thereby reducing the plasma cholesterol level in the subject.

[0091] In one embodiment, the methods and uses of the present invention further comprise administering to the subject an additional therapeutic agent. The additional therapeutic agent may be a statin.

[0092] In certain embodiments, the subject can be administered with a therapeutic amount of dsRNA such as about 0.01mg / kg to about 200mg / kg.In other embodiments, the subject can be administered with a therapeutic amount of dsRNA such as about 0.01mg / kg to about 500mg / kg.In still other embodiments, the subject can be administered with a therapeutic amount of dsRNA of about 500mg / kg or more.

[0093] 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.

[0094] The agent is administered to the subject intravenously, intramuscularly, or subcutaneously. In one embodiment, the agent is administered to the subject subcutaneously.

[0095] In one embodiment, the methods and uses of the present invention further comprise determining the level of MYLIP in the subject.

[0096] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a duplex region, wherein the sense strand comprises the nucleotide sequence of any one of the agents in Table 3 or 4, and the antisense strand comprises the nucleotide sequence of any one of the agents in Table 3 or 4, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and wherein the dsRNA agent is conjugated to a ligand. DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention provides an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the MYLIP gene. The MYLIP gene can be located in a cell, for example, in a cell of 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 the MYLIP gene and to treat subjects who would benefit from inhibiting or reducing the expression of the MYLIP gene, for example, subjects suffering from or susceptible to MYLIP-related diseases, disorders or conditions, for example, subjects suffering from or susceptible to lipid imbalance (e.g., hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, etc.) or pathological conditions related thereto (e.g., atherosclerosis, coronary heart disease, other cardiovascular disorders, etc.).

[0098] The iRNA of the present invention targeting MYLIP may be 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, 19-26, 19-27, 19-28, 19-29 ...30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-30, 19-3 The RNA strand may comprise 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, which is substantially complementary to at least a portion of an mRNA transcript of the MYLIP gene.

[0099] 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 a MYLIP 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.

[0100] The use of the iRNA agents described herein allows for the targeted degradation of the mRNA of the MYLIP gene in mammals.

[0101] In particular, very low doses of iRNAs can specifically and efficiently mediate RNA interference (RNAi) to significantly inhibit the expression of the MYLIP gene. Thus, methods and compositions comprising these iRNAs are useful for treating subjects who would benefit from inhibiting or reducing the expression of the MYLIP gene, such as subjects suffering from or susceptible to a MYLIP-related disease, disorder, or condition, such as subjects suffering from or susceptible to a lipid imbalance (e.g., hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, etc.) or a pathological condition related thereto (e.g., atherosclerosis, coronary heart disease, other cardiovascular disorders, etc.).

[0102] The following detailed description discloses how to make and use compositions containing iRNA to inhibit expression of the MYLIP gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibiting and / or reducing expression of this gene.

[0103] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Additionally, 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.

[0104] 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.

[0105] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0106] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0107] The term "about" is used herein to mean within a range that is typical 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 number or range in the series.

[0108] 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 specified property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each number in the series or range.

[0109] As used herein, "no more than" or "or less" refers to the value next to the phrase and to zero, as the logical lower value or integer, as is logical from the context. For example, a duplex with an overhang of "two or fewer nucleotides" has 2, 1, or 0 nucleotides overhang. When "or less" is present before a series of numbers or a range, it is understood that "or less" can modify each number in the series or range. As used herein, a range includes both upper and lower limits.

[0110] As used herein, the term "at least about," when referring to measurable values ​​such as parameters and amounts, encompasses variations of + / -20%, such as + / -10%, + / -5%, or + / -1% from the specified value, so long as such variations are appropriate for practicing the disclosed invention. For example, inhibition of expression of the MYLIP gene by "at least about 25%" means that inhibition of expression of the MYLIP gene can be measured to be any value + / -20% of the specified 25%, i.e., 20%, 30%, or any intermediate value between 20% and 30%.

[0111] 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 the same non-modulated cell, tissue or system as 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 the expression of the gene, RNA and / or protein described above that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, or 5-fold or more than that observed in the absence of the RNAi agent.The percentage and / or fold difference can be expressed relative to the control level, for example,

[0112]

number

[0113] 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.

[0114] In the event of a conflict between a shown target site and the nucleotide sequence for the sense or antisense strand, the shown sequence takes precedence.

[0115] In the event of a conflict between a chemical structure and a chemical name, the chemical structure shall prevail.

[0116] Unless otherwise specified, the term "MYLIP," also known as "IDOL (Inducible Degrader of the LDL receptor)," and "MIR (Modulator of Immune Recognition)," refers to the well-known gene encoding myosin regulatory light chain-interacting protein from any vertebrate or mammalian source, including, without limitation, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig.

[0117] The term also refers to fragments and variants of native MYLIP that maintain at least one in vivo or in vitro activity of native MYLIP. The term encompasses the full-length, unprocessed precursor form of MYLIP, as well as mature forms that result from post-translational cleavage of the signal peptide and forms that result from proteolytic processing.

[0118] The human MYLIP gene has eight exons. The nucleotide and amino acid sequence of the human MYLIP transcript can be found, for example, in GenBank reference sequence NM_013262.4 (SEQ ID NO: 1, reverse complement SEQ ID NO: 2).

[0119] The MYLIP gene is located in chromosomal region 6p22.3. The nucleotide sequence of the genomic region of a human chromosome containing the MYLIP gene can be found, for example, in Genome Reference Consortium Human Build 38 (also referred to as human genome build38 or GRCh38), available at GenBank. The nucleotide sequence of the genomic region of human chromosome 6 containing the MYLIP gene can also be found, for example, in GenBank accession number NC_000006.12, which corresponds to nucleotides 16,129,086 to 16,151,015 of human chromosome 6.

[0120] The nucleotide and amino acid sequence of the mouse MYLIP transcript can be found, for example, in GenBank reference sequence NM_153789.3 (SEQ ID NO: 3, reverse complement SEQ ID NO: 4).

[0121] The nucleotide and amino acid sequence of the rat MYLIP transcript can be found, for example, in GenBank reference sequence NM_001107344.2 (SEQ ID NO: 5, reverse complement SEQ ID NO: 6).

[0122] The nucleotide and amino acid sequence of the rhesus monkey MYLIP transcript can be found, for example, in GenBank reference sequence NM_001261795.2 (SEQ ID NO: 7, reverse complement SEQ ID NO: 8).

[0123] There are two predicted transcript variants of the cynomolgus monkey MYLIP gene. The nucleotide and amino acid sequence of the cynomolgus monkey MYLIP transcript variant X1 can be found, for example, in GenBank reference sequence XM_005553979.2 (SEQ ID NO: 9, reverse complement SEQ ID NO: 10). The nucleotide and amino acid sequence of the cynomolgus monkey MYLIP transcript variant X2 can be found, for example, in GenBank reference sequence XM_005553980.2 (SEQ ID NO: 11, reverse complement SEQ ID NO: 12).

[0124] Further examples of MYLIP mRNA sequences can be easily accessed using publicly available databases, such as GenBank, UniProt and OMIM. Further information about MYLIP can be found, for example, at https: / / www.ncbi.nlm.nih.gov / gene / 29116. The term MYLIP as used herein also refers to the MYLIP gene variants listed in clinical variant databases, such as http: / / www.ncbi.nlm.nih.gov / clinvar / ?term=MYLIP[gene].

[0125] The term "MYLIP," as used herein, also refers to a specific polypeptide expressed in a cell due to naturally occurring DNA sequence variations of the MYLIP gene, for example, a single nucleotide polymorphism in the MYLIP gene. Many SNPs within the MYLIP gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0126] Myosin regulatory light chain-interacting protein (MYLIP) is an E3 ubiquitin ligase that directly binds to the cytoplasmic tail of the low-density lipoprotein (LDL) receptor (LDLR) and promotes its ubiquitination by the UBE2D1 / E1 complex (Hong, et al. (2014) Cell Metab. 20(5):910-918 (doi: 10.1016 / j.cmet.2014.10.001)). Upon ubiquitination, LDLR enters the multivesicular body (MVB) protein sorting pathway and is shuttled to lysosomes for degradation. LDLR is a cell surface receptor primarily expressed in the liver that recognizes apoprotein B100 embedded in the outer phospholipid layer of LDL particles and mediates the endocytosis of cholesterol-rich LDL. This receptor also recognizes apoE proteins found on chylomicron and VLDL remnants (IDL) and promotes their degradation by APOER2 and VLDLR, suggesting a possible role for MYLIP in neuronal development and function. MYLIP is expressed in various tissues, including the thyroid gland, placenta, uterus, cervix, bone marrow, and some arteries, but at lower levels in the liver. MYLIP transcription is induced by liver X receptors (LXRs), thyroid hormone receptor-like nuclear receptors known for their role in regulating cholesterol, fatty acid, and glucose homeostasis. Induction of hepatic lipogenesis has been identified as an undesirable side effect of potential LXR agonist therapeutics. Genome-wide association studies have shown that polymorphisms in the MYLIP locus are associated with plasma LDL cholesterol levels in humans, and loss-of-function (LOF) mutations are associated with reduced LDL cholesterol levels, suggesting enhanced LDL clearance via the LDLR. (Hong et al (2014) Cell Metab. 20(5):910-918 (doi:10.1016 / j.cmet.2014.10.001)) Along with MYLIP, the proprotein convertase subtilisin / kexin type 9 (PCSK9) pathway, which promotes LDLR degradation, and the sterol regulatory element-binding protein (SREBP) pathway, which induces LDLR transcription, are also known to affect LDLR expression.(Zhang et al. (2012) Arterioscler Thromb Vasc Biol.32(11):2541-6(doi:10.1161 / ATVBAHA.112.250571)).

[0127] MYLIP is a 45-kDa cytoplasmic protein possessing two distinct protein domains: an N-terminal FERM domain and a C-terminal RING domain (Martinelli et al. (2020) J Biol Chem. 295(39):13570-13583 (doi: 10.1074 / jbc.RA120.014349)). The FERM domain interacts with the intracellular tail of the LDLR at the plasma membrane via a putative helix that coordinates the interaction of MYLIP with a conserved LDLR motif. The RING domain recruits E2 ubiquitin-conjugating enzymes to MYLIP, and both E2s from the UBC13 and UBE2D families have been shown to interact with MYLIP during LDLR degradation. The endocytic pathway followed by ubiquitinated LDLR is clathrin- and ARH-independent, distinct from the pathway utilized by PCSK9. (Zhang et al. (2012) Arterioscler Thromb Vasc Biol. 32(11):2541-6 (doi: 10.1161 / ATVBAHA.112.250571)).

[0128] Acute hepatic overexpression of MYLIP in mice has been shown to reduce LDLR protein levels and increase plasma cholesterol levels, whereas targeted deletion of MYLIP in cultured mouse cells increased LDLR protein levels and LDL uptake, which was additive to the effects of statin treatment. However, LXR activation has been shown to increase plasma LDL levels in primates but not in mice, indicating a stronger level of MYLIP upregulation in primates than in mice. (Zelcer et al. (2009) Science. 3;325(5936):100-4 (doi: 10.1126 / science.1168974); Hong, et al. (2014) Cell Metab. 20(5):910-918 (doi: 10.1016 / j.cmet.2014.10.001)).

[0129] As used herein, "target sequence" refers to a contiguous portion of a nucleotide sequence in an mRNA molecule formed upon transcription of a MYLIP gene, e.g., an mRNA that is the product of RNA processing of a 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 an mRNA molecule formed upon transcription of a MYLIP gene.

[0130] The target sequence of the MYLIP gene may be about 9 to 36 nucleotides in length, e.g., 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.

[0131] As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0132] " G ", " C ", " A ", " T " and " U " generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as 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 parts (see, for example, Table 2).Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other parts without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement part.For example, but not limited to, the nucleotide that contains inosine as its base can form base pairs with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA that is characterized in the present invention. In another example, adenine and cytosine in any of the oligonucleotides can be substituted with guanine and uracil, respectively, to form G-U Wobble base pairs with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured in the present invention.

[0133] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," used interchangeably herein, refer to an agent that contains RNA, as 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 inhibits the expression of the MYLIP gene in cells, for example, in cells within a subject, for example, a mammalian subject.

[0134] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a MYLIP 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, an RNase 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 the siRNA duplex, allowing complementary antisense strands to induce target recognition [Nykanen, et al., (2001) Cell 107:309].When 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].Therefore, in one aspect, the present invention relates to the single-stranded RNA (sssiRNA) that is produced in cells, promotes the formation of RISC complex, and thereby silences target gene, i.e., MYLIP gene.Therefore, the term " siRNA " is used herein to also mean the RNAi described above.

[0135] 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 and then cleave the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAi agents are 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 the single-stranded siRNA described herein or as the single-stranded siRNA chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0136] 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 a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel, substantially complementary nucleic acid strands, said to have a "sense" or "antisense" orientation with respect to the target RNA, i.e., the MYLIP 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.

[0137] Generally, most of the nucleotides in each strand of dsRNA molecule are ribonucleotides, but as described herein in detail, each strand or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.In addition, as used herein, " RNAi agent " can include ribonucleotides with chemical modifications; RNAi agent can contain 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.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional groups or atoms, etc., in internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present invention include all types of modifications disclosed herein or known in the art.Any such modifications used in siRNA type molecules are encompassed by " RNAi agent " for the purpose of this specification and claims.

[0138] The duplex region may be of any length that allows for specific degradation of the desired target RNA through the RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15 to 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 to 30, 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, or 20 base pairs in length. , 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 above-listed ranges and lengths are also contemplated as part of the invention.

[0139] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and can 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, the connecting RNA is called a "hairpin loop." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can 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 can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0140] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and these molecules can, but do not necessarily, be covalently linked. When the two strands are covalently linked by means other than an uninterrupted chain 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 also contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide. In another embodiment, at least one strand comprises 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 still other embodiments, the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0141] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which contains fewer than 30 nucleotides, e.g., 17-27, 19-27, 17-25, 19-25, or 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a MYLIP target mRNA sequence, to induce cleavage of the target RNA. In another embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which contains 19-23 nucleotides that interacts with a target RNA sequence, e.g., a MYLIP target mRNA sequence, to induce cleavage of the target RNA. In one embodiment, the sense strand is 21 nucleotides in length. In another embodiment, the antisense strand is 23 nucleotides in length.

[0142] 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, if the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa, a nucleotide overhang exists.A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides.A nucleotide overhang can comprise 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 at the 5'-end, 3'-end, or both of the antisense strand or sense strand of dsRNA.

[0143] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and / or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and / or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0144] In certain embodiments, the overhang on the sense strand, the antisense strand, or both strands may comprise an extended length of more than 10 nucleotides, e.g., 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on 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 on 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.

[0145] The term "blunt" or "blunt-ended" as used herein in reference to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA can be blunt. When 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 are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0146] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., a MYLIP mRNA.

[0147] As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a MYLIP nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, the mismatch may be in an internal region or a terminal region of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., at the 5' and / or 3' ends of the iRNA. In some embodiments, a double-stranded RNA agent of the present invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the present invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the present invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the present invention contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within five, four, or three nucleotides from the 3' end of the iRNA. In other embodiments, the nucleotide mismatch is, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not present in the seed region.

[0148] 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.

[0149] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.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 that is bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0150] 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, 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 the second nucleotide sequence under certain conditions, as understood by those skilled in the art. Such conditions may be, for example, "stringent conditions," including, but not limited to, 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by a wash (see, e.g., "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 while minimizing the number of other sequences with which it hybridizes. Stringent conditions are sequence-dependent and vary under different circumstances, and the "stringent conditions" that antisense compounds hybridize to target sequences are determined by the nature and composition of antisense compounds and the assay that they are examined.Other conditions, such as physiologically relevant conditions that can be encountered in living organisms, can also be applied.Those skilled in the art will be able to determine the most suitable set of conditions for testing the complementarity of two sequences according to the final application of hybridized nucleotide.

[0151] The complementary sequence in iRNA, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the length of one or both nucleotide sequences.Such sequences can be referred to herein as " completely complementary " with respect to each other.However, when referred to herein as " substantially complementary " between the first sequence and the second sequence, the two sequences can be completely complementary, or can form one or more mismatched base pairs, generally 5, 4, 3 or 2 or less, when hybridizing for up to 30 double strands. In some embodiments, a "substantially complementary" sequence disclosed herein comprises a contiguous nucleotide sequence that is at least about 80% complementary to the corresponding region of the target MYLIP sequence throughout its entire length, e.g., 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, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is completely complementary to the shorter oligonucleotide, can still be considered "completely complementary" for the purposes described herein.

[0152] "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-naturally occurring 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.

[0153] The terms "complementary," "fully complementary," and "substantially complementary" are used herein as understood in connection with their use 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.

[0154] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding MYLIP). For example, a polynucleotide is complementary to at least a portion of a MYLIP mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding MYLIP.

[0155] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to a target MYLIP sequence (e.g., a human MYLIP sequence). In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target MYLIP sequence (e.g., a human MYLIP sequence) and comprise 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:1 or the equivalent region of a fragment of SEQ ID NO:1, throughout its entire length.

[0156] In one embodiment, the RNAi agent of the present invention is substantially complementary to an antisense polynucleotide that is further complementary to a target MYLIP sequence (e.g., a human MYLIP 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 or the equivalent region of any one of the fragments of SEQ ID NO:2, throughout its entire length.

[0157] In some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target mouse MYLIP sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to mouse MYLIP and comprise 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: 3 or the equivalent region of any one of the fragments of SEQ ID NO: 3, throughout its entire length.

[0158] In one embodiment, the RNAi agent of the present invention is substantially complementary to an antisense polynucleotide that is further complementary to a mouse MYLIP 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: 4 or the equivalent region of any one of the fragments of SEQ ID NO: 4, throughout its entire length.

[0159] In some embodiments, the iRNA of the invention comprises an antisense strand that is substantially complementary to a target MYLIP 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 Table 3 or 4, or the equivalent region of a fragment of any one of the sense strands in Table 3 or 4, throughout its length.

[0160] The term "inhibiting," as used herein, is used synonymously with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0161] The phrase "inhibiting expression of the MYLIP gene", as used herein, includes inhibition of expression of any MYLIP gene (e.g., mouse MYLIP gene, rat MYLIP gene, monkey MYLIP gene or human MYLIP gene), as well as variants or mutants of the MYLIP gene that encode a MYLIP protein.

[0162] "Inhibiting expression of the MYLIP gene" includes any level of inhibition of the MYLIP gene, e.g., at least partial suppression of the MYLIP gene, e.g., at least about 20% inhibition. In certain embodiments, 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.

[0163] The expression of the MYLIP gene may be evaluated based on the level of any variable associated with the expression of the MYLIP gene, for example, the MYLIP mRNA level or the MYLIP protein level. The expression of the MYLIP gene may be evaluated indirectly, for example, based on the enzyme activity of MYLIP in a tissue sample (e.g., GTPase-activating protein (GAP) activity) or the level of MYLIP-mediated signal transduction in a tissue sample, such as a liver sample. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level available in the art, for example, a pre-administration baseline level or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive drug control).

[0164] In one embodiment, at least partial suppression of expression of the MYLIP gene is assessed by a reduction in the amount of MYLIP mRNA, which can be isolated from or detected in a first cell or group of cells in which the MYLIP gene is transcribed and which has been treated or has been treated to inhibit expression of the MYLIP gene, 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.

[0165] The degree of inhibition may be expressed by the following formula:

[0166]

number

[0167] As used herein, the phrase " contacting cells with RNAi agent " such as dsRNA includes contacting cells by any possible means.Contacting cells with RNAi agent includes contacting cells with iRNA in vitro or contacting cells with iRNA in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cells by separately carrying out a method, or RNAi agent can be placed in a situation that can allow or cause it to contact cells later.

[0168] For example, cell can be contacted in vitro by incubating cell with RNAi agent.For example, cell can be contacted in vivo by injecting RNAi agent into the tissue where cell is located or nearby, or by injecting into another area, for example, bloodstream (i.e., intravenous) or subcutaneous cavity, so that the agent can reach the tissue where the cell to be contacted is located.For example, RNAi agent can comprise and / or be coupled to a ligand, such as GalNAc3, that directs RNAi agent to specific site, for example, liver.It is also possible to combine in vitro and in vivo methods for contacting.For example, cell can be contacted with RNAi agent in vitro, and then transferred to subject.

[0169] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into a cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. Introducing the 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 beta-glucan delivery systems, 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 can include methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below and / or known in the art.

[0170] The term "lipid-soluble substance" or "lipophilic moiety" 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 by the octanol / water partition coefficient, log K ow It 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 also be predicted by using coefficients attributed 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), 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 calculated based on its logK ow is 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 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.

[0171] The lipophilicity of a molecule can be varied 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.

[0172] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with 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, and then it can be positively correlated with the silencing activity of double-stranded RNAi agent.

[0173] In one embodiment, the plasma protein binding assay evaluated is an electrophoretic mobility shift assay (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 duplex was incubated with human serum albumin and the unbound fraction was determined. An exemplary assay protocol involves diluting the duplex at a stock concentration of 10 μM in 1×PBS containing 0, 20, or 90% serum to a final concentration of 0.5 μM (total volume of 20 μL). The samples are mixed, centrifuged for 30 seconds, and then incubated at room temperature for 10 minutes. After the incubation step is complete, 4 μL of 6×EMSA gel loading solution is 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). The gel 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 washed, 5 μL of SYBR Gold can be added to the gel, which is then incubated at room temperature for 10 minutes. The gel can then be washed again with 50 mL of 10% TBE. In this exemplary assay, the gel can be read using a 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 photograph of the gel is obtained, the image can be processed using Image Lab 5.2. Lanes and bands can be manually configured and band intensities can be measured. Band intensities for each sample can be normalized to PBS to obtain the fraction of unbound siRNA. This measurement allows for the determination of relative hydrophobicity.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.

[0174] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides increased hydrophobicity for enhanced in vivo delivery of the siRNA.

[0175] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., 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.

[0176] 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 a monkey or chimpanzee), 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).

[0177] In embodiments, the subject is a human, for example, a human being treated or assessed for a disease, disorder, or condition that would be expected to benefit from reduced MYLIP expression; a human being at risk for a disease, disorder, or condition that would be expected to benefit from reduced MYLIP expression; a human being with a disease, disorder, or condition that would be expected to benefit from reduced MYLIP expression; and / or a human being treated for a disease, disorder, or condition that would be expected to benefit from reduced MYLIP expression as described herein.

[0178] In another embodiment, the subject is homozygous for the MYLIP gene. Each allele of the gene may encode a functional MYLIP protein. In yet another embodiment, the subject is heterozygous for the MYLIP gene. The subject may have an allele that encodes a functional MYLIP protein and an allele that encodes a loss-of-function mutant of MYLIP. As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, including, without limitation, the alleviation or amelioration of one or more symptoms associated with MYLIP gene expression and / or MYLIP protein production. In some embodiments, a condition associated with MYLIP gene expression and / or MYLIP protein production may be a symptom of a disease or disorder whose pathology or cause is independent of MYLIP expression and / or MYLIP protein production, but which nonetheless can be compensated for / treated / countered by inhibiting MYLIP gene expression and / or MYLIP protein production, e.g., a MYLIP-associated disease, such as a lipid imbalance (e.g., hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, etc.) or a pathological condition related thereto (e.g., atherosclerosis, coronary heart disease, other cardiovascular disorders, etc.). "Treatment" can also mean prolonging survival as compared to expected survival if no treatment is administered.

[0179] The term "lower" in the context of the level of MYLIP gene expression or MYLIP protein production in a subject, or a disease marker or symptom, refers to a statistically significant decrease in the level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or below the level of detection in the relevant cells or tissue, e.g., liver cells, or other subject sample, e.g., blood, serum, or urine derived therefrom, relative to the detection method. In certain embodiments, the decrease is at least 20%.

[0180] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of the MYLIP gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with the disease, disorder, or condition, e.g., symptoms of MYLIP gene expression, such as lipid imbalance (e.g., hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, etc.) or pathological conditions associated therewith (e.g., atherosclerosis, coronary heart disease, other cardiovascular disorders, etc.). Failure to develop the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% of a magnitude clinically acceptable for the disease or disorder), or a delayed (e.g., a delay of days, weeks, months, or years) onset of delayed symptoms (e.g., reduced lipid accumulation in the liver and / or spread of lipid droplets in the liver) is considered effective prevention.

[0181] As used herein, the term "MYLIP-associated disease" refers to a disease or disorder caused by or associated with MYLIP gene expression or MYLIP protein production. The term "MYLIP-associated disease" includes diseases, disorders, or conditions that would benefit from reduced MYLIP gene expression or protein activity. For example, "MYLIP-associated disease" includes diseases or disorders that do not result from MYLIP gene expression and / or MYLIP protein production, but in which reduced MYLIP gene expression and / or MYLIP protein production can nonetheless alleviate the symptoms of the disease or disorder or prevent or correct its deleterious physiological effects. Subjects with or at risk for a MYLIP-associated disease or disorder include those who exhibit wild-type MYLIP gene expression and / or instead exhibit normal / healthy levels of MYLIP gene expression and MYLIP protein production. MYLIP-associated disease further includes diseases in which subjects harbor missense mutations and / or deletions in the MYLIP gene, or diseases in subjects with reduced MYLIP expression who would otherwise benefit from further reductions in MYLIP expression.

[0182] In one embodiment, a "MYLIP-related disorder" is a lipid imbalance (i.e., dyslipidemia). Dyslipidemia refers to abnormal amounts of lipids (e.g., triglycerides, cholesterol, and / or fatty phospholipids) in the blood, and includes, for example, hypercholesterolemia, hyperlipidemia, and hypertriglyceridemia.

[0183] "MYLIP-related disorders" 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, chylomya, mixed dyslipidemia (such as obesity, metabolic syndrome, diabetes), lipodystrophy, subcutaneous lipoatrophy, and dyslipidemias, including, for example, decreased LPL activity and / or LPL deficiency, decreased LDL receptor activity and / or LDL receptor deficiency, altered 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 conditions.

[0184] Other MYLIP-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 pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction or peripheral vascular disease.

[0185] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a subject with a MYLIP-related disease, disorder, or condition, is sufficient to effectively treat the disease (e.g., by attenuating, ameliorating, or maintaining the existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0186] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA that, when administered to a subject with a MYLIP-related disease, disorder, or condition, is sufficient to prevent or ameliorate the disease, or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the iRNA, how the agent is administered, the degree of risk of the disease, and other individual characteristics of the patient being treated, such as 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.

[0187] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses the 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 amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0188] The phrase "pharmaceutically 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 excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0189] The phrase "pharmaceutically acceptable carrier," as used herein, refers to a pharmaceutically 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 and not deleterious to the subject being treated. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, 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 wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, pepper, and the like. (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.

[0190] The term "sample," as used herein, encompasses similar fluids, cells, or tissues isolated from a subject, as well as collections of 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 can include samples from tissues, organs, or localized regions. For example, samples can 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 specific part of the liver, or a specific type of cell in the liver, such as hepatocytes). In some embodiments, a "sample derived from a subject" refers to blood or plasma obtained from a subject.

[0191] 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, but not limited to, 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.

[0192] The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains, which may be straight 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 comprise a C6-C18 alkyl hydrocarbon chain.

[0193] 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., [(CH2) n ] (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 from 1 to 6 carbon atoms in a branched arrangement, such as, for example, [(CH2CH2CH2CH2CH(CH3)], [(CH2CH2CH2CH2C(CH3)2], and [(CH2C(CH3)2CH(CH3))]. The term "alkylenedioxo" refers to a divalent species of the structure -ORO- (R represents alkylene).

[0194] The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0195] The term "halo" refers to any radical of fluorine, chlorine, bromine, or iodine. "Halogen" and "halo" are used interchangeably herein.

[0196] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. 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, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, and cyclohexyl. 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.

[0197] 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.

[0198] As used herein, the term "alkynyl" refers to a hydrocarbon radical, straight-chain 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" refers to 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-chain or branched portions of the alkynyl group may contain triple bonds, as normal valence permits, and may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as normal valence permits.

[0199] 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, "(C-C)alkoxy" includes methoxy, ethoxy, and propoxy. For example, "(C-C)alkoxy" is intended to include C, C, C, C, C, C, C, C, and C alkoxy groups. For example, "(C-C)alkoxy" is intended to include C, C, C, C, C, C, C, C, and C 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, monosubstituted, or disubstituted. 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).

[0200] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbon ring having seven or fewer 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, it is understood that attachment is 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.

[0201] "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 heteroatoms.

[0202] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic ring containing up to seven atoms in each ring, at least one of which is aromatic, and containing one to four heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, 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 normal valence allows.

[0203] As used herein, the term "heterocycle," "heterocyclic," or "heterocyclyl" refers to a 3- to 14-membered aromatic or non-aromatic heterocycle containing 1 to 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 heteroaryls as 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 oxooxazolidinyl. yl, 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, dihydrothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of heterocyclyl substituents 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.

[0204] "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, and tetrahydrofuran.

[0205] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms for monocyclic, bicyclic, or tricyclic, respectively), in which 0, 1, 2, 3, or 4 atoms in each ring may be substituted. 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.

[0206] 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 can be optionally further substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0207] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted with substituents.

[0208] 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.

[0209] 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, propynoyl, butynoyl, pentinoyl, hexynoyl), aryloyl (e.g., benzoyl), and heteroaryloyl (e.g., pyrroloyl, imidazoloyl, quinolinoyl, pyridinoyl).

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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 OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to encompass 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.

[0215] II. iRNAs of the Invention The present specification describes an iRNA that inhibits the expression of a target gene.In one embodiment, the iRNA inhibits the expression of the MYLIP gene.In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the MYLIP gene in cells, such as hepatocytes, for example, hepatocytes in a subject, such as a mammal, for example, a human with obesity, metabolic disorder, or MYLIP-related disorder.

[0216] The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the MYLIP gene. The complementary region 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 cells that express 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 similar cells that are not contacted with the RNAi agent or an RNAi agent that is not complementary to the MYLIP gene. Gene expression can be assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry. 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.

[0217] 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 complementary region that is substantially complementary or completely complementary to the target sequence. The target sequence can be derived from the sequence of mRNA formed during the expression of MYLIP 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 sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, so as to be opposite each other on separate oligonucleotides.

[0218] Generally, the duplex structure is 15-30 base pairs 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, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19-6 21-23, 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.

[0219] Similarly, the region of complementarity to the target sequence may be 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.

[0220] 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 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 In some embodiments, the dsRNA is about 15 to about 23 nucleotides in length, or 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 almost always a part of a longer RNA molecule, often an mRNA molecule.In relevant cases, the "part" of the mRNA target is a continuous sequence of the mRNA target that is long enough to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0221] Those skilled in the art will appreciate that the double-stranded region of dsRNA, the main functional portion, for example, about 9 to 36 base pairs, for example, 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 Those skilled in the art will also recognize that a 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 is also a dsRNA. Thus, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting MYLIP expression is not generated in the target cell by cleavage of a larger dsNRNA.

[0222] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Compared with their blunt-end counterparts, the dsRNA with at least one nucleotide overhang can have unexpectedly superior inhibitory properties.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs such as deoxynucleotide / nucleoside.The overhang can be on the sense strand, on the antisense strand, or any combination thereof.In addition, the nucleotide of the overhang can be on the 5'-end, 3'-end, or both of the antisense strand or the sense strand of dsRNA.

[0223] 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.

[0224] The iRNA compounds of the present invention can be prepared using a two-step method. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the dsRNA compound can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis has the advantage that it is easy to prepare oligonucleotide strands containing unnatural nucleotides or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0225] 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 3 or 4, and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences in Table 3 or 4. 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 produced upon expression of the MYLIP gene. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide described as the sense strand (passenger strand) in Table 3 or 4, and the second oligonucleotide described as the corresponding antisense strand (guide strand) to the sense strand in Table 3 or 4. In one embodiment, the substantially complementary sequences to the dsRNA are comprised in separate oligonucleotides. In another embodiment, the substantially complementary sequences to the dsRNA are comprised in a single oligonucleotide.

[0226] Although the sequences in Tables 3 or 4 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 3 or 4 that is unmodified, unconjugated, and / or modified and / or conjugated differently than described.

[0227] Those skilled in the art are well aware that dsRNAs having a duplex structure of about 20-23 base pairs, for example, 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 can also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the above-described embodiment, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise 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, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, or 20 or more contiguous nucleotides derived from one of the sequences presented herein and whose ability to inhibit expression of the MYLIP gene compared to control levels differs from a dsRNA comprising the entire 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 are contemplated as being within the scope of the present invention.

[0228] In addition, the RNA agents described in Tables 3 or 4 identify sites in MYLIP mRNA transcripts that are susceptible to RISC-mediated cleavage. Therefore, the present invention further features iRNAs that target within these sites. As used herein, an iRNA is said to "target within" a particular site of an mRNA transcript if it promotes cleavage of the mRNA transcript anywhere within the particular site. Such iRNAs will generally comprise at least about 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from regions adjacent to the selected sequence in the gene.

[0229] Target sequences are generally about 15-30 nucleotides in length, although there is considerable variation in the suitability of particular sequences within this range for directing cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for identifying optimal targets for any given gene target, an empirical approach can also be taken: literally or figuratively (including, for example, in silico) placing a "window" or "mask" of a predetermined size (21 nucleotides, as a non-limiting example) over the RNA sequence to identify sequences within a size range that can serve as target sequences. By incrementally shifting the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences 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 identified sequences (using assays described herein or known in the art) to identify optimally performing sequences, 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 the given sequence to identify sequences with equivalent or better inhibitory properties.

[0230] Further optimization can be achieved for any sequence identified herein by systematically adding or removing nucleotides to generate longer or shorter sequences, and then testing the resulting sequences by moving windows of longer or shorter size up or down the target RNA from that point. Furthermore, coupling this approach to generating new candidate targets with testing the effectiveness of iRNAs based on those target sequences in inhibition assays known in the art and / or described herein can result 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 modifying 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 circulatory half-life, improving thermostability, enhancing transmembrane delivery, targeting to specific locations or cell types, enhancing interaction with silencing pathway enzymes, or increasing release from endosomes).

[0231] The iRNA agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be limited to within the last five nucleotides from the 5' or 3' end of the region of complementarity, as appropriate. For example, in such an embodiment, in a 23-nucleotide RNAi agent, the strand complementary to a region of the MYLIP 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 the target sequence is effective in inhibiting expression of the MYLIP gene. For example, Jackson et al. (Nat. Biotechnol. 2003; 21: 635-637) described that the expression of a small set of genes that shared sequence identity with MAPK14 siRNA in only 12–18 nt of the sense strand was downregulated by MAPK14 with similar kinetics. Similarly, using qPCR and reporter assays, Lin et al. (Nucleic Acids Res. 2005; 33(14): 4527-4535) showed that 7 nt of complementarity between siRNA and target was sufficient to trigger target mRNA degradation. It is important to consider the efficacy of mismatched iRNAs in inhibiting MYLIP gene expression, especially when specific regions of complementarity in the MYLIP gene are known to have polymorphic sequence variation within the population.

[0232] An RNA target may have a region or span of the target RNA nucleotide sequence that is relatively more susceptible or apt to mediated cleavage of the RNA target via RNA interference induced by binding of an RNAi agent to that region than other regions of the RNA target. Increased susceptibility to RNA interference within such a "hotspot region" (or simply "hotspot") means that an iRNA agent targeting that region is likely to be more effective at inducing iRNA interference than an iRNA agent targeting another region 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 forming on 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.

[0233] 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 specific 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 a specific embodiment of the present invention, hotspot region may comprise a limited region of target RNA, in some cases, for example, 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 other hand, other regions to which hotspots are 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.

[0234] The compared regions of the target RNA can 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 can 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.). Generally, 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 can be fully characterized by observing the efficacy of iRNA agents that cumulatively span at least about 60% of the target region identified as a hotspot, for example, 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 within 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 (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) for a region may be identified as efficacious.

[0235] Quantitative comparison of inhibition measurements across different regions of defined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nt) can be used to assess the suitability of RNA regions for targeting. For example, an average level of inhibition can be determined for each region, and the averages for each region can be compared. The average level of inhibition within hotspot regions can be substantially higher than the average of the averages for all evaluated regions. According to some embodiments, the average level of inhibition in hotspot regions can 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 hotspot regions can 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 amount (e.g., p<0.05). According to some embodiments, each measured inhibition 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 measured inhibition within a region may be substantially higher than the average of all measured inhibition across all measured regions. For example, each measured inhibition in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all measured inhibition. According to some embodiments, each measured inhibition 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 measured inhibition. Each measured inhibition may be higher than all measured inhibition by a statistically significant amount (e.g., p<0.05). Criteria for assessing hotspots may include various combinations of the above criteria where compatible (e.g., an average level of inhibition with no measured inhibition below a threshold level of a second amount at least approximately equal to a first amount and less than the first amount).

[0236] Therefore, it is explicitly contemplated that any iRNA agent, including the specific exemplary iRNA agents described herein, that targets a hotspot region of a target RNA may be preferably selected to induce RNA interference of a target mRNA, since targeting the hotspot region is more likely to exhibit a stronger inhibitory response than targeting a region that is not a hotspot region. RNAi agents that target 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, is completely within a hotspot region, may be considered to target a hotspot region. Hotspot regions of RNA targets of the present invention may include any region that the data disclosed herein show is more frequently targeted by effective RNAi agents, including any of the standards described elsewhere herein, regardless of whether the scope of the hotspot region is explicitly specified.

[0237] In various embodiments, a dsRNA agent of the invention targets a hotspot region of the mRNA encoding MYLIP. In one embodiment, the hotspot region comprises nucleotides 341-417 of SEQ ID NO: 1. The dsRNA agent may be selected from the group consisting of AD-1753624, AD-1753557, AD-1753536, and AD-1753520.

[0238] 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. An 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 nucleotide.

[0239] In some aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides with 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 includes 4 or fewer nucleotides with 2'-fluoro modifications (e.g., 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other embodiments, the antisense strand includes 6 or fewer nucleotides with 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).

[0240] 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 include 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).

[0241] 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 5'-vinyl phosphate (5'-VP). In one embodiment, the phosphate mimic is 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).

[0242] 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 a 2' phosphate, such as G2p, C2p, A2p, or U2p, and vinyl-phosphonate nucleotides; and combinations thereof. In other embodiments, each of the duplexes in Tables 3 and 4 may be individually modified to provide a separate double-stranded iRNA agent of the present disclosure. In one example, 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, 5'-N1-...-N n-2 N n-1 N n L963' The three 3'-terminal nucleotides (N) of the sense sequence of 5'-N1-...-N n-2 sN n -1sN n 3' may be replaced with That is, for example, the antisense sequence remains unchanged, AD-1753226, the sense sequence: csgscagaGfcUfGfCfagccuucgauL96 (SEQ ID NO: 287), csgscagaGfcUfGfCfagccuucgsasu (SEQ ID NO: 692) may be substituted to provide another double-stranded iRNA agent of this disclosure.

[0243] 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, S.L. et al. (Eds.), 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 sugar replacement, 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 containing modified backbones and no natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have phosphorus atoms in backbones can be considered.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atoms in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified iRNA has phosphorus atoms in its internucleoside backbone.

[0244] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their analogs linked in 2'-5', and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. 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.

[0245] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent 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 5,286,712. 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 , 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.

[0246] 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 (formed in part 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 others with mixed N, O, S, and CH2 constituent moieties.

[0247] Representative United States patents that 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.

[0248] In other embodiments, suitable RNA mimetics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with alternative groups. The nucleobase units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic known 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, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. 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.

[0249] Some embodiments featured in the present invention include RNAs with phosphorothioate backbones, as well as oligonucleotides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or NMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Pat. No. 5,489,677, and amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Pat. No. 5,034,506. A natural phosphodiester backbone can be represented as --OP(O)(OH)--OCH2--.

[0250] 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 C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In another embodiment, the dsRNA is 10The 2'-position may include one of the following substituents: alkyl, substituted alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, acceptor group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic properties of iRNA, and similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0251] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of 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 a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent 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, and 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 commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0252] The iRNAs of the present invention may also contain 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), and 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 ... These include 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, particularly when combined with 2'-O-methoxyethyl sugar modification.

[0253] Representative U.S. patents that 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. , 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.

[0254] 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 a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo 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].

[0255] The iRNAs 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 containing 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 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present invention can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of a 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 comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides comprising 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, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs, see e.g., U.S. Pat. No. 8,399,845). 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.

[0256] Additional representative U.S. 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.

[0257] For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0258] The iRNAs of the invention can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing 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."

[0259] The iRNA of the present invention may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0260] 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.

[0261] In some embodiments, the iRNA of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of the sugar linkages have been removed to form unlocked "sugar" residues. In one example, UNAs also encompass monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0262] 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.

[0263] RNAi agents of the present disclosure may also include one or more "cyclohexene nucleic acids" or ("CeNAs"). CeNAs are nucleotide analogs that replace the furanose moiety of DNA with a cyclohexene ring. The inclusion of cyclohexenyl nucleosides in DNA strands increases the stability of DNA / RNA hybrids. CeNAs are stable to degradation in serum, and CeNA / RNA hybrids can activate Escherichia coli RNase H, resulting in cleavage of the RNA strand (see Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595-8602, incorporated herein by reference).

[0264] Potentially stabilizing modifications to the ends of RNA molecules 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.

[0265] Other modifications of the iRNA of the present invention include 5' phosphate or 5' phosphate mimic, such as 5' terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent.Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0266] In certain embodiments, the RNAi agent of the invention is an agent that inhibits expression of the MYLIP gene selected from the group of agents set forth in Tables 3 or 4. Any of these agents may further comprise a ligand.

[0267] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the invention, double-stranded RNAi agents of the invention include agents having chemical modifications disclosed, for example, in WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.

[0268] Thus, the present invention provides double-stranded RNAi agents capable of inhibiting expression of a target gene (i.e., the MYLIP gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be in the range of 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.

[0269] 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 an RNAi agent can be 12 to 30 nucleotide pairs in length. For example, the duplex region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 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.

[0270] 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 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. 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 mismatches with the target mRNA, may be complementary to the targeted gene sequence, or may be a different sequence. The first and second strands may also be joined by additional bases, e.g., to form a hairpin, or by other non-basic linkers.

[0271] In one embodiment, the nucleotides in the overhang region of the RNAi agent can be independently modified or unmodified nucleotides, including but not limited to 2'-sugar modified nucleotides, 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.

[0272] 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) contain 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.

[0273] RNAi agents can contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3' end of the sense strand or at the 3' end of the antisense strand.RNAi can also have a blunt end located at the 5' end of the antisense strand (i.e., the 3' end of the sense strand), or vice versa.Generally, 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 the asymmetric antisense strand and the 3' end overhang of the antisense strand are favorable for the guide strand loading into the RISC process.

[0274] 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.

[0275] 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. 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.

[0276] In yet another embodiment, the RNAi agent is 21 nucleotides in length 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. 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.

[0277] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, 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, and one end of the RNAi agent is blunt and the other end comprises a two-nucleotide overhang. The two-nucleotide overhang may be present at the 3' end of the antisense strand.

[0278] When a two-nucleotide overhang is at the 3'-end of the antisense strand, there can 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 terminal nucleotides 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 strand and antisense strand of the RNAi agent, including the nucleotide that is part of the motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 2'-fluoro, for example, in the alternating motif.The RNAi agent can further comprise a ligand (for example, GalNAc3).

[0279] In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein 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 six 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 double-stranded nucleic acid comprises at least one nucleotide of the sense strand, at least one nucleotide of the 5'-terminal and 3'-terminal sense strand, which is base-paired with a nucleotide of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands. The antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, and reduces target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell. The sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs being at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0280] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of 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 forming a blunt end, the second strand being 1-4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides in length, the second strand being sufficiently complementary to a target mRNA along the length of the second strand of at least 19 nucleotides, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially producing siRNAs comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent may further comprise a ligand.

[0281] 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.

[0282] 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.

[0283] For RNAi agents having a duplex region 17-23 nucleotides long, the cleavage sites in the antisense strand are typically 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.

[0284] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be arranged so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0285] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif located in another part of the strand, away from the motif at or near the cleavage site of the same strand. The wing modifications are adjacent to the first motif or are 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. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be located at one end of the first motif located at or near the cleavage site, or on either side of the lead motif.

[0286] Similar to the sense strand, the antisense strand of 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 break site of the strand.This antisense strand may also contain one or more wing modifications with the same sequence as the wing modifications that may be present on the sense strand.

[0287] 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.

[0288] In another embodiment, 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.

[0289] 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.

[0290] 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 two modifications from one strand each occupy one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand each occupy the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; and two modifications from one strand occupy either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0291] 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.

[0292] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties or non-linked Os at phosphate moieties. In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur. For example, modifications may occur only at the 3' or 5' terminal position, or only in terminal regions, such as at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

[0293] 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 part of the bases in the 3' or 5' overhang can be modified, for example, with the modifications described herein.Modifications can 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, instead of the ribosugar of the nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0294] 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. The strands 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.

[0295] At least two different modifications are usually present on the sense and antisense strands, and the two modifications can be 2'-O-methyl or 2'-fluoro modifications, or other modifications.

[0296] 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, with each modification occurring on alternating nucleotides of a strand. The 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 could be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AABBBAAABBB...," or "ABCABCABCABC...," etc.

[0297] The types of modifications contained within the alternating motifs can be the same or different. For example, if A, B, C, and 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 for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0298] In one embodiment, the RNAi agent of the present invention comprises an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides 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 within 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 within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0299] In one embodiment, RNAi agent comprises the pattern of alternating motifs of 2'-O-methyl modification, and the 2'-F modification on sense strand has the pattern of alternating motifs of 2'-O-methyl modification initially and the pattern of alternating motifs of 2'-F modification initially on antisense strand, i.e., the 2'-O-methyl modified nucleotide on sense strand base pair with the 2'-F modified nucleotide on antisense strand, and vice versa.The 1st position of sense strand can start with 2'-F modification, and the 1st position of antisense strand can start with 2'-O-methyl modification.

[0300] 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, and unexpectedly, enhances the gene silencing activity of target gene.

[0301] 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.

[0302] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present at any nucleotide in the sense strand, the antisense strand, or both strands, at any position in the strand. For example, the internucleotide linkage modification may be present at every nucleotide in 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 contain 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 that of 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 to 8 phosphorothioate internucleotide linkages. In one embodiment, 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 comprises at least two phosphorothioate internucleotide linkages at the 5' end or the 3' end.

[0303] In one embodiment, RNAi comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, the overhang region can comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between these two nucleotides.Internucleotide linkage modification can also be such that in the double-stranded region, overhang nucleotide is linked to the terminal paired nucleotide.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be additional phosphorothioate or methylphosphonate internucleotide linkage that connects 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, where two of the three nucleotides 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' end of the antisense strand.

[0304] 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.

[0305] In one embodiment, the RNAi agent comprises a mismatch (or mismatches) in the double strand with the target or a combination thereof. Mismatches can be present in the overhang region or the double-stranded 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 simplest approach is to examine pairs on an individual 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 pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings containing universal bases are preferred over canonical pairings.

[0306] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within 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 containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0307] 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.

[0308] 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 deoxythymidine nucleotides, e.g., two dT nucleotides, at the 3' end of the sense and / or antisense strands.

[0309] 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 each independently represent 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 represent 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.

[0310] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0311] 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), and the number can start from the first nucleotide from the 5' end, or, optionally, the number can start from the first paired nucleotide within the duplex region from the 5' end.

[0312] 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:

[0313] 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0314] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0315] 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 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0316] Each of X, Y and Z may be the same as or different from one another.

[0317] 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:

[0318] When the sense strand is represented by formula (Ia), each N a may independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0319] In one embodiment, the antisense strand sequence of the RNAi has 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 represent 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing 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; 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:

[0320] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0321] 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 to 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, or 13.

[0322] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0323] 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.

[0324] 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:

[0325] When the antisense strand is represented by formula (If), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0326] 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.

[0327] 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.

[0328] 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:

[0329] 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.

[0330] Each of X', Y' and Z' may be the same as or different from one another.

[0331] 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' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0332] 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, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, and 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.

[0333] In one embodiment, the antisense strand may contain a Y'Y'Y' motif present at positions 11, 12, 13 of the strand, where the number starts from the first nucleotide from the 5' end, or, where appropriate, the number may start from the 5' end with the first paired nucleotide in the duplex region, and 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.

[0334] 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 formulas (Ie), (If), (Ig) and (Ih), respectively.

[0335] 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 represent 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 represent 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. is expressed by

[0336] 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.

[0337] 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.

[0338] 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.

[0339] When the RNAi agent is represented by formula (Ik), each N b independently represent 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.

[0340] 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.

[0341] When an RNAi agent is represented as formula (Im), 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, Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0342] In formulae (Ii), (Ij), (Ik), (Il) and (Im), X, Y and Z may be the same or different from one another.

[0343] When an 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 the corresponding Y' nucleotide, or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0344] When the RNAi agent is represented by formula (Ik) or (Im), at least one of the Z nucleotides can 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.

[0345] When the RNAi agent is represented by formula (II) or (Im), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides can be base-paired with the corresponding X' nucleotide, or all three of the X nucleotides can be base-paired with the corresponding X' nucleotide.

[0346] 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.

[0347] 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 modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p np' is linked to an adjacent nucleotide via a phosphorothioate linkage. In yet another embodiment, when an RNAi agent is represented by formula (Im), 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, 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 an RNAi agent is represented by formula (Im), 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, 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).

[0348] 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.

[0349] In one embodiment, the RNAi agent is a multimer containing 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 comprise a ligand. Each duplex may target the same gene, or may target two different genes, or each duplex may target the same gene at two different target sites.

[0350] 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 comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0351] 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 may be conjugated to a ligand. Each of the agents may target the same gene, or may target two different genes, or each of the agents may target the same gene at two different target sites.

[0352] In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or fewer nucleotides with 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 6 nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 2 nucleotides with 2'-fluoro modifications in the antisense strand.

[0353] In other embodiments, the iRNA agent of the present invention may contain very few nucleotides containing 2'-fluoro modifications, for example, two or less nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain two, one, or zero nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent may contain two nucleotides with 2'-fluoro modifications, for example, zero nucleotides with 2'-fluoro modifications in the sense strand and two nucleotides with 2'-fluoro modifications in the antisense strand.

[0354] Various publications describe the multimeric RNAi agent that can be used in the method of the present invention.Such publications include WO2007 / 091269, United States Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of whose entire contents is incorporated herein by reference.

[0355] As described in more detail below, RNAi agents that include 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, such as 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 has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The 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, such as nitrogen, oxygen, or sulfur. The heterocyclic carrier can be a monocyclic ring system or can contain two or more double bonds, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0356] Ligands can be attached to polynucleotides via carriers. The carriers include (i) at least one "backbone attachment point," e.g., two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., 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 heteroatom (separate from the atom providing 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, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often include a functional group, e.g., an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the constituent ring.

[0357] RNAi agent can 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 skeleton or diethanolamine skeleton.

[0358] In another embodiment of the invention, the iRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent has the formula (L):

[0359] [ka] It may be represented by:

[0360] 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.

[0361] C1 is a thermolabile nucleotide located at the 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 located in the sense strand at a position that pairs with the nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide has a thermolabile modification that may include an abasic modification, a mismatch with the opposing nucleotide in the duplex, and a sugar modification, such as a 2'-deoxy modification or an acyclic nucleotide, such as 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)

[0362] [ka] and iii) an abasic modification selected from the group consisting of:

[0363] [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a 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, where optionally, at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermolabile modification in C1 is GNA, or

[0364] [ka] is.

[0365] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with a steric volume less than or equal to that of a 2'-OMe modification. Steric volume refers to the sum of the steric effects of the modifications. Methods for determining the steric effect of a nucleotide modification 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 to or equivalent to the 2' position of the ribose sugar, providing the nucleotide with a steric volume less than or equal to that of a 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In 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.

[0366] n 1 , n 3 and q 1 are independently 4 to 15 nucleotides in length.

[0367] n 5 , q 3 and q 7 are independently 1 to 6 nucleotides in length.

[0368] n 4 , q 2 and q 6 are independently 1 to 3 nucleotides in length, or n 4 is 0.

[0369] q 5 are independently 0 to 10 nucleotides in length.

[0370] n 2 and q4 are independently 0 to 3 nucleotides in length.

[0371] Or, n 4 is 0 to 3 nucleotides in length.

[0372] 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).

[0373] In certain embodiments, n 4 , q 2 and q 6 are 1 respectively.

[0374] In certain embodiments, n 2 , n 4 , q 2 , q 4 and q 6 are 1 respectively.

[0375] In certain embodiments, C1 is a sequence in which the sense strand is 19 to 22 nucleotides in length and n 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.

[0376] 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 q 6 is equal to 1.

[0377] 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.

[0378] 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.

[0379] In certain embodiments, T1' and T3' are 11 nucleotides apart in length (ie, not counting T1' and T3').

[0380] 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.

[0381] In certain embodiments, 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.

[0382] 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, and n 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 If T1 is 1, then T1 is at the cleavage site of the sense strand at position 11 from the 5' end of the sense strand.

[0383] 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 q 4 is 1.

[0384] In one exemplary embodiment, the sense strand is 19 to 22 nucleotides in length, and 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, and 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.

[0385] 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.

[0386] 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 q 4 is 1.

[0387] 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).

[0388] In certain embodiments, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 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).

[0389] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0390] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, 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).

[0391] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0392] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 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).

[0393] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0394] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 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).

[0395] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, and there may be at least two additional TTs at the 3' end of the antisense strand.

[0396] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 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), 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).

[0397] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0398] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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).

[0399] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and 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.

[0400] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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).

[0401] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 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.

[0402] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 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] The RNAi agent can contain a phosphorus-containing group at the 5'-end of the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl (

[0404] [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).

[0405] [ka] ), 5'-Z-VP isomer (i.e., cis-vinylphosphonate

[0406] [ka] ), or a mixture thereof.

[0407] In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand. In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0408] In certain embodiments, the RNAi agent comprises a 5'-P. In certain embodiments, the RNAi agent comprises a 5'-P in the antisense strand.

[0409] In certain embodiments, the RNAi agent comprises a 5'-PS. In certain embodiments, the RNAi agent comprises a 5'-PS on the antisense strand.

[0410] 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.

[0411] In certain embodiments, the RNAi agent comprises a 5'-PS2. In certain embodiments, the RNAi agent comprises a 5'-PS2 on the antisense strand.

[0412] In certain embodiments, the RNAi agent comprises a 5'-PS2. In certain embodiments, the RNAi agent comprises a 5'-deoxy-5'-C-malonyl in the antisense strand.

[0413] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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.

[0414] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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.

[0415] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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.

[0416] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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'-PS2.

[0417] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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.

[0418] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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). The RNAi agent also includes a 5'-P.

[0419] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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 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). The RNAi agent also includes a 5'-PS.

[0420] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). The RNAi agent also includes a 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0421] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 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). The RNAi agent also includes a 5'-PS2.

[0422] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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 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). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0423] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0424] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0425] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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.

[0426] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-PS2.

[0427] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0428] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5is 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.

[0429] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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.

[0430] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0431] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 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'-PS2.

[0432] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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 5'-deoxy-5'-C-malonyl.

[0433] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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.

[0434] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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.

[0435] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 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.

[0436] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA RNAi agent also includes a 5'-PS2.

[0437] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0438] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). The RNAi agent also includes a 5'-P.

[0439] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). The RNAi agent also includes a 5'-PS.

[0440] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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 (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). The RNAi agent also includes a 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0441] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). The RNAi agent also includes a 5'-PS2.

[0442] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0443] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 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.

[0444] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0445] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 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.

[0446] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-PS2.

[0447] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0448] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 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). The RNAi agent also includes a 5'-P.

[0449] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 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). The RNAi agent also includes a 5'-PS.

[0450] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 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). The RNAi agent also includes a 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0451] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 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). The RNAi agent also includes a 5'-PS2.

[0452] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5is 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). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0453] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). 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.

[0454] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). 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.

[0455] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0456] 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.

[0457] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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 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). The RNAi agent also includes a 5'-PS2 and a targeting agent. In certain embodiments, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0458] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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 internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at 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 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.

[0459] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 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 at 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). 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.

[0460] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 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 at 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). 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.

[0461] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 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 at 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). 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.

[0462] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 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'-PS2 and a targeting agent. In certain embodiments, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0463] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 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 at 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). 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.

[0464] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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). 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.

[0465] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4, 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). 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.

[0466] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, 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). 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.

[0467] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, 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). The RNAi agent also includes a 5'-PS2 and a targeting agent. In certain embodiments, the 5'-PS2 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, 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 internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at 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 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.

[0469] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 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). 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.

[0470] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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). 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.

[0471] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 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). 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.

[0472] In certain embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'-F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, 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). The RNAi agent also includes a 5'-PS2 and a targeting agent. In certain embodiments, the 5'-PS2 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, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, and includes two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at 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 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.

[0474] In certain embodiments, the RNAi agents of the invention include: (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached to its 3'-terminus, the ASGPR ligand comprising three GalNAc derivatives attached through 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 (counted 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.

[0475] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) a sense strand (counted from the 5' end) 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; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted 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 nucleotides 22 and 23 (counted 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.

[0476] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11 (counting 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0477] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0478] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0479] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0480] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0481] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0482] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) a 3'-terminally attached 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 (counted 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 nucleotides 22 and 23 (counted 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.

[0483] In another specific embodiment, the RNAi agent of the invention is (a)(i) 19 nucleotides in length; (ii) a 3'-terminally attached ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19, and 2'-F modifications at positions 5 and 7-9, and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end); (b)(i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotides 20 and 21 (counted 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.

[0484] In certain embodiments, an iRNA agent for use in the methods of the invention is an agent selected from those listed in Tables 3 or 4. These agents may further comprise a ligand.

[0485] In one embodiment, the drug is AD-1753226、AD-1753250、AD-1753259、AD-1753324、AD-1753346、AD-1753413、AD-1753439、AD-1753456、AD-1753520、AD-1753536、 AD-1753557、AD-1753624、AD-1753651、AD-1753661、AD-1753725、AD-1753744、AD-1753813、AD-1753827、AD-1753844、AD-1753860、AD-1753917、AD- 1753949、AD-1754026、AD-1754053、AD-1754117、AD-1754133、AD-1754147、AD-1754162、AD-1754226、AD-1754240、AD-1754255、AD-1754327、AD-175 4342、AD-1754353、AD-1754420、AD-1754434、AD-1754450、AD-1754530、AD-1754550、AD-1754618、AD-1754637、AD-1754661、AD-1754735、AD-175474 9、AD-1754818、AD-1754838、AD-1754862、AD-1754939、AD-1755013、AD-1755046、AD-1755120、AD-1755141、AD-1755155、AD-1755219、AD-1755326、A D-1755340、AD-1755358、AD-1755435、AD-1755451、AD-1755515、AD-1755539、AD-1755554、AD-1755621、AD-1755636、AD-1755650、AD-1755714、AD-1 755734、AD-1755747、AD-1755761、AD-1755827、AD-1755830、AD-1755845、AD-1755859、AD-1755923、AD-1755943、AD-1756028、AD-1756044、AD-1756 131、AD-1756146、AD-1756219、AD-1756236、AD-1756319、AD-1756335、AD-1756354、AD-1756429、AD-1756439、AD-1756514、AD-1756533、AD-1756552、AD-1756616, AD-1756631, AD-1756653, AD-1756718, AD-1756749, AD-1756814, AD-1756834, AD-1756858, AD-1756915, AD-1756932, AD-1756947, AD-175 7019, AD-1757032, AD-1757052,...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the antisense sequence of AD-1753624, AD-1753557, AD-1753536, or AD-1753520, or a sequence selected from the group consisting of a nucleotide sequence shown in the table below. Table 1 Table 2 10. A dsRNA agent comprising a region of complementarity to an mRNA encoding MYLIP, the region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences set forth in claim 1. (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 comprise 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, the at least one modified nucleotide being 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'-methoxyethyl and / or are selected from the group consisting of 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; (i) the dsRNA agent includes at least one modified nucleotide, wherein at least one modified nucleotide is 2'-O-methyl and / or 2'-fluoro modified; The dsRNA agent of claim 1. (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) each of the sense and antisense strands is 30 nucleotides or less in length; (d) each strand of the sense and antisense strands is independently 19-30, 19-25, or 21-23 nucleotides in length; (e) at least one strand of the sense strand or the antisense strand comprises a 3' overhang of at least one nucleotide; (f) at least one strand of the sense strand or the antisense strand comprises a 3' overhang of at least two nucleotides; (g) the region of complementarity comprises any one of the antisense sequences in the table of claim 1; (h) the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of any one of the agents listed in the table of claim 1; (k) the dsRNA agent is selected from the group consisting of AD-1753624, AD-1753557, AD-1753536, and AD-1753520; (i) the dsRNA agent targets a hotspot region of the mRNA encoding MYLIP, and / or (j) the dsRNA agent targets a hotspot region of an mRNA encoding MYLIP, wherein the hotspot region comprises nucleotides 341-417 of SEQ ID NO:1; The dsRNA agent of claim 1. (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 includes a ligand, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. (d) the dsRNA agent comprises a ligand, wherein the ligand is 【Chemistry 1】 and / or (e) The dsRNA agent has the following structure: 【Chemistry 2】 wherein X is O or S. conjugated to a ligand as shown in The dsRNA agent of claim 1.

5. 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of myosin regulatory light chain-interacting protein (MYLIP) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand contains at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the sense sequence of AD-1753624, AD-1753557, AD-1753536, or AD-1753520, or at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense sequences listed in the table below, and the antisense strand contains at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the antisense sequence of AD-1753624, AD-1753557, AD-1753536, or AD-1753520, or at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense sequences listed in the table below. Table 3 Table 4 and wherein the antisense sequences comprise at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences set forth in 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; the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached through a monovalent, divalent, or trivalent branched linker; dsRNA agents. (a) all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides, and / or (b) the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequence of any one of the dsRNA agents listed in the Table of Claim 5; The dsRNA agent of claim 5. (a) the dsRNA agent targets a hotspot region of the mRNA encoding MYLIP; (b) the dsRNA agent targets a hotspot region of the mRNA encoding MYLIP that includes nucleotides 341-417 of SEQ ID NO:1, and / or (c) the dsRNA agent is selected from the group consisting of AD-1753624, AD-1753557, AD-1753536, and AD-1753520; The dsRNA agent of claim 1.

8. A cell comprising the dsRNA agent of any one of claims 1 to 7.

9. A vector encoding at least one strand of the dsRNA agent of any one of claims 1-7.

10. A pharmaceutical composition for inhibiting the expression of myosin regulatory light chain-interacting protein (MYLIP), comprising the dsRNA agent of any one of claims 1 to 7. (a) the dsRNA agent is formulated in an unbuffered solution. (b) the dsRNA agent is formulated in an unbuffered solution, the unbuffered solution being saline or water; (c) the dsRNA agent is formulated in a buffer solution; (d) the dsRNA agent is formulated in a buffer solution, the buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof; and / or (e) the dsRNA agent is formulated in a buffer solution, and the buffer solution is phosphate buffered saline (PBS); The pharmaceutical composition of claim 10.

12. 10. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 7 for use in a method of inhibiting myosin regulatory light chain-interacting protein (MYLIP) expression in a cell. (a) the cell is in a subject; (b) the cell is in a human subject; (c) MYLIP expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or is inhibited to below the level of detection of MYLIP expression; (d) the cell is in a human subject suffering from a MYLIP-associated disease, disorder, or condition. (e) the cell is in a human subject suffering from a MYLIP-associated disease, disorder, or condition, wherein the MYLIP-associated disease, disorder, or condition is a lipid imbalance; (f) the cell is in a human subject suffering from a lipid imbalance, including hypercholesterolemia, hyperlipidemia, or hypertriglyceridemia. (g) the cell is in a human subject suffering from hypercholesterolemia, including hyper-LDL cholesterolemia. (h) the cell is in a human subject suffering from a MYLIP-associated disease, disorder, or condition, wherein the MYLIP-associated disease, disorder, or condition is a pathological condition associated with lipid imbalance; (i) the cell is in a human subject suffering from a MYLIP-associated disease, disorder, or condition, and the MYLIP-associated disease, disorder, or condition is cardiovascular disease; and / or (j) the cell is in a human subject suffering from cardiovascular disease, wherein the cardiovascular disease is atherosclerosis or coronary heart disease; The pharmaceutical composition of claim 12.

14. (a) The subject is obese. (b) the method comprises administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject. (c) the method includes administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject, wherein the additional therapeutic agent comprises a statin. (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. (e) the dsRNA agent is administered to the subject intravenously, intramuscularly, or subcutaneously, and / or (f) the method further comprises determining the level of MYLIP in the subject. The pharmaceutical composition of claim 13. (a) inhibiting expression of MYLIP in a subject; and / or (b) preventing at least one symptom in a subject having a disease, disorder, or condition that would benefit from reduced expression of the MYLIP gene; A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 7 for use in a method.

16. (a) The subject is obese. (b) the method comprises administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject. (c) the method includes administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject, wherein the additional therapeutic agent comprises a statin. (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. (e) the dsRNA agent is administered to the subject intravenously, intramuscularly, or subcutaneously, and / or (f) the method further comprises determining the level of MYLIP in the subject.

16. The pharmaceutical composition of claim 15.

17. 10. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-7 for use in a method of treating a subject suffering from a MYLIP-related disease, disorder or condition.

18. (a) The MYLIP-associated disease, disorder, or condition is a lipid imbalance. (b) the MYLIP-related disease, disorder, or condition is a lipid imbalance, including hypercholesterolemia, hyperlipidemia, or hypertriglyceridemia; (c) the MYLIP-related disease, disorder, or condition is hypercholesterolemia, including hyper-LDL cholesterolemia; (d) the MYLIP-related disease, disorder, or condition is a pathological condition associated with lipid imbalance; (e) the MYLIP-related disease, disorder, or condition is a cardiovascular disease; (f) the MYLIP-related disease, disorder, or condition is a cardiovascular disease, and the cardiovascular disease is atherosclerosis or coronary heart disease; (g) the subject is obese; (h) the method comprises administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject. (i) the method includes administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject, wherein the additional therapeutic agent comprises a statin; (j) 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. (k) the dsRNA agent is administered to the subject intravenously, intramuscularly, or subcutaneously, and / or (l) the method further comprises determining the level of MYLIP in the subject; 18. The pharmaceutical composition of claim 17.

19. (a) reducing the risk of developing or worsening cardiovascular disease in a subject; (b) reducing the risk of developing atherosclerosis or worsening atherosclerosis in a subject; and / or (c) reducing plasma levels of cholesterol in subjects with elevated plasma low-density lipoprotein (LDL) cholesterol. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 7 for use in a method.

20. (a) The subject is obese. (b) the method comprises administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject. (c) the method includes administering the pharmaceutical composition to a subject and administering an additional therapeutic agent to the subject, wherein the additional therapeutic agent comprises a statin. (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. (e) the dsRNA agent is administered to the subject intravenously, intramuscularly, or subcutaneously, and / or (f) the method further comprises determining the level of MYLIP in the subject.

20. The pharmaceutical composition of claim 19.

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