Plasminogen (PLG) iRNA compositions and methods of use thereof

Abstract

The present invention relates to double-stranded ribonucleic acid (dsRNA) compositions that target the plasminogen (PLG) gene, and methods of using the dsRNA compositions to inhibit the expression of PLG and to treat subjects who would benefit from reduced expression of PLG, e.g., subjects with a PLG-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 Application No. 63 / 378,731, filed October 7, 2022, and U.S. Provisional Application No. 63 / 587,546, filed October 3, 2023, the entire contents of which are hereby incorporated by reference herein.

[0002] Reference to sequence listing An official copy of the Sequence Listing has been submitted electronically herewith as an XML Sequence Listing with the file name ALN492WO_SeqList.xml, created on October 3, 2023, and 43,460,748 bytes in size. The Sequence Listing contained in this XML document is a part of this specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates generally to RNAi agents and methods that target plasminogen (PLG). [Background technology]

[0004] Plasminogen (PLG) is the precursor of the enzyme plasmin, a serine protease that acts to break down fibrin and dissolve blood clots (fibrinolysis). PLG is primarily synthesized in the liver and released into the systemic circulation at high plasma concentrations (1.5–2 μM). The two main physiological activators of plasminogen to plasmin are tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Plasminogen activator inhibitor-1 (PAI-1) is an endogenous negative regulator that inhibits the activity of tPA and uPA, limiting the activation of plasminogen to plasmin and subsequent fibrinolysis.

[0005] Menorrhagia (HMB) is excessive menstrual bleeding that impairs a woman's physical, social, emotional, and / or material quality of life. HMB affects approximately 30% of women of reproductive age and represents a significant burden for over 10 million American women each year. HMB has been associated with iron deficiency anemia, fatigue, and absenteeism from school / work / activities. Approximately 60-90% of women with bleeding disorders have HMB. Over $1 billion is spent annually on HMB treatment.

[0006] Current standard treatments for HMB include the use of the antifibrinolytic plasminogen activator inhibitor small molecule tranexamic acid (TXA), oral contraceptives (OCPs), and / or hormone-releasing intrauterine devices (IUDs). However, side effects, high drug burden, and lack of efficacy frequently lead to discontinuation of these therapies. Therefore, additional treatments for HMB are needed.

[0007] Women with HMB have been shown to have increased uterine fibrinolytic activity, including elevated levels of PLG, increased levels of plasminogen activator t-PA, and delayed levels of PAI-1.

[0008] Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes excessive bleeding and affects men and women of all ages and ethnic backgrounds. Patients with HHT have abnormally fragile blood vessels, bleed easily, and have localized elevated fibrinolytic activity and vascular malformations, such as telangiectasias and arteriovenous malformations. Approximately 90% of individuals with HHT experience recurrent nosebleeds, which are accompanied by gastrointestinal bleeding, HMB, anemia, and frequent iron supplements / blood transfusions. Currently, there are no FDA-approved medications for the treatment of HHT, and TXA is used off-label in patients with HHT. Given the central role of PLG in mediating fibrinolysis, suppressing PLG gene expression is a potential target for reducing excessive mucocutaneous bleeding in disorders such as HHT and HMB, as well as other types of bleeding associated with bleeding disorders, including nosebleeds and easy bruising. Summary of the Invention

[0009] The present invention provides an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the plasminogen (PLG) gene. The PLG 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 inhibit the expression of the PLG gene and / or to treat a subject who would benefit from inhibiting or reducing the expression of the PLG gene, for example, a subject suffering from or susceptible to a PLG-related disease, such as a bleeding disorder.

[0010] Thus, in one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) 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 or 3, 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 or 4. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or 3, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or 4.

[0011] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) 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 PLG, comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B by no more than 1, 2, or 3 nucleotides. 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 PLG, comprising at least 15 consecutive nucleotides of any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.

[0012] In other embodiments, the region of complementarity is selected from the group consisting of 3 to 25, 90 to 112, 126 to 148, 153 to 175, 185 to 207, 202 to 224, 221 to 243, 247 to 269, 267 to 289, 287 to 309, 303 to 325, 323 to 345, 353 to 375, 372 to 394, 396 to 418, 460 to 482, 513 to 535, 550 to 572, 575 to 597, 592 to 614, 607 to 629, 623 to 645, 642 to 664, 703 to 725, 718 to 740, 740 to 762, 787 to 809, 818 to 840, 850 to 860, 861 to 870, 871 to 880, 872 to 882, 873 to 884, 875 to 886, 876 to 888, 877 to 889, 889 to 890, 900 to 900, 901 to 902, 903 to 904, 905 to 906, 907 to 908, 909 to 910, 911 to 912, 913 to 914, 915 to 916, 917 to 918, 918 to 920, 919 to 921, 922 to 923 872, 870-892, 885-907, 910-932, 982-1004, 1012-1034, 1029-1051, 1061-1083, 1094-1116, 1239-1261, 1254-1276, 1313-1335, 1339-1361, 1364-1376 86, 1396-1418, 1426-1448, 1452-1474, 1469-1491, 1489-1511, 1592-1614, 1607-1629, 1651-1673, 1685-1707, 1700-1722, 1716-1738, 1841-1863, 18 56~1878, 1919~1941, 1978~2000, 1998~2020, 2047~2069, 2062~2084, 2086~2108, 2150~2172, 2173~2195, 2223~2245, 2248~2270, 2263~2285, 2343~2352 365, 2433~2455, 2473~2495, 2511~2533, 2553~2575, 2578~2600, 2605~2627, 2621~2643, 2646~2668, 2661~2683, 2701~2723, 2727~2749, 2742~2764, 2 771~2793, 2804~2826, 2826~2848, 2874~2896, 2890~2912, 2914~2936, 2929~2951, 2947~2969, 2962~2984, 2987~3009, 3006~3028, 3025~3047, 3068~ 3090, 3097~3119, 3116~3138, 3143~3165, 3168~3190, 3183~3205, 3209~3231, 3236~3258, 3269~3291, 3289~3311, 3310~3332, 3330~3352, 3391~3413,3407-3429, 3422-3444, 3447-3469, 3468-3490, 3485-3507; or 8-30, 45-67, 519-541, 534-556, 617-639, 653-675, 669-691, 702-724, 717-739, 760-782, 776-798, 798-820, 851-873, 870-892, 88 and at least 15 consecutive nucleotides that differ by no more than 1, 2, or 3 nucleotides from any one of nucleotides 5 to 907, 904 to 926, 929 to 951, 962 to 984, 984 to 1006, 1000 to 1022, 1016 to 1038, 1055 to 1077, 1070 to 1092, 1113 to 1135, 1128 to 1150, or 1159 to 1181. In some embodiments, the region of complementarity is selected from the group consisting of 3 to 25, 90 to 112, 126 to 148, 153 to 175, 185 to 207, 202 to 224, 221 to 243, 247 to 269, 267 to 289, 287 to 309, 303 to 325, 323 to 345, 353 to 375, 372 to 394, 396 to 418, 460 to 482, 513 to 535, 550 to 57 of SEQ ID NO: 1. 2, 575-597, 592-614, 607-629, 623-645, 642-664, 703-725, 718-740, 740-762, 787-809, 818-840, 850-872, 870-892, 885-907, 910-932, 982-1004, 1012-1034, 1029-1051, 1061-1083, 1094-1116, 1 239~1261, 1254~1276, 1313~1335, 1339~1361, 1364~1386, 1396~1418, 1426~1448, 1452~1474, 1469~1491, 1489~1511, 1592~1614, 1607~1629, 1651~1673, 1685~1707, 1700~1722, 1716~1738, 1841~ 1863, 1856~1878, 1919~1941, 1978~2000, 1998~2020, 2047~2069, 2062~2084, 2086~2108, 2150~2172, 2173~2195, 2223~2245, 2248~2270, 2263~2285, 2343~2365, 2433~2455, 2473~2495, 2511~2533,2553~2575, 2578~2600, 2605~2627, 2621~2643, 2646~2668, 2661~2683, 2701~2723, 2727~2749, 2742~2764, 2771~2793, 2804~2826, 2826~2848, 2874~2896, 2890~2912, 2914~2936, 2929~2951, 2947 ~2969, 2962~2984, 2987~3009, 3006~3028, 3025~3047, 3068~3090, 3097~3119, 3116~3138, 3143~3165, 3168~3190, 3183~3205, 3209~3231, 3236~3258, 3269~3291, 3289~3311, 3310~3332, 3330~335 2, 3391-3413, 3407-3429, 3422-3444, 3447-3469, 3468-3490, 3485-3507; or 8-30, 45-67, 519-541, 534-556, 617-639, 653-675, 669-691, 702-724, 717-739, 760-782, 776-798, 798-820, 85 It contains at least 15 consecutive nucleotides of any one of nucleotides 1 to 873, 870 to 892, 885 to 907, 904 to 926, 929 to 951, 962 to 984, 984 to 1006, 1000 to 1022, 1016 to 1038, 1055 to 1077, 1070 to 1092, 1113 to 1135, 1128 to 1150, or 1159 to 1181.

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

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

[0015] In one embodiment, the antisense strand of the dsRNA agent is selected from the antisense strand of AD-2315878 (SEQ ID NO: 1291), AD-2315874 (SEQ ID NO: 1287), or AD-2315875 (SEQ ID NO: 1288). In another embodiment, the sense strand is selected from the sense strand of AD-2315878 (SEQ ID NO: 1277), AD-2315874 (SEQ ID NO: 1273), or AD-2315875 (SEQ ID NO: 1274). In a further embodiment, the dsRNA agent is AD-2315878, AD-2315874, or AD-2315875.

[0016] In one embodiment, (a) the sense strand of the dsRNA agent comprises the sequence of SEQ ID NO:881 and all of its modifications, and the antisense strand of the dsRNA agent comprises the sequence of SEQ ID NO:1265 and all of its modifications; (b) the sense strand of the dsRNA agent comprises the sequence of SEQ ID NO:914 and all of its modifications, and the antisense strand of the dsRNA agent comprises the sequence of SEQ ID NO:1266 and all of its modifications; or (c) the sense strand of the dsRNA agent comprises the sequence of SEQ ID NO:907 and all of its modifications, and the antisense strand of the dsRNA agent comprises the sequence of SEQ ID NO:1272 and all of its modifications.

[0017] In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in cells 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 or 3 by 1, 2 or 3 or less, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 or 4 by 1, 2 or 3 or less, 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 or 3, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or 4, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0018] 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.

[0019] 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 modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a phosphorothioate ...tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol The nucleotide modification is selected from the group consisting of nucleotides containing a phosphate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5' phosphate, nucleotides containing a 5' phosphate mimic, glycol-modified nucleotides, 2-O-(N-methylacetamide)-modified nucleotides, nucleotides containing vinylphosphonic acid, nucleotides containing glycol nucleic acids (GNAs) (e.g., adenosine-glycol nucleic acids), nucleotides containing glycol nucleic acid S-isomers (S-GNAs) (e.g., thymidine-glycol nucleic acid S-isomers), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked ribonucleotides (3'-RNA), and terminal nucleotides linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group, and combinations thereof. In one embodiment, the nucleotide modification is 2'-O-methyl and / or 2'-fluoro.

[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 any one of Tables 3, 4, 5, 6, 7, 8A or 8B.

[0032] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 PLG, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having the formula (III): 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'(III) [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 (III) is represented by Formula (IIIa): 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'(IIIa) is expressed by

[0037] In another embodiment, Formula (III) is represented by Formula (IIIb): Sense: 5' n p -N a -YYY-N b -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'(IIIb) [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 (III) is Formula (IIIc): 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'(IIIc) [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 (III) is formula (IIId): 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'(IIId) [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, 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 one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 PLG, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having the formula (III): 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'(III) [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.

[0065] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 PLG, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having the formula (III): 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'(III) [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 represent 0 to 6; n p'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage; each Na and Na' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides that are 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.

[0066] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 PLG, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having the formula (III): 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'(III) [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 represent 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 ' is different from the modification on Y'], and the sense strand is conjugated to at least one ligand, which is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0067] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 PLG, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having the formula (III): 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'(III) [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', 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.

[0068] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 PLG, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent having the formula (III): 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'(IIIa) [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 represent 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.

[0069] In one embodiment, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) 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 or 3, 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 or 4, wherein substantially all of the nucleotides in 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 in 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 or 3, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 2 or 4, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end and substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, 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.

[0070] 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.

[0071] In one embodiment, the region of complementarity comprises any one of the antisense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.

[0072] 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 any one of Tables 3, 4, 5, 6, 7, 8A or 8B.

[0073] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of plasminogen (PLG) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region.The sense strand comprises the nucleotide sequence of any one of the agents in Table 3, 4, 5, 6, 7, 8A or 8B, and the antisense strand comprises the nucleotide sequence of any one of the agents in Table 3, 4, 5, 6, 7, 8A or 8B.Substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the dsRNA agent is conjugated to a ligand.

[0074] In various embodiments of the dsRNA agent, the dsRNA agent targets a hotspot region of the mRNA encoding PLG.

[0075] In another embodiment, the invention provides dsRNA agents that target hotspot regions of plasminogen (PLG) mRNA.

[0076] 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).

[0077] In one aspect, the present invention provides a method for inhibiting the expression of plasminogen (PLG) in a cell, comprising contacting the cell with a dsRNA agent or pharmaceutical composition of the present invention to inhibit the expression of PLG in the cell.

[0078] The cell may be in a subject, such as a human subject.

[0079] In one embodiment, PLG expression is inhibited by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to below the level of detection of PLG expression. In another embodiment, PLG expression is inhibited by about 50% or less. In a specific embodiment, PLG expression is inhibited by about 50%.

[0080] In one embodiment, the human subject is suffering from a PLG-associated disease, disorder, or condition. In one embodiment, the PLG-associated disease, disorder, or condition is a bleeding disorder such as hereditary hemorrhagic telangiectasia (HHT). In one embodiment, the symptom of the bleeding disorder is menorrhagia (HMB). In one embodiment, the bleeding disorder is selected from the group consisting of PAI-1 deficiency, hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenenia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum bleeding, and excessive bleeding after surgery. In one embodiment, the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT). In one embodiment, the PLG-related disease, disorder or condition is a surgical procedure such as cardiac surgery, oral surgery, liver surgery, nephrolithotomy, orthopedic surgery, gynecological surgery, trauma treatment, tooth extraction, or dermatological surgery.In one embodiment, the subject is treated to prevent excessive bleeding before surgery.In another embodiment, the PLG-related disease, disorder or condition is melasma or hyperpigmentation of the skin.

[0081] In one aspect, the present invention provides a method for inhibiting the expression of PLG in a subject. The method comprises administering a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the present invention to the subject, thereby inhibiting the expression of PLG in the subject.

[0082] In another aspect, the present invention provides a method for treating a subject suffering from PLG-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 PLG-related disease, disorder or condition.

[0083] In another embodiment, the present invention provides the method for preventing at least one symptom in the subject with disease, disorder or condition, which will benefit from the reduction of the expression of PLG gene.Method comprises administering to subject the preventively effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby preventing at least one symptom in the subject with disease, disorder or condition, which will benefit from the reduction of the expression of PLG gene.

[0084] In one embodiment, administering the dsRNA agent or pharmaceutical composition to a subject reduces PLG activity, decreases PLG protein accumulation, and / or reduces excessive bleeding in the subject.

[0085] In one embodiment, the PLG-related disease, disorder, or condition is a bleeding disorder.

[0086] In one embodiment, the symptom of a bleeding disorder is menorrhagia (HMB).

[0087] In one embodiment, the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT).

[0088] In one embodiment, the bleeding disorder is selected from the group consisting of hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum hemorrhage, and excessive bleeding after surgery.

[0089] In one embodiment, the PLG-associated disease, disorder, or condition is a surgical procedure such as cardiac surgery, oral surgery, liver surgery, nephrolithotomy, orthopedic surgery, gynecological surgery, trauma treatment, tooth extraction, or dermatological surgery.

[0090] In one embodiment, the PLG-related disease, disorder, or condition is melasma or hyperpigmentation of the skin.

[0091] In one embodiment, a subject is treated prior to surgery to prevent excessive bleeding.

[0092] In one embodiment, the bleeding disorder is a mucocutaneous bleeding disorder (MCB), hi one embodiment, the mucocutaneous bleeding disorder is selected from the group consisting of inherited platelet disorders (IPD), hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndrome (EDS), and von Willebrand disease (VWD).

[0093] In one embodiment, the methods and uses of the invention further comprise administering to the subject an additional therapeutic agent.

[0094] 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.

[0095] The agent is administered to the subject intravenously, intramuscularly, or subcutaneously. In one embodiment, the agent is administered to the subject subcutaneously.

[0096] In one embodiment, the methods and uses of the present invention further comprise determining the level of PLG in the subject.

[0097] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of plasminogen (PLG) 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 any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, and the antisense strand comprises the nucleotide sequence of any one of the agents in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, 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.

[0098] In one embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium salts, calcium salts, lithium salts, potassium salts, ammonium salts, magnesium salts, and mixtures thereof. Those skilled in the art will appreciate that the RNAi agent may be provided as a polycationic salt having one cation for each free acid group of the optionally modified phosphodiester backbone and / or any other acidic modifications (e.g., 5'-terminal phosphonate group). For example, an oligonucleotide of "n" nucleotides in length contains "n-1" optionally modified phosphodiesters, so a 21-nt oligonucleotide may be provided as a salt having up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a 21-nt sense strand and a 23-nt antisense strand may be provided as a salt having up to 42 cations (e.g., 42 sodium cations). In the preceding example, if the RNAi agent includes a 5'-terminal phosphate or 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (eg, 44 sodium cations). [Brief explanation of the drawings]

[0099] [Figure 1] Figure 1 shows the results of a multi-dose in vitro screen using 1,100 PLG siRNA duplexes in transfected primary human hepatocytes (PHHs). The final duplex concentrations were 10 nM (diamonds), 1 nM (squares), or 0.1 nM (triangles). The results are plotted against the position of the duplex on the human NM_000301 transcript. [Figure 2] Figure 2 shows the percentage of plasma PLG antigen remaining (measured by ELISA) in an in vivo single-dose study in PXB mice. Exemplary PLG siRNA duplexes were administered at 0.5 mg / kg, and PLG levels were measured in plasma at the indicated time points. [Figure 3] Figure 3 shows the percentage of remaining plasma human PLG antigen (measured by ELISA) in an in vivo single-dose study in PXB mice. Exemplary PLG siRNA duplexes were administered at 1 mg / kg and 3 mg / kg, and PLG levels were measured in plasma at the indicated time points. [Figure 4] Figure 4 shows the percentage of remaining plasma human PLG antigen (measured by ELISA) in an in vivo single-dose study in PXB mice. Exemplary PLG siRNA duplexes were administered at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, and PLG levels were measured in plasma at the indicated time points. [Figure 5] Figure 5 shows the percentage of remaining plasma human PLG antigen (measured by ELISA) in an in vivo single-dose study in PXB mice. Exemplary PLG siRNA duplexes were administered at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, and PLG levels were measured in plasma at the indicated time points. DETAILED DESCRIPTION OF THE INVENTION

[0100] The present invention provides iRNA compositions that carry out RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PLG gene. The PLG gene can be in a cell, for example, in a cell of a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention to inhibit expression of the PLG gene and to treat a subject who would benefit from inhibiting or reducing expression of the PLG gene, e.g., a subject who would benefit from reduced bleeding, e.g., a subject suffering from or susceptible to a PLG-related disease, disorder, or condition, e.g., a subject suffering from or susceptible to a bleeding disorder (i.e., menorrhagia), such as a subject suffering from hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenenia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum hemorrhage, or excessive bleeding after surgery.

[0101] The iRNA of the present invention targeting PLG 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 ... 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 and that is substantially complementary to at least a portion of an mRNA transcript of the PLG gene.

[0102] 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 PLG 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.

[0103] The use of the iRNA agents described herein allows for the targeted degradation of the mRNA of the PLG gene in a mammal.

[0104] In particular, very low doses of iRNA can specifically and efficiently mediate RNA interference (RNAi) to significantly inhibit the expression of PLG genes. Thus, methods and compositions comprising these iRNAs are useful for treating subjects who would benefit from inhibiting or reducing expression of the PLG gene, e.g., subjects who would benefit from reduced bleeding, e.g., subjects suffering from or susceptible to a PLG-related disease, disorder, or condition, e.g., subjects suffering from or susceptible to a bleeding disorder, such as hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenenia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum hemorrhage, or excessive bleeding after surgery.

[0105] The following detailed description discloses how to make and use compositions containing iRNA to inhibit expression of the PLG gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibition and / or reduction of expression of this gene.

[0106] 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.

[0107] 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.

[0108] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0109] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0110] 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.

[0111] Unless otherwise specified, the term "PLG," also known as "plasminogen," "plasmin," "HAE4," "EC 3.4.21.7," and "EC 3.4.21," refers to the well-known gene encoding the PLG protein from any vertebrate or mammalian source, including, without limitation, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig.

[0112] The term also refers to fragments and variants of native PLG that maintain at least one in vivo or in vitro activity of native PLG.

[0113] Plasminogen (PLG) is a serine protease that mediates fibrinolysis, or the dissolution of fibrin clots. PLG is highly expressed in the liver and, to a lesser extent, in the kidney. Plasminogen is released from the liver into the systemic circulation. Because plasminogen is the primary driver of fibrinolysis, reducing plasminogen levels or activity in patients with bleeding disorders may be beneficial. Non-limiting examples of bleeding disorders include hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum bleeding, or excessive bleeding after surgery.

[0114] In one embodiment, the bleeding disorder is a mucocutaneous bleeding disorder (MCB). The biology of MCB is less well understood than other bleeding disorders, such as hemophilia, and diagnosis can take a long time. Symptoms of MCB include nosebleeds (epistaxis), menorrhagia, postpartum bleeding, gastrointestinal bleeding, easy bruising, persistent bleeding, and gum bleeding. TXA is used as a treatment to reduce bleeding in patients with MCB. Non-limiting examples of mucosal bleeding disorders include inherited platelet disorders (IPD), hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndrome (EDS), and von Willebrand disease (VWD).

[0115] Nucleotide and amino acid sequences of exemplary PLGs can be found, for example, in GenBank Accession No. NM_000301.5 (SEQ ID NO: 1; reverse complement SEQ ID NO: 2) and GenBank Accession No. NM_001168338.1 (SEQ ID NO: 3; reverse complement SEQ ID NO: 4) for human (Homo sapiens) PLG; GenBank Accession No. XM_005551498.2 (SEQ ID NO: 685; reverse complement SEQ ID NO: 686) for cynomolgus monkey (Macaca fascicularis) PLG; GenBank Accession No. NM_008877.3 (SEQ ID NO: 687; reverse complement SEQ ID NO: 688) for mouse (Mus musculus) PLG; and GenBank Accession No. NM_053491.2 (SEQ ID NO: 689; reverse complement SEQ ID NO: 690) for rat (Rattus norvegicus) PLG.

[0116] Additional examples of PLG mRNA sequences are readily available from public databases such as GenBank, UniProt, and OMIM.

[0117] Additional information regarding PLG is provided, for example, in the NCBI Gene database (http: / / www.ncbi.nlm.nih.gov / gene / 5340).

[0118] The term "PLG" as used herein also refers to a specific polypeptide expressed in a cell due to naturally occurring DNA sequence variations of the PLG gene, for example, a single nucleotide polymorphism in the PLG gene. Many SNPs within the PLG gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0119] In some embodiments, iRNAs substantially complementary to regions of human PLG mRNA cross-react with mouse PLG mRNA. In some embodiments, iRNAs substantially complementary to regions of mouse PLG mRNA cross-react with human PLG mRNA, making them potential candidates for human targeting. In some embodiments, iRNAs substantially complementary to regions of mouse or human PLG mRNA cross-react with rat, monkey, and / or rabbit PLG mRNA.

[0120] As used herein, "target sequence" refers to a contiguous portion of a nucleotide sequence in an mRNA molecule formed upon transcription of a PLG 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 PLG gene.

[0121] The target sequence of the PLG gene may be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence may be about 15 to 30 nucleotides in length, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 1 It may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0122] 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.

[0123] " 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.

[0124] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," used interchangeably herein, refer to agents that contain RNA, as those terms are defined herein, and mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, e.g., inhibits, the expression of the PLG gene in cells, e.g., cells within a subject, such as a mammalian subject.

[0125] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, e.g., a PLG 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 the 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 (ssRNA) that is produced in cells, promotes the formation of RISC complex, and thereby silences target gene, i.e., PLG gene.Therefore, the term " siRNA " is used herein to also mean the RNAi described above.

[0126] 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.

[0127] 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 containing two antiparallel, substantially complementary nucleic acid strands, said to have a "sense" or "antisense" orientation with respect to the target RNA, i.e., the PLG gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0128] 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.

[0129] 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.

[0130] The two strands forming the duplex structure can be different parts of one larger RNA molecule, or they can be separate RNA molecules. The two strands are part of a larger molecule, and are connected by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming the duplex structure, and the connecting RNA strands are called "hairpin loops." 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.

[0131] 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 nucleotide chain 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 dsRNA minus all overhangs that exist in the duplex.In addition to the duplex structure, RNAi can also contain one or more nucleotide overhangs.

[0132] 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 PLG 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 PLG 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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., PLG mRNA.

[0138] As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence, for example, a PLG nucleotide sequence, as defined herein. If the region of complementarity is not completely complementary to the target sequence, the mismatch can be in the internal or terminal region of the molecule. Generally, the most tolerable mismatch is within the terminal region, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' end of the iRNA.

[0139] 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.

[0140] 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.

[0141] 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 the 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 of skill in the art. Such conditions may be "stringent conditions," which may include, for example, 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). Other conditions, such as physiologically relevant conditions encountered within an organism, may also be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0142] 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 entire length of one or both nucleotide sequences.Such sequences can be referred to herein as "fully complementary" with respect to each other.However, when the first sequence is referred to herein as "substantially complementary" with respect to the second sequence, the two sequences can be completely complementary, or can form one or more mismatched base pairs, generally 5, 4, 3 or 2 or less, during hybridization for a duplex of up to 30 base pairs, while maintaining hybridization ability under the conditions most relevant to the final application, such as inhibiting gene expression through the RISC pathway.However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these 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 perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0143] "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 Hoogstein base pairing.

[0144] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as understood in connection with their use.

[0145] 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 PLG). For example, a polynucleotide is complementary to at least a portion of a PLG mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PLG.

[0146] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target PLG sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target PLG sequence, and comprise a contiguous nucleotide sequence that is at least about 80% complementary, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the nucleotide sequence of SEQ ID NO: 1 or 3 or the equivalent region of a fragment of SEQ ID NO: 1 or 3 throughout its entire length.

[0147] In one embodiment, the RNAi agent of the present invention is substantially complementary to an antisense polynucleotide that is further complementary to a target PLG 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 4 or an equivalent region of a fragment of any one of SEQ ID NO: 2 or 4, throughout its entire length.

[0148] In some embodiments, the iRNA of the invention comprises an antisense strand that is substantially complementary to a target PLG 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, over its entire length to the nucleotide sequence of any one of the sense strands in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, or the equivalent region of a fragment of any one of the sense strands in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.

[0149] The term "inhibiting," as used herein, is used synonymously with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0150] The phrase "inhibiting expression of the PLG gene," as used herein, includes inhibition of expression of any PLG gene (e.g., mouse PLG gene, rat PLG gene, monkey PLG gene, or human PLG gene), as well as variants or mutants of the PLG gene that encode PLG proteins.

[0151] "Inhibiting expression of the PLG gene" includes any level of inhibition of the PLG gene, e.g., at least partial suppression of the PLG 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%. In some embodiments, inhibition is at least about 50%.

[0152] The expression of PLG gene can be evaluated based on the level of any variable associated with the expression of PLG gene, for example, PLG mRNA level or PLG protein level.The expression of PLG gene can be evaluated indirectly, for example, based on the PLG activity level in tissue samples, for example, liver samples.Inhibition can be evaluated by the absolute or relative reduction of one or more of these variables compared with the control level.The control level can be any kind of control level used 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 (for example, a buffer-only control or an inactive drug control).

[0153] In one embodiment, at least partial suppression of PLG gene expression is assessed by a reduction in the amount of PLG mRNA, which can be isolated from or detected in a first cell or group of cells in which the PLG gene is transcribed and which has been treated or has been treated to inhibit expression of the PLG gene, and compared with 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.

[0154] The degree of inhibition may be expressed by the following formula:

[0155]

number

[0156] 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.

[0157] 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 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.

[0158] 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.

[0159] 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.

[0160] 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).

[0161] In embodiments, the subject is a human, for example, a human being treated or evaluated for a disease, disorder, or condition that is expected to benefit from reduced PLG expression; a human being at risk for a disease, disorder, or condition that is expected to benefit from reduced PLG expression; a human being with a disease, disorder, or condition that is expected to benefit from reduced PLG expression; and / or a human being treated for a disease, disorder, or condition that is expected to benefit from reduced PLG expression as described herein.

[0162] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, including, but not limited to, alleviation or amelioration of one or more symptoms associated with PLG gene expression and / or PLG protein production, e.g., a PLG-related disorder, e.g., bleeding disorders (i.e., menorrhagia) and melasma. In one embodiment, the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT). In one embodiment, the bleeding disorder is menorrhagia (HMB). In another embodiment, the bleeding disorder is selected from the group consisting of PAI-1 deficiency, hereditary hemorrhagic telangiectasia (HHT), von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenenia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum hemorrhage, and excessive bleeding after surgery. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0163] The term "lower" in the context of a PLG-related disorder 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 more. In certain embodiments, the decrease is at least 20%. "Lower" in the context of PLG levels in a subject is preferably a decrease to a level within the range considered normal for individuals without the disorder.

[0164] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of the PLG gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with the disease, disorder, or condition, such as symptoms of PLG gene expression, such as excessive bleeding. Not developing 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% on a scale clinically acceptable for the disease or disorder), or a delay (e.g., a delay of several days, weeks, months, or years) in the onset of delayed symptoms (e.g., reduced bleeding) is considered effective prevention.

[0165] As used herein, the term "PLG-associated disease" refers to a disease or disorder caused by or associated with PLG gene expression or PLG protein production. The term "PLG-associated disease" includes diseases, disorders, or conditions that would benefit from reduced PLG gene expression or protein activity.

[0166] In one embodiment, the "PLG-related disease" is a bleeding disorder. A "bleeding disorder" is any disease, disorder, or condition associated with severe or excessive bleeding. Non-limiting examples of bleeding disorders include hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum bleeding, and excessive bleeding after surgery.

[0167] In one embodiment, the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT). HHT is an inherited vascular disorder that causes excessive bleeding and affects men and women of all ages and ethnic backgrounds. Approximately 70,000 people in the United States have HHT, and 1.4 million people worldwide suffer from HHT. Three genetic mutations in the TGFβ pathway are known to cause HHT, including mutations in the endoglinin, Smad4, and ALK1 genes, which are involved in endothelial cell migration in angiogenesis and vascular remodeling. HHT patients bleed easily and have abnormally fragile blood vessels exhibiting increased localized fibrinolysis and vascular malformations, such as telangiectasia and arteriovenous malformations. Approximately 90% of HHT patients experience recurrent nosebleeds of varying frequency and severity, with more than five nosebleeds per week and nosebleeds lasting more than five hours, which may require emergency room visits and blood transfusions. Patients with HHT may also experience gastrointestinal bleeding, menorrhagia (HMB), anemia, and frequent iron / blood transfusions.

[0168] Currently, there are no FDA-approved medications for the treatment of HHT. Tranexamic acid (TXA) is an oral antifibrinolytic drug used off-label in patients with HHT, but it is not an ideal treatment due to its low bioavailability, high drug burden (two large tablets taken three to four times daily), and off-target side effects. Therefore, there is a need for treatments with sustained efficacy for HHT. In one embodiment, the symptom of the bleeding disorder can be menorrhagia (HMB).

[0169] HMB is excessive menstrual bleeding that impairs a woman's physical, social, emotional, and / or material quality of life. One in five women ages 30-55 perceive their menstrual bleeding as abnormal. HMB poses a significant burden to over 10 million American women each year. HMB has been associated with iron deficiency anemia, fatigue, and missed school / work / activities. Over $1 billion is spent annually on HMB treatment.

[0170] Causes of HMB are classified under the acronym PALM-COEIN: polyps, adenomyosis, leiomyomas, malignancies, coagulation disorders, ovulatory disorders, endometriosis, iatrogenic (e.g., copper IUD intrauterine contraceptive systems), and other (e.g., cesarean section scar abnormalities). Uterine fibroids and polyps are the most commonly reported drivers of HMB. Approximately 30% of women with HMB have a known bleeding disorder, such as von Willebrand disease, low factor XI, or platelet abnormalities. Approximately 50% of women with HMB have no pathological evidence of the cause.

[0171] Standard treatment for HMB includes the plasminogen activator inhibitor tranexamic acid (TXA), an intrauterine device (IUD), such as Mirena, and / or oral contraceptives (OCPs). TXA is typically used in women who do not want or cannot tolerate hormones. Treatment may include combination therapy with an OCP or TXA and an IUD.

[0172] However, these treatments have side effects that can lead to discontinuation. Approximately 40% of women with HMB discontinue the Mirena IUD within two years due to lack of effectiveness (60%), hormonal side effects (20%), and irregular bleeding. 15% of women with IUDs required additional TXA. Other side effects include uterine wall perforation, IUD expulsion or migration, intermenstrual bleeding, headache, acne, and breast pain. Side effects of OCPs include nausea, breast pain, headache, decreased libido, and thrombosis. TXA inhibits PLG activity, resulting in side effects such as menstrual pain, headache, lower back pain, nausea, vomiting, and musculoskeletal pain. TXA inhibits GABA in the spinal cord. A Off-target inhibition of γ-aminobutyric acid type A and glycine receptors may lead to dysregulation of pain processing and increase the risk of seizures (Ohashi et al., 2015, Sci Rep. 5:13458). Therefore, alternative treatments to HMB are needed.

[0173] The dsRNA agent provided herein that inhibits plasminogen expression can be used to treat bleeding disorders such as HMB.The potential advantages of the plasminogen-reducing siRNA approach in HMB over current standard treatments are that siRNA is a non-hormonal option, which can avoid the hormonal side effects of OCP and Mirena IUD, avoid the side effects of TXA, and can be administered less frequently, potentially reducing the risk of thrombosis.

[0174] Patients with genetic disorders resulting in plasminogen deficiency (e.g., type I or type II plasminogen deficiency) are not at increased risk for thrombosis (Schuster V. et al., 2007, J Thromb Haemost. 5: 2315-22). However, because these patients have little or no PLG activity, they experience fibrinous lesions due to fibrin deposition, such as fibrinous conjunctivitis, fibrinous gingivitis, fibrinous cervicitis, and fibrinous endometritis. Treatment for patients with plasminogen deficiency involves intravenous infusion of purified human plasma donor-derived glutamic acid (administered every 2–4 days). Studies on TXA have shown that lower doses of TXA can be as effective as standard doses in treating HMB (Minimum Effective Dose of Tranexamic Acid in Women with Menorrhagia: 24th Congress of the International Society on Thrombosis and Hemostasis, 2013). Therefore, a lower plasminogen suppression potency of about 50% or less may be sufficient. Thus, to minimize side effects due to PLG deficiency, such as the occurrence of pathology, the knockdown of PLG by the dsRNA agent provided herein can be selected or designed to achieve a knockdown of PLG of 50% or less. In some embodiments, the dsRNA agent provided herein reduces the expression of PLG by about 50%, 45%, 40%, 45%, 30%, 25%, 20%, 15%, 10%, 5% or less.

[0175] In one embodiment, the person who will benefit from reducing PLG gene expression or protein activity is the woman with uterine fibroids.In some embodiments, the person who will benefit from reducing PLG gene expression or protein activity is the woman with HMB and uterine fibroids, the woman with known bleeding disorder, the woman with unexplained bleeding who is insufficient with IUD or OCP, the woman who is already taking tranexamic acid (TXA), the woman who cannot tolerate hormone therapy (IUD or OCP), the woman who cannot tolerate TXA, or the woman who does not choose surgery or hormone option.

[0176] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a PLG-related disease, disorder, or condition, is sufficient to effectively treat the disease (e.g., by attenuating, ameliorating, or maintaining an 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.

[0177] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA that, when administered to a subject with a PLG-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 for 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 characteristics of the patient being treated.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] II. iRNAs of the Invention The iRNA described herein inhibits the expression of target gene.In one embodiment, the iRNA inhibits the expression of PLG gene.In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of PLG gene in cells, for example, liver cells, for example, liver cells in the body of a subject, for example, a mammal, for example, a human with bleeding disorder or condition.

[0183] dsRNA comprises an antisense strand with a complementary region that is complementary to at least a portion of the mRNA formed in the expression of PLG gene.The complementary region is about 30 nucleotides or less (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19 or 18 nucleotides or less in length).When iRNA contacts with the cell that expresses target gene, it inhibits the expression of target gene (for example, human, primate, non-primate or rodent target gene) by at least about 10%, as determined by, for example, PCR or branched DNA (bDNA)-based method, or by protein-based method, for example, immunofluorescence analysis using Western blotting or flow cell technology.

[0184] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (antisense strand) comprises a region of complementarity that is substantially complementary, generally completely complementary, to the target sequence. The target sequence can be derived from the sequence of mRNA formed upon expression of the PLG gene. The other strand (sense strand) comprises a region 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, such that they are opposite each other on separate oligonucleotides.

[0185] 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.

[0186] 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.

[0187] 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).

[0188] 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 PLG expression is not generated in the target cell by cleavage of a larger dsNRA.

[0189] 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.

[0190] 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.

[0191] 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 siRNA compounds 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.

[0192] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, sense sequence and antisense sequence.Sense strand is selected from the group of sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A or 8B, and the corresponding nucleotide sequence of the antisense strand of sense strand is selected from the group of sequences listed in any one of Tables 3, 4, 5, 6, 7, 8A or 8B.In this embodiment, one of the two sequences is complementary to the other of the two sequences, and in this case, one of the sequences is substantially complementary to the sequence of the mRNA produced during the expression of PLG gene.Therefore, in this embodiment, dsRNA will comprise two oligonucleotides, one oligonucleotide being described as sense strand (passenger strand) in any one of Tables 3, 4, 5, 6, 7, 8A or 8B, and the second oligonucleotide being described as the corresponding antisense strand (guide strand) of sense strand in any one of Tables 3, 4, 5, 6, 7, 8A or 8B. In one embodiment, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides, hi another embodiment, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0193] Although the sequences in Tables 3, 4, 5, 6, 7, 8A, or 8B 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 any one of Tables 3, 4, 5, 6, 7, 8A, or 8B that is unmodified, unconjugated, and / or modified and / or conjugated differently than described.

[0194] 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 PLG gene differs from dsRNA containing the entire sequence by no more than about 5, 10, 15, 20, 25, or 30% inhibition, are contemplated to be within the scope of the present invention.

[0195] In addition, the RNAs listed in any one of Tables 3, 4, 5, 6, 7, 8A or 8B identify a site in the PLG transcript that is susceptible to RISC-mediated cleavage.Therefore, the present invention further features an iRNA that targets within this site.As used herein, an iRNA is said to target a specific site within an RNA transcript if it promotes the cleavage of the transcript at any location within the specific site.Such an iRNA will generally comprise at least about 15 consecutive nucleotides from one of the sequences provided herein, coupled with additional nucleotide sequences taken from the region adjacent to the selected sequence in the gene.

[0196] 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.

[0197] 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).

[0198] The iRNA agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches to the target sequence, it is preferable that the region of mismatch is not located in the center of the region of complementarity. When the antisense strand of an iRNA contains mismatches to the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, in a 23-nucleotide iRNA agent, the strand complementary to a region of the PLG gene generally does not contain mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an iRNA containing mismatches to a target sequence is effective in inhibiting PLG gene expression. It is important to consider the efficacy of an iRNA with mismatches in inhibiting PLG gene expression, especially if a particular region of complementarity in the PLG gene is known to have polymorphic sequence variation within the population.

[0199] 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.

[0200] 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.

[0201] 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) to the region may be identified as efficacious.

[0202] 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).

[0203] 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.

[0204] In various embodiments, a dsRNA agent of the invention targets a hotspot region of the mRNA encoding PLG.

[0205] III. Modified iRNAs of the Invention In one embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified, e.g., does not contain 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.

[0206] 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).

[0207] 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).

[0208] 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. In some embodiments, the 5' end of the antisense strand of the double-stranded iRNA agent does not comprise a 5'-vinyl phosphonate (VP).

[0209] 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, 4, 5, 6, 7, 8A, or 8B may be individually modified to provide another 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, a structure of the formula: 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-2042815, the sense sequence: gsuscaacAfaCfAfUfccugggauuuL96 (SEQ ID NO: 279), gsuscaacAfaCfAfUfccugggaususu (SEQ ID NO: 684) may be substituted to provide another double-stranded iRNA agent of this disclosure.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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 new groups. The base 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.

[0216] 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 the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2--- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) of the above-referenced U.S. Patent No. 5,489,677, and amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506.

[0217] 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: lower alkyl, substituted lower 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, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic properties of iRNA, and other substituents with 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(CH2)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).

[0218] 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.

[0219] 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.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L. ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (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 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 substitutions have 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 are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0220] Representative United States 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, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672 and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0221] 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].

[0222] 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.

[0223] 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.

[0224] 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).

[0225] 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."

[0226] 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.

[0227] 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.

[0228] 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).

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In certain embodiments, the RNAi agent of the present invention is an agent that inhibits expression of the PLG gene selected from the group of agents set forth in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B. Any of these agents may further comprise a ligand.

[0233] 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 International Publication No. WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.

[0234] Thus, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., a PLG 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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 (or 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 guide strand loading into the RISC process.

[0240] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, wherein 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, 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.

[0241] In another embodiment, the RNAi agent is a 20-nucleotide double-ended bluntmer, 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.

[0242] In yet another embodiment, the RNAi agent is a double-ended bluntmer 21 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0243] 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. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand.

[0244] When a two-nucleotide overhang is at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, 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 3'-fluoro, for example, in the alternating motif.The RNAi agent may further comprise a ligand (preferably GalNAc3).

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] For RNAi agents with 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, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 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 agent from the 5' end.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.Strands can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0261] 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.

[0262] 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.

[0263] 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...".

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] In another embodiment, the 3'-terminal nucleotide of the sense strand is deoxythymine (dT). In another embodiment, the 3'-terminal nucleotide of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, at the 3'-end of the sense and / or antisense strands.

[0275] 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. Preferably, all of YYY are 2'-F modified nucleotides.

[0276] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0277] 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.

[0278] 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:

[0279] 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.

[0280] When the sense strand is represented by formula (Ic), N brepresents 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.

[0281] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 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.

[0282] Each of X, Y and Z may be the same as or different from one another.

[0283] 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:

[0284] 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.

[0285] In one embodiment, the antisense strand sequence of the RNAi has formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -np '3' (II) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides, each n p ' and n q ' independently represent an overhanging nucleotide, 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:

[0286] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0287] 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. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0288] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0289] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0290] Thus, the antisense strand has the following formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb); 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc); or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3'(IId).

[0291] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0292] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0293] When the antisense strand is represented by formula (IId), each N bEach 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 ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0294] 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:

[0295] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0296] Each of X', Y' and Z' may be the same as or different from one another.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc) and (IId), respectively.

[0301] 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 (III): Sense: 5' np -N a -(XXX)i -Nb- YYY -Nb -(ZZZ)j-Na-nq 3' Antisense: 3' np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq ’ 5' (III) [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 Na and Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb and Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each np', np, nq', and nq independently represents an overhanging nucleotide, 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

[0302] 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.

[0303] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following formula: 5' np - Na -YYY -Na-nq 3' 3' np'-Na'-Y'Y'Y' -Na'nq' 5' (IIIa) 5' np -Na -YYY -Nb -ZZZ -Na-nq 3' 3' np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5' (IIIb) 5' np-Na- XXX -Nb -YYY - Na-nq 3' 3' np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq' 5' (IIIc) 5' np -Na -XXX -Nb-Y YY -Nb- ZZZ -Na-nq 3' 3' np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq ’ 5' (IIId) Includes.

[0304] When the RNAi agent is represented by Formula (IIIa), each Na independently represents an oligonucleotide sequence comprising from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0305] When an RNAi agent is represented by Formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0306] When an RNAi agent is represented by Formula (IIIc), 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.

[0307] When an RNAi agent is represented as Formula (IIId), 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.

[0308] In formulae (III), (IIIa), (IIIb), (IIIc) and (IIId), X, Y and Z may be the same or different from one another.

[0309] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), 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.

[0310] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides, or at least two of the Z nucleotides can be base-paired with the corresponding Z' nucleotide, or all three of the Z nucleotides can be base-paired with the corresponding Z' nucleotide.

[0311] When the RNAi agent is represented by formula (IIIc) or (IIId), 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.

[0312] 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.

[0313] In one embodiment, when the RNAi agent is represented by formula (IIId), N aThe modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), 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 (IIId), 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 (IIId), 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).

[0314] In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, 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 bivalent or trivalent branched linker.

[0315] In one embodiment, the RNAi agent is a multimer containing at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), 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.

[0316] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), 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.

[0317] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at the 5' end, and one or both of the 3' ends 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] As described in more detail below, RNAi agents that include one or more carbohydrate moieties conjugated to the RNAi agent can optimize 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 (preferably 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.

[0322] Ligands can be attached to polynucleotides via carriers. The carrier comprises (i) at least one "backbone attachment point," preferably two, 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 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 contain functional groups, e.g., amino groups, or generally provide a bond suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the ring.

[0323] The RNAi agent may be conjugated to the ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0324] 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):

[0325] [ka] It may be represented by:

[0326] 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.

[0327] 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)

[0328] [ka] and iii) an abasic modification selected from the group consisting of:

[0329] [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

[0330] [ka] is.

[0331] 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.

[0332] n 1 , n 3 and q 1 are independently 4 to 15 nucleotides in length.

[0333] n 5 , q 3 and q 7 are independently 1 to 6 nucleotides in length.

[0334] n 4 , q 2 and q 6 are independently 1 to 3 nucleotides in length, or n 4 is 0.

[0335] q 5 are independently 0 to 10 nucleotides in length.

[0336] n 2 and q4 are independently 0 to 3 nucleotides in length.

[0337] Or, n 4 is 0 to 3 nucleotides in length.

[0338] 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).

[0339] In certain embodiments, n 4 , q 2 and q 6 are 1 respectively.

[0340] In certain embodiments, n 2 , n 4 , q 2 , q 4 and q 6 are 1 respectively.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] In certain embodiments, T1' and T3' are 11 nucleotides apart in length (ie, not counting T1' and T3').

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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).

[0354] 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).

[0355] 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.

[0356] 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).

[0357] 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.

[0358] 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).

[0359] 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.

[0360] 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).

[0361] 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.

[0362] 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).

[0363] 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.

[0364] 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).

[0365] 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.

[0366] 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).

[0367] 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.

[0368] 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).

[0369] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications to the RNAi agents described herein. In exemplary embodiments, the vinyl phosphonates of the present disclosure have the following structure:

[0370] [ka] It has.

[0371] In an exemplary embodiment, the 5' vinylphosphonate modified nucleotide of the disclosure has the following structure:

[0372] [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy); R5' is =C(H)-P(O)(OH)2, and the C5' carbon and R 5 The double bond between is in the E or Z orientation (e.g., E orientation), B is a nucleotide base or a modified nucleobase, and B can be adenine, guanine, cytosine, thymine, or uracil.

[0373] In one embodiment, R 5 In another embodiment, R is methoxy and R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E orientation. 5 In another embodiment, X is S, R is methoxy, and R is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E orientation. 5 is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E orientation.

[0374] The vinyl phosphonate of the present disclosure can be attached to either the antisense strand or the sense strand of the dsRNA of the present disclosure. In certain preferred embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA, optionally at the 5'-end of the antisense strand of the dsRNA. The dsRNA agent can include a phosphorus-containing group at the 5'-end of the sense strand or the 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 vinyl phosphonate (5'-VP), 5'-terminal methyl phosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. When the 5'-terminal phosphorus-containing group is 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate).

[0375] [ka] ), 5'-Z-VP isomer (i.e., cis-vinylphosphonate

[0376] [ka] ), or a mixture thereof.

[0377] Vinyl phosphate modifications are also contemplated in the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures are as follows:

[0378] [ka] is.

[0379] Another exemplary vinyl phosphate structure is the one shown above with R 5’ is =C(H)-OP(O)(OH)2, and the C5' carbon and R 5’The double bond between may be in the E or Z orientation (e.g., E orientation). For example, if the phosphate mimic is 5'-vinyl phosphate, the 5'-terminal nucleotide may have the immediately preceding structure in which the phosphonate group is replaced with a phosphate.

[0380] 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.

[0381] In certain embodiments, the RNAi agent comprises a 5'-P. In certain embodiments, the RNAi agent comprises a 5'-P in the antisense strand.

[0382] In certain embodiments, the RNAi agent comprises a 5'-PS. In certain embodiments, the RNAi agent comprises a 5'-PS on the antisense strand.

[0383] 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.

[0384] In certain embodiments, the RNAi agent comprises a 5'-PS2. In certain embodiments, the RNAi agent comprises a 5'-PS2 on the antisense strand.

[0385] 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.

[0386] 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. The RNAi agent also contains a 5'-PS.

[0387] 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'-P.

[0388] 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. 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.

[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. The RNAi agent also includes a 5'-PS2.

[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 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0391] 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.

[0392] 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.

[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 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). The RNAi agent also includes a 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[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 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.

[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 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 5'-deoxy-5'-C-malonyl.

[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 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 contains a 5'-P.

[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 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'-PS.

[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 q6 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.

[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 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'-PS2.

[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 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[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 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'-P.

[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 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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.

[0403] 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'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0404] 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'-PS2.

[0405] 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 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.

[0406] 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'-P.

[0407] 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.

[0408] 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 includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0409] 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.

[0410] 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0411] 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.

[0412] 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'-PS.

[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'-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.

[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 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.

[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 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 5'-deoxy-5'-C-malonyl.

[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 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'-P.

[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 q2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[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 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'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[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 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. The RNAi agent also includes a 5'-PS2.

[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 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0421] 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'-P.

[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 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.

[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'-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.

[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 q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 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.

[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'-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.

[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, 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.

[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 3is 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.

[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, 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'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0429] 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.

[0430] 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.

[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, 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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 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.

[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, 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.

[0447] 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.

[0448] In another specific embodiment, the RNAi agent of the invention is (a)(i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached through a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iv) 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.

[0449] 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 desoxynucleotide (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.

[0450] 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.

[0451] 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.

[0452] 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.

[0453] 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, and desoxy-nucleotides (e.g., dT) at positions 24 and 25 (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.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] In certain embodiments, the iRNA for use in the methods of the invention is an agent selected from those listed in Tables 3, 4, 5, 6, 7, 8A, or 8B. These agents may further comprise a ligand.

[0458] IV. Ligand-Conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking the RNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118, Kabanov et al., (1990) FEBS Lett., 259:327-330, Svinarchuk et al., (1993) Biochimie, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654, Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973) or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0459] In one embodiment, a ligand alters the distribution, targeting, or lifetime of an iRNA agent into which it is incorporated. In a preferred embodiment, a ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cellular or organ compartment, a tissue, an organ, or a region of the body, e.g., as compared to a species in which the ligand is not present. Preferred ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0460] Ligands can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0461] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, for example, an antibody that binds to a specific cell type, such as a kidney cell. The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0462] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable cleavage inhibitors include cleavage inhibitors (e.g., oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0463] The ligand can be a protein, such as a glycoprotein or peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specific cell type, such as a hepatocyte. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0464] The ligand can be a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent, e.g., by disrupting the cytoskeleton of a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, e.g., taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0465] In some embodiments, the ligand attached to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0466] Ligand-conjugated oligonucleotides of the invention can be synthesized by using oligonucleotides bearing pendant reactive functionality, e.g., resulting from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, ligands that have been synthesized with any of a variety of protecting groups, or ligands that have a linking moiety attached to them.

[0467] The oligonucleotides used in the conjugate of the present invention can be conveniently and routinely produced by known techniques of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, California).Any other means for such synthesis known in the art can also or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, for example, phosphorothioate and alkylated derivatives.

[0468] For the ligand-conjugated oligonucleotides and molecules having ligand-sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0469] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0470] A. Lipid Conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. The lipid or lipid-based molecule preferably binds to serum proteins, such as human serum albumin (HSA). The HSA-binding ligand allows the conjugate to be distributed to target tissues in the body, such as non-renal target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport into target cells or cell membranes, and / or (c) adjust the binding to serum proteins, such as HSA.

[0471] Lipid-based ligands can be used to inhibit, for example, control, the binding of conjugate to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney, and therefore is less likely to be eliminated from the body.The lipid or lipid-based ligand that binds less strongly to HSA can be used to target conjugate to the kidney.

[0472] In a preferred embodiment, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues, but preferably not so strong that the HSA-ligand binding is irreversible.

[0473] In other preferred embodiments, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate is preferably distributed to the kidney. Other moieties that target kidney cells can be used instead of, or in addition to, the lipid-based ligand.

[0474] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant types, for example, cancer cells.Exemplary vitamins include vitamins A, E and K.Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, such as hepatocytes.Also included are HSA and low-density lipoprotein (LDL).

[0475] B. Cell-penetrating agents In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. Exemplary agents are peptides, such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and lipophobic phase.

[0476] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0477] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 5). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 6)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 7)) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 8)) have been found to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Examples of peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units for cellular targeting include arginine-glycine-aspartic acid (RGD)-peptides or RGD mimics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications that, for example, increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0478] The RGD peptide for use in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0479] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0480] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars, and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0481] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the invention is

[0482] [ka] [ka] [ka] [ka] [ka] [ka] [wherein Y is O or S, and n is 3 to 6 (Formula XXIV)];

[0483] [ka] [wherein Y is O or S, and n is 3 to 6 (Formula XXV)];

[0484] [ka] [wherein X is O or S. (Formula XXVII)];

[0485] [ka] [ka] [ka] is selected from the group consisting of:

[0486] In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, e.g.,

[0487] [ka] is.

[0488] Other exemplary carbohydrates for use in the embodiments described herein include:

[0489] [ka] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0490] In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker.

[0491] In one embodiment, a standard RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent, e.g., the 3' or 5' end of the sense strand of a dsRNA agent described herein. In another embodiment, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent through multiple monovalent linkers.

[0492] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0493] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0494] Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0495] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0496] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., attaches two parts of a compound by a covalent bond.A linker is typically a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH or a group including, but not limited to, a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl, alkylheteroarylalkynyl

[0044] In some embodiments, R8 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In one embodiment, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 17, or 8 to 16 atoms.

[0497] A cleavable linking group is one that is sufficiently stable outside a cell, but that, upon entry into a target cell, is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0498] Cleavable linking groups are susceptible to the influence of cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more common or found at higher levels or activity inside cells than in serum or blood.Examples of such degrading agents include redox agents that are selected for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that produce a pH of 5 or less, general acids, peptidases (can be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0499] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0500] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can vary depending on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid via a linker that comprises ester group.Liver cell is rich in esterase, therefore, linker is cleaved more efficiently in liver cell than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testis cell.

[0501] When targeting cell types that are rich in peptidases, such as liver cells and synovial cells, linkers containing peptide bonds can be used.

[0502] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first and a second condition can be determined, with the first being selected to exhibit cleavage in target cells, and the second being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be carried out in a cell-free system, in cells, in cell culture, in organs or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0503] i. Redox-cleavable linking group In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some cases, candidate compounds are cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media c...

Claims

1. 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding PLG that comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A or 8B.

2. 10. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide.

3. 3. The dsRNA agent of claim 1 or 2, wherein substantially all of the nucleotides of the sense strand comprise a modification.

4. 3. The dsRNA agent of claim 1 or 2, wherein substantially all of the nucleotides of the antisense strand comprise a modification.

5. 3. The dsRNA agent of claim 1 or 2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification.

6. 6. The dsRNA agent of any one of claims 1 to 5, wherein the antisense strand is selected from the antisense strand of AD-2315878 (SEQ ID NO: 1291), AD-2315874 (SEQ ID NO: 1287), or AD-2315875 (SEQ ID NO: 1288).

7. The dsRNA agent of any one of claims 1 to 6, wherein the sense strand is selected from the sense strand of AD-2315878 (SEQ ID NO: 1277), AD-2315874 (SEQ ID NO: 1273), or AD-2315875 (SEQ ID NO: 1274).

8. 8. The dsRNA of any one of claims 1 to 7, wherein the dsRNA agent is AD-2315878, AD-2315874, or AD-2315875.

9. A double-stranded RNA (dsRNA) agent for inhibiting expression of plasminogen (PLG) in a cell, the double-stranded RNA agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; A dsRNA agent wherein the sense strand is conjugated to a ligand attached at the 3'-end.

10. 10. The dsRNA agent of claim 9, wherein all of the nucleotides of the sense strand comprise a modification.

11. 10. The dsRNA agent of claim 9, wherein all nucleotides in the antisense strand comprise a modification.

12. 10. The dsRNA agent of claim 9, wherein every nucleotide in the sense strand and every nucleotide in the antisense strand comprises a modification.

13. 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 modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified 13. The dsRNA agent of any one of claims 2 to 12, wherein the nucleotide is selected from the group consisting of a 5'-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5' phosphate, a nucleotide containing a 5' phosphate mimic, a glycol-modified nucleotide, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

14. 14. The dsRNA agent of claim 13, wherein the nucleotide modifications are 2'-O-methyl and / or 2'-fluoro modifications.

15. The dsRNA agent of any one of claims 1 to 14, wherein the region of complementarity is at least 17 nucleotides in length.

16. The dsRNA agent of any one of claims 1 to 15, wherein the region of complementarity is 19 to 30 nucleotides in length.

17. 17. The dsRNA agent of claim 16, wherein the region of complementarity is 19 to 25 nucleotides in length.

18. 18. The dsRNA agent of claim 17, wherein the region of complementarity is 21 to 23 nucleotides in length.

19. The dsRNA agent of any one of claims 1-18, wherein each strand is independently 30 or fewer nucleotides in length.

20. 20. The dsRNA agent of any one of claims 1-19, wherein each strand is independently 19 to 30 nucleotides in length.

21. 21. The dsRNA agent of claim 20, wherein each strand is independently 19 to 25 nucleotides in length.

22. 21. The dsRNA agent of claim 20, wherein each strand is independently 21 to 23 nucleotides in length.

23. The dsRNA agent of any one of claims 1 to 22, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

24. 24. The dsRNA agent of claim 23, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

25. The dsRNA agent of any one of claims 1 to 8 and 13 to 24, further comprising a ligand.

26. 26. The dsRNA agent of claim 25, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

27. 27. The dsRNA agent of claim 9 or 26, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

28. The ligand is 【Chemical 1】 28. The dsRNA agent of claim 27, wherein:

29. A dsRNA agent may have the following structure: 【Chemistry 2】 wherein X is O or S.

29. The dsRNA agent of claim 28, wherein the dsRNA agent is conjugated to a ligand as shown in

30. 30. The dsRNA agent of claim 29, wherein X is O.

31. The dsRNA agent of any one of claims 1-8 or 15-30, wherein the region of complementarity comprises any one of the antisense sequences in any one of Tables 3, 4, 5, 6, 7, 8A or 8B.

32. (a) the sense strand comprises the sequence of SEQ ID NO: 881 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO: 1265 and all modifications; (b) the sense strand comprises the sequence of SEQ ID NO: 914 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO: 1266 and all modifications; or (c) the sense strand comprises the sequence of SEQ ID NO: 907 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO: 1272 and all modifications.

33. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A or 8B, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A or 8B; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand contains two phosphorothioate internucleotide linkages at the 5' end; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus; A dsRNA agent wherein the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached through a monovalent, divalent, or trivalent branched linker.

34. 34. The dsRNA agent of claim 33, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

35. 35. The dsRNA agent of claim 33 or 34, wherein the antisense sequence comprises any one of the antisense sequences set forth in any one of Tables 3, 4, 5, 6, 7, 8A or 8B.

36. 36. The dsRNA agent of any one of claims 1-35, wherein 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 agents listed in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B.

37. 37. The dsRNA of any one of claims 1 to 36, wherein the dsRNA agent targets a hotspot region of the mRNA encoding PLG.

38. dsRNA agents targeting hotspot regions of plasminogen (PLG) mRNA.

39. 39. A cell comprising the dsRNA agent of any one of claims 1-38.

40. A vector encoding at least one strand of the dsRNA agent of any one of claims 1-38.

41. 39. A pharmaceutical composition for inhibiting the expression of the plasminogen (PLG) gene, comprising the dsRNA agent of any one of claims 1 to 38.

42. 42. The pharmaceutical composition of claim 41, wherein the agent is formulated in an unbuffered solution.

43. 43. The pharmaceutical composition of claim 42, wherein the unbuffered solution is saline or water.

44. 42. The pharmaceutical composition of claim 41, wherein the agent is formulated in a buffer solution.

45. 45. The pharmaceutical composition of claim 44, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

46. 45. The pharmaceutical composition of claim 44, wherein the buffer solution is phosphate buffered saline (PBS).

47. A method for inhibiting plasminogen (PLG) expression in a cell, comprising contacting the cell with an agent described in any one of claims 1 to 38 or a pharmaceutical composition described in any one of claims 41 to 46, thereby inhibiting the expression of PLG in the cell.

48. 48. The method of claim 47, wherein the cell is in a subject.

49. 49. The method of claim 48, wherein the subject is a human.

50. 50. The method of any one of claims 47 to 49, wherein PLG expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or is inhibited to below the level of detection of PLG expression.

51. 51. The method of claim 50, wherein the human subject is suffering from a PLG-associated disease, disorder or condition.

52. 52. The method of claim 51, wherein the PLG-associated disease, disorder or condition is a bleeding disorder.

53. 53. The method of claim 52, wherein the bleeding disorder is hereditary hemorrhagic telangiectasia (HHT).

54. 52. The method of claim 51, wherein the PLG-associated disease, disorder, or condition is melasma or hyperpigmentation of the skin, or the subject is treated preoperatively to prevent excessive bleeding.

55. 53. The method of claim 52, wherein the bleeding disorder is selected from the group consisting of menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum hemorrhage, and excessive bleeding after surgery.

56. 53. The method of claim 52, wherein the bleeding disorder is a mucocutaneous bleeding disorder (MCB).

57. 57. The method of claim 56, wherein the mucocutaneous bleeding disorder is selected from the group consisting of inherited platelet disorders (IPD), hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndrome (EDS), and von Willebrand disease (VWD).

58. 10. A method of inhibiting expression of PLG in a subject, comprising inhibiting expression of PLG in the subject by administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-38, or the pharmaceutical composition of any one of claims 41-46.

59. A method for treating a subject suffering from a PLG-associated disease, disorder, or condition, comprising administering to the subject a therapeutically effective amount of the agent of any one of claims 1 to 38, or the pharmaceutical composition of any one of claims 41 to 46, thereby treating the subject suffering from a PLG-associated disease, disorder, or condition.

60. A method for preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduced expression of the PLG gene, comprising preventing at least one symptom in a subject having a disease, disorder or condition that would benefit from reduced expression of the PLG gene by administering to the subject a prophylactically effective amount of an agent described in any one of claims 1 to 38 or a pharmaceutical composition described in any one of claims 41 to 46.

61. 61. The method of any one of claims 58 to 60, wherein the PLG-associated disease, disorder or condition is a bleeding disorder.

62. 62. The method of claim 61, wherein the symptom of the bleeding disorder is heavy menstrual bleeding (HMB).

63. 62. The method of claim 61, wherein the bleeding disorder is selected from the group consisting of hereditary hemorrhagic telangiectasia (HHT), menorrhagia (HMB), PAI-1 deficiency, von Willebrand disease, low factor XI, platelet abnormalities, hemophilia A, hemophilia B, afibrinogenemia, parahemophilia, low factor VIII, low factor IX, low factor VII, low factor XIII, low factor X, low factor V, low factor II, nosebleeds, bleeding gums, easy bruising, postpartum hemorrhage, and excessive bleeding after surgery.

64. 61. The method of claim 59 or 60, wherein the PLG-associated disease, disorder, or condition is melasma or hyperpigmentation of the skin, or the subject is treated preoperatively to prevent excessive bleeding.

65. 62. The method of claim 61, wherein the bleeding disorder is a mucocutaneous bleeding disorder (MCB).

66. 66. The method of claim 65, wherein the mucocutaneous bleeding disorder is selected from the group consisting of inherited platelet disorders (IPD), hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndrome (EDS), and von Willebrand disease (VWD).

67. 67. The method of any one of claims 48 to 66, further comprising administering to the subject an additional therapeutic agent.

68. 68. The method of any one of claims 48-67, wherein 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.

69. 69. The method of any one of claims 48 to 68, wherein the agent is administered to the subject intravenously, intramuscularly, or subcutaneously.

70. 70. The method of any one of claims 48 to 69, further comprising determining the level of PLG in the subject.

71. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of plasminogen (PLG) in a cell, the dsRNA agent comprising a sense strand and an antisense strand which form a double-stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the agents in any one of Tables 3, 4, 5, 6, 7, 8A, or 8B, and the antisense strand comprises the nucleotide sequence of any one of the agents in Tables 3, 4, 5, 6, 7, 8A, or 8B; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; The dsRNA agent is conjugated to a ligand.

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