Transmembrane protease, serine 6 (TMPRSS6) iRNA compositions and methods of use thereof

Abstract

The present invention relates to RNAi agents, e.g., double-stranded RNA (dsRNA) agents targeting the transmembrane protease, serine 6 (TMPRSS6) gene. The present invention also relates to methods of using such RNAi agents to inhibit expression of the TMPRSS6 gene, and to methods of preventing and treating TMPRSS6-related disorders, e.g., iron overload-related disorders and / or ineffective erythropoiesis disorders, e.g., hereditary hemochromatosis, β-thalassemia (e.g., β-thalassemia major and β-thalassemia intermedia), polycythemia vera, myelodysplastic syndromes, congenital dyserythroplastic anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.

Description

[Background technology]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 179,607, filed April 26, 2021, and U.S. Provisional Patent Application No. 63 / 278,227, filed November 11, 2021. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 18, 2022, is named 121301_15420_SL.TXT, and is 673,291 bytes in size.

[0003] TMPRSS6 (transmembrane protease, serine 6), also known as matriptase-2, is a type II serine protease. TMPRSS6 mRNA is primarily expressed in the liver, but is also found at high levels in the kidney, at lower levels in the uterus, and in much smaller amounts in many other tissues (Beliveau et al., 2019, Cell Chemical Biology 26, 1559-1572). TMPRSS6 plays a key role in iron homeostasis through the regulation of hepcidin expression. Hepcidin, a liver-derived peptide hormone, is known as a central regulator of whole-body iron homeostasis, and its imbalanced production contributes to the pathogenesis of various iron disorders. Hepcidin functions by blocking dietary iron absorption from the intestine, as well as iron release from macrophages and hepatocytes (Ganz T. 2011, Blood, vol. 117, 17, 4425-4433). Hepcidin gene expression can be stimulated in response to iron through a BMP / SMAD-dependent signaling cascade mediated by the BMP coreceptor hemojuvelin (HJV). TMPRSS6 inhibits BMP-mediated upregulation of hepcidin by cleaving the BMP coreceptor HJV, inhibiting BMP signaling, SMAD translocation to the nucleus, and hepcidin transcriptional activation, which causes downregulation of hepcidin levels (Finberg, KE, et al., 2010, Blood 115, 3817-3826; Wang, CY, et al., 2014 Front. Pharmacol. 5, 114).

[0004] Therefore, inhibition of TMPRSS6 leads to an increase in hepcidin levels, making it an attractive pharmacological target for disorders associated with iron overload and inappropriately low hepcidin, or disorders in which iron restriction is desirable.Many disorders, such as thalassemia, hemochromatosis, and certain types of myelodysplastic syndrome (MDS), are associated with iron overload, a condition characterized by increased iron levels.Iron overload can lead to excess iron deposition in various tissues, ultimately resulting in tissue and organ damage.In addition, iron restriction is desirable in certain disorders, such as polycythemia vera.

[0005] Current treatments for iron overload-related disorders and disorders for which iron restriction is desirable (such as polycythemia vera) include bloodletting or phlebotomy, splenectomy, iron chelation therapy, and diet, which are treatments for removing iron-rich blood from the body.However, these treatments are not always effective.Therefore, there is a need in the art for alternative treatments for subjects with iron overload-related disorders. Summary of the Invention

[0006] The present invention provides an iRNA composition that affects the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the gene encoding transmembrane protease, serine 6 (TMPRSS6). The TMPRSS6 gene can be present in a cell, for example, in a cell of a subject, such as a human subject. The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of the TMPRSS6 gene and / or to treat a subject that would benefit from the inhibition or reduction of the expression of the TMPRSS6 gene, for example, a subject suffering from or susceptible to a TMPRSS6-related disorder, for example, an iron overload-related disorder and / or an ineffective erythropoiesis disorder, for example, thalassemia, for example, β-thalassemia, hemochromatosis, myelodysplastic syndrome (MDS), or polycythemia vera.

[0007] Thus, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) that inhibits expression of transmembrane protease, serine 6 (TMPRSS6) in a cell, said dsRNA comprising a sense strand and an antisense strand that form a double-stranded region, said antisense strand comprising a region of complementarity to an mRNA encoding TMPRSS6, said region of complementarity comprising at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences of any one of Tables 2-7.

[0008] In one embodiment, the dsRNA agent comprises a sense strand that includes a contiguous nucleotide sequence having at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity over its entire length to any one of the nucleotide sequences of the sense strand of any one of Tables 2-7, and an antisense strand that includes a contiguous nucleotide sequence having at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity over its entire length to any one of the nucleotide sequences of the antisense strand of any one of Tables 2-7.

[0009] In one embodiment, the dsRNA agent comprises a sense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of the sense strand of any one of Tables 2-7, and an antisense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of the antisense strand of any one of Tables 2-7.

[0010] In one embodiment, the dsRNA agent comprises a sense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than two nucleotides from any one of the nucleotide sequences of the sense strand of any one of Tables 2-7, and an antisense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than two nucleotides from any one of the nucleotide sequences of the antisense strand of any one of Tables 2-7.

[0011] In one embodiment, the dsRNA agent comprises a sense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than a single nucleotide from any one of the nucleotide sequences of the sense strand of any one of Tables 2-7, and an antisense strand that includes at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than a single nucleotide from any one of the nucleotide sequences of the antisense strand of any one of Tables 2-7.

[0012] In one embodiment, the dsRNA agent comprises a sense strand that comprises a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences of the sense strand in any one of Tables 2-7, and an antisense strand that comprises a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences of the antisense strand in any one of Tables 2-7.

[0013] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transmembrane protease, serine 6 (TMPRSS6) in a cell, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand being selected from nucleotides 187-210; 227-254; 322-363; 362-390; 398-420; 404-429; 410-435; 439-461; 443-467; 448-474; 460-483; 466-487; 468-489; 470-482; 472-473; 474-475; 476-481; 478-482; 478-483; 479-484; 485-486; 487-488; 489-490; 490-500; 50 ... 8;496~519;519~542;526~548;557~593;641~671;652~676;687~713;725~762;757~794;886~908;921~951;956~987;1051~1082;1233~1269;1279~1313;1313~1341;1327~1351;1415~1439;1447~1480;1464~1486;1486~1509;1559~1589;1571~1595;1579~160 9;1707~1735;1738~1764;1806~1828;1864~1886;1934~1966;1967~1991;2008~2031;2015~2043;2042~2072;2287~2311;2297~2354;2336~2361;2360~2384;2416~2438;2481~2510;2496~2527;2526~2558;2665~2693;2693~2719;2707~2729;2799~2821;2851 The antisense strand comprises at least 15 contiguous nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, that differ from any one of the nucleotide sequences of SEQ ID NO: 1-2874; 2971-2999; 2981-3006 and 3155-3195 by three or less, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides, which differ from the corresponding nucleotide sequence of SEQ ID NO: 2 by three or less, e.g., 3, 2, 1, or 0 nucleotides.

[0014] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of transmembrane protease, serine 6 (TMPRSS6) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand being selected from the group consisting of nucleotides 230-252, 324-346, 560-578, 560-582, 2338-2360, 3163-3185, 3169-3191, and 3172-3194 of SEQ ID NO:1. The antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:2 by no more than three, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from the corresponding nucleotide sequence of SEQ ID NO:2 by no more than three, e.g., 3, 2, 1, or 0 nucleotides.

[0015] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of transmembrane protease, serine 6 (TMPRSS6) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3, e.g., 3, 2, 1, or 0 nucleotides from any one of the nucleotide sequences of nucleotides 560-578, 2338-2360, and 3169-3191 of SEQ ID NO:1, and the antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3, e.g., 3, 2, 1, or 0 nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0016] In some embodiments, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by three or less, e.g., 3, 2, 1, or 0 nucleotides, from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1556360, AD-1571158, AD-1571033, AD-1554875, AD-1571160, AD-1555117, AD-1554911, and AD-1556915.

[0017] In some embodiments, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by three or less, e.g., 3, 2, 1, or 0 nucleotides, from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1556360, AD-1571158, and AD-1571033.

[0018] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0019] In one embodiment, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand comprise a modification, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand comprise a modification.

[0020] In one embodiment, every nucleotide in the sense strand contains a modification, every nucleotide in the antisense strand contains a modification, or every nucleotide in the sense strand and every nucleotide in the antisense strand contain a modification.

[0021] 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'-C-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl ... The nucleotide sequence is selected from the group consisting of modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermally destabilized nucleotides, glycol modified nucleotides (GNAs), nucleotides containing 2' phosphates, and 2-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0022] In one embodiment, the modification to the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol, and combinations thereof.

[0023] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, a nucleotide having a 2' phosphate, e.g., G2p, C2p, A2p, U2p, and a vinyl-phosphonate nucleotide, and combinations thereof.

[0024] In some embodiments, the modified nucleotides include a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0025] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at both the 5' end and the 3' end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand.

[0026] The double-stranded region can be 19-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 23-27 nucleotide pairs in length, or 21-23 nucleotide pairs in length.

[0027] In one embodiment, each strand is independently no more than 30 nucleotides in length.

[0028] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0029] The region of complementarity can be at least 17 nucleotides in length, 19-23 nucleotides in length, or 19 nucleotides in length.

[0030] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0031] In one embodiment, the dsRNA agent further comprises a ligand.

[0032] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of a dsRNA agent.

[0033] In one embodiment, the ligand is conjugated to the 5' end of the sense strand of a dsRNA agent.

[0034] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0035] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0036] In one embodiment, the ligand is: [ka]

[0037] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] In the formula, X is O or S.

[0038] In one embodiment, X is O.

[0039] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below. [ka]

[0040] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0041] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand, eg, the antisense strand or the sense strand.

[0042] In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand, for example, the antisense strand or the sense strand.

[0043] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi one embodiment, the strand is the antisense strand.

[0044] In one embodiment, the first base pair at the 5'-end of the antisense strand of the duplex is an AU base pair.

[0045] In one embodiment, the sense strand comprises at least 17 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-GACGCCACGCAUGCUGUGUGU-3' (SEQ ID NO: 119).

[0046] In one embodiment, the sense strand comprises at least 19 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-GACGCCACGCAUGCUGUGUGU-3' (SEQ ID NO: 119).

[0047] In one embodiment, the sense strand comprises or consists of the nucleotide sequence 5'-GACGCCACGCAUGCUGUGUGU-3' (SEQ ID NO: 119).

[0048] In one embodiment, the antisense strand comprises at least 17 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-ACACACAGCAUGCGUGGCGUCAC-3' (SEQ ID NO:245).

[0049] In one embodiment, the antisense strand comprises at least 19 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-ACACACAGCAUGCGUGGCGUCAC-3' (SEQ ID NO:245).

[0050] In one embodiment, the antisense strand comprises at least 21 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-ACACACAGCAUGCGUGGCGUCAC-3' (SEQ ID NO:245).

[0051] In one embodiment, the antisense strand comprises or consists of the nucleotide sequence 5'-ACACACAGCAUGCGUGGCGUCAC -3' (SEQ ID NO:245).

[0052] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-GACGCCACGCAUGCUGUGUGU-3' (SEQ ID NO: 119) and the antisense strand comprises the nucleotide sequence of 5'-ACACACAGCAUGCGUGGCGUCAC-3' (SEQ ID NO: 245).

[0053] In one embodiment, the sense strand differs from the nucleotide sequence 5'-gsascgccacGfCfAfugcugugugu-3' (SEQ ID NO: 371) by no more than 3, e.g., no more than 0, 1, 2, or 3 modified nucleotides, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; and s is a phosphorothioate linkage.

[0054] In one embodiment, the antisense strand differs from the nucleotide sequence of 5'-asdCsacdAcdAgcaudGcGfuggcgucsasc-3' (SEQ ID NO:497) by 3 or less, e.g., 0, 1, 2, or 3 modified nucleotides, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; where dA, dG, dC are 2'-deoxyadenosine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate, and 2'-deoxycytidine-3'-phosphate, respectively; and s is a phosphorothioate linkage.

[0055] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-gsascgccacGfCfAfugcugugugu-3' (SEQ ID NO: 371) and the antisense strand comprises the nucleotide sequence of 5'-asdCsacdAcdAgcaudGcGfuggcgucsasc-3' (SEQ ID NO: 497), where a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U, respectively; Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U, respectively; where dA, dG, dC are 2'-deoxyadenosine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate, and 2'-deoxycytidine-3'-phosphate, respectively; and s is a phosphorothioate linkage.

[0056] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-gsascgccacGfCfAfugcuguguguL96-3' (SEQ ID NO: 371) and the antisense strand comprises the nucleotide sequence of 5'-asdCsacdAcdAgcaudGcGfuggcgucsasc-3' (SEQ ID NO: 497), where a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, and Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U, respectively, where dA, dG, dC are 2'-deoxyadenosine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate and 2'-deoxycytidine-3'-phosphate, respectively, s is a phosphorothioate linkage and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0057] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-gsascgccacGfCfAfugcugugugu-3' (SEQ ID NO:371) and the antisense strand comprises the nucleotide sequence of 5'-asdCsacdAcdAgcaudGcGfuggcgucsasc-3' (SEQ ID NO:497), where a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U, respectively; Af, Gf, Cf and Uf are 2'-fluoro A, G, C and U, respectively; where dA, dG, dC are 2'-deoxyadenosine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate, and 2'-deoxycytidine-3'-phosphate, respectively; and s is a phosphorothioate linkage; and the 3' end of the sense strand is conjugated to a ligand as shown in the diagram below: [ka] In the formula, X is O.

[0058] In one embodiment, the sense strand comprises at least 17 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-CCUUUGGAAUAAAGCUGCCUU -3' (SEQ ID NO:844).

[0059] In one embodiment, the sense strand comprises at least 19 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-CCUUUGGAAUAAAGCUGCCUU -3' (SEQ ID NO:844).

[0060] In one embodiment, the sense strand comprises or consists of the nucleotide sequence 5'-CCUUUGGAAUAAAGCUGCCUU -3' (SEQ ID NO:844).

[0061] In one embodiment, the antisense strand comprises at least 17 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-AAGGCAGCUUUAUUCCAAAGGGC-3' (SEQ ID NO: 1868).

[0062] In one embodiment, the antisense strand comprises at least 19 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-AAGGCAGCUUUAUUCCAAAGGGC-3' (SEQ ID NO: 1868).

[0063] In one embodiment, the antisense strand comprises at least 21 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-AAGGCAGCUUUAUUCCAAAGGGC-3' (SEQ ID NO: 1868).

[0064] In one embodiment, the antisense strand comprises or consists of the nucleotide sequence 5'-AAGGCAGCUUUAUUCCAAAGGGC-3' (SEQ ID NO: 1868).

[0065] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-CCUUUGGAAUAAAGCUGCCUU-3' (SEQ ID NO: 844) and the antisense strand comprises the nucleotide sequence of 5'-AAGGCAGCUUUAUUCCAAAGGGC-3' (SEQ ID NO: 1868).

[0066] In one embodiment, the sense strand differs from the nucleotide sequence 5'-cscsuuugGfaAfUfAfaagcugccuu-3' (SEQ ID NO: 2095) by 3 or less, e.g., 0, 1, 2, or 3 modified nucleotides, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; and s is a phosphorothioate linkage.

[0067] In one embodiment, the antisense strand differs from the nucleotide sequence 5'-asAfsggdCa(G2p)cuuuauUfcCfaaaggsgsc-3' (SEQ ID NO: 2324) by 3 or less, e.g., 0, 1, 2, or 3 modified nucleotides, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; G2p is guanosine-2'-phosphate; and s is a phosphorothioate linkage.

[0068] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-cscsuuugGfaAfUfAfaagcugccuu-3' (SEQ ID NO: 2095) and the antisense strand comprises the nucleotide sequence of 5'-asAfsggdCa(G2p)cuuuauUfcCfaaaggsgsc-3' (SEQ ID NO: 2324), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; G2p is guanosine-2'-phosphate; and s is a phosphorothioate linkage.

[0069] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-cscsuuugGfaAfUfAfaagcugccuuL96-3' (SEQ ID NO: 2095) and the antisense strand comprises the nucleotide sequence of 5'-asAfsggdCa(G2p)cuuuauUfcCfaaaggsgsc-3' (SEQ ID NO: 2324), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; G2p is guanosine-2'-phosphate; s is a phosphorothioate linkage; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0070] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-cscsuuugGfaAfUfAfaagcugccuu-3' (SEQ ID NO: 2095) and the antisense strand comprises the nucleotide sequence of 5'-asAfsggdCa(G2p)cuuuauUfcCfaaaggsgsc-3' (SEQ ID NO: 2324), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; G2p is guanosine-2'-phosphate; and s is a phosphorothioate linkage; and the 3' end of the sense strand is conjugated to a ligand as shown in the diagram below: [ka] In the formula, X is O.

[0071] In one embodiment, the sense strand comprises at least 17 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-UCACCUGCUUCUUCUGGUU-3' (SEQ ID NO: 1686).

[0072] In one embodiment, the sense strand comprises or consists of the nucleotide sequence 5'-UCACCUGCUUCUUCUGGUU-3' (SEQ ID NO: 1686).

[0073] In one embodiment, the antisense strand comprises at least 17 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence 5'-AACCAGAAGAAGCAGGUGA-3' (SEQ ID NO: 1790).

[0074] In one embodiment, the antisense strand comprises or consists of the nucleotide sequence 5'-AACCAGAAGAAGCAGGUGA-3' (SEQ ID NO: 1790).

[0075] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-UCACCUGCUUCUUCUGGUU-3' (SEQ ID NO: 1686) and the antisense strand comprises the nucleotide sequence of 5'-AACCAGAAGAAGCAGGUGA-3' (SEQ ID NO: 1790).

[0076] In one embodiment, the sense strand differs from the nucleotide sequence of 5'-UfcAfcCfuGfcUfuCfuUfcUfgGfsusUf-3' (SEQ ID NO: 1974) by 3 or less, e.g., 0, 1, 2, or 3 modified nucleotides, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; and s is a phosphorothioate linkage.

[0077] In one embodiment, the antisense strand differs from the nucleotide sequence 5'-asAfscCfaGfaAfgAfaGfcAfgGfusGfsa-3' (sequence number), by 3 or less, e.g., 0, 1, 2, or 3 modified nucleotides, where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; and s is a phosphorothioate linkage.

[0078] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-UfcAfcCfuGfcUfuCfuUfcUfgGfsusUf-3' (SEQ ID NO: 2203) and the antisense strand comprises the nucleotide sequence of 5'-asAfscCfaGfaAfgAfaGfcAfgGfusGfsa-3' (SEQ ID NO:), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; and s is a phosphorothioate linkage.

[0079] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-Q191sUfcAfcCfuGfcUfuCfuUfcUfgGfsusUf-3' (SEQ ID NO: 1974) and the antisense strand comprises the nucleotide sequence of 5'-asAfscCfaGfaAfgAfaGfcAfgGfusGfsa-3' (SEQ ID NO: 2203), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; s is a phosphorothioate linkage; and Q191 is N-[tris(GalNAc-alkyl)-amidododecanoyl]-(S)-pyrrolidin-3-ol-phosphorothioate (p-C12-(GalNAc-alkyl)3).

[0080] In one embodiment, the sense strand comprises the nucleotide sequence of 5'-UfcAfcCfuGfcUfuCfuUfcUfgGfsusUf-3' (SEQ ID NO: 1974), and the antisense strand comprises the nucleotide sequence of 5'-asAfscCfaGfaAfgAfaGfcAfgGfusGfsa-3' (SEQ ID NO: 2203), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, and U, respectively; and s is a phosphorothioate linkage; and the 5' end of the sense strand is conjugated to a ligand as shown in the diagram below. [ka]

[0081] In another embodiment, the RNAi agent is a pharma- ceutically acceptable salt thereof. The "pharma- ceutically acceptable salt" of each of the RNAi agents herein includes, but is not limited to, sodium, calcium, lithium, potassium, ammonium, magnesium salts, and mixtures thereof. One skilled in the art will understand that the RNAi agent, when provided as a polycationic salt, has one cation per free acid group of the optionally modified phosophodiester backbone and / or any other acidic modification (e.g., phosphonate group at the 5' end). For example, an oligonucleotide that is "n" nucleotides in length contains n-1 optionally modified phosophodiesters, such that an oligonucleotide that is 21 nt in length can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a sense strand that is 21 nt in length and an antisense strand that is 23 nt in length can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the preceding examples, where the RNAi agent also includes a 5'-terminal phosphate group or a 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (e.g., 44 sodium cations).

[0082] The invention also provides cells containing any of the dsRNA agents of the invention, and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

[0083] A pharmaceutical composition of the invention may comprise a dsRNA agent in an unbuffered solution, e.g., saline or water, or a pharmaceutical composition of the invention may comprise a dsRNA agent in a buffered solution, e.g., a buffered solution comprising acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, or phosphate buffered saline (PBS).

[0084] In one aspect, the present invention provides a method for inhibiting the expression of transmembrane protease, serine 6 (TMPRSS6) gene in a cell, comprising contacting the cell with any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby inhibiting the expression of the TMPRSS6 gene in the cell.

[0085] In one embodiment, the cell is within a subject, such as a human subject, having a transmembrane protease, serine 6 (TMPRSS6) associated disorder, such as, for example, an iron overload associated disorder and / or an ineffective erythropoiesis disorder, e.g., hereditary hemochromatosis, beta thalassemia (e.g., beta thalassemia major and beta thalassemia intermedia), polycythemia vera, myelodysplastic syndrome, congenital dyserythroid anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.

[0086] In some embodiments, the TMPRSS6-related disorder is β-thalassemia. In one embodiment, the TMPRSS6-related disorder is β-thalassemia major. In another embodiment, the TMPRSS6-related disorder is β-thalassemia intermedia. In some embodiments, the TMPRSS6-related disorder is polycythemia vera.

[0087] In certain embodiments, TMPRSS6 expression is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In one embodiment, inhibiting expression of TMPRSS6 reduces TMPRSS6 protein levels in the serum of the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0088] In certain embodiments, contacting cells with dsRNA agent increases hepcidin expression by at least 50%, 60%, 70%, 80%, 90% or 95%.In one embodiment, increasing hepcidin expression increases hepcidin protein level in the serum of subject by at least 50%, 60%, 70%, 80%, 90% or 95%.

[0089] In one aspect, the present invention provides a method for treating a subject with a disorder that would benefit from reduced transmembrane protease, serine 6 (TMPRSS6) expression.The method comprises administering to the subject a therapeutically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby treating the subject with a disorder that would benefit from reduced TMPRSS6 expression.

[0090] In another aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from a decrease in transmembrane protease, serine 6 (TMPRSS6) expression. The method comprises administering to the subject a prophylactically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby preventing at least one symptom in a subject with a disorder that would benefit from a decrease in TMPRSS6 expression.

[0091] In certain embodiments, the disorder is a transmembrane protease, serine 6 (TMPRSS6) associated disorder, e.g., an iron overload associated disorder and / or an ineffective erythropoiesis disorder, e.g., hereditary hemochromatosis, beta thalassemia (e.g., beta thalassemia major and beta thalassemia intermedia), polycythemia vera, myelodysplastic syndrome, congenital dyserythroid anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.

[0092] In some embodiments, the TMPRSS6-related disorder is β-thalassemia. In one embodiment, the TMPRSS6-related disorder is β-thalassemia major. In another embodiment, the TMPRSS6-related disorder is β-thalassemia intermedia. In some embodiments, the TMPRSS6-related disorder is polycythemia vera.

[0093] In certain embodiments, administering dsRNA to subject causes the iron level, ferritin level, and / or transferrin saturation level to decrease in subject, and / or the TMPRSS6 protein accumulation to decrease.In some embodiments, administering dsRNA to subject causes the hemoglobin level and / or hematocrit level to increase in subject.

[0094] In a further aspect, the present invention also provides a method for inhibiting expression of TMPRSS6 in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the dsRNAs provided herein, thereby inhibiting expression of TMPRSS6 in the subject.

[0095] In one embodiment, the subject is a human.

[0096] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0097] In one embodiment, the dsRNA agent is administered to the subject subcutaneously or intravenously.

[0098] In one embodiment, the method of the invention further comprises determining the level of TMPRSS6 in a sample from the subject.

[0099] In one embodiment, the TMPRSS6 level in the subject's sample is the TMPRSS6 protein level in a blood, serum, or liver sample.

[0100] In one embodiment, the method of the invention further comprises determining the level of iron and / or hepcidin in a sample from the subject.

[0101] In certain embodiments, the methods of the invention further comprise administering to the subject an additional therapeutic agent, hi one embodiment, the methods of the invention further comprise administering to the subject an iron chelator, e.g., deferiprone, deferoxamine, and deferasirox.

[0102] The present invention also provides a kit comprising any of the dsRNA of the present invention or any of the pharmaceutical compositions of the present invention, and optionally instructions for use.In one embodiment, the present invention provides a kit for carrying out the method of inhibiting the expression of TMPRSS6 gene in cells by contacting cells with the double-stranded RNAi agent of the present invention in an amount effective to inhibit the expression of TMPRSS6 in cells.The kit comprises the RNAi agent, instructions for use, and optionally comprises a means for administering the RNAi agent to a subject.

[0103] The invention also provides an RNA-induced silencing complex (RISC) comprising the antisense strand of any of the dsRNA agents of the invention. [Brief description of the drawings]

[0104] [Figure 1] FIG. 1 is a schematic diagram showing the study design for determining the efficacy of dsRNA agents disclosed herein in vivo in cynomolgus monkeys. [Diagram 2]2 is a graph showing the percentage of serum TMPRSS6 mRNA remaining in cynomolgus monkeys (n=3 per group) administered a single 3 mg / kg or 10 mg / kg dose of the indicated dsRNA duplex subcutaneously at days 21, 22, 57, and 85 after administration. TMPRSS6 mRNA levels are shown relative to control levels obtained from cynomolgus monkeys administered PBS as a control. [Diagram 3] FIG. 3 is a graph showing plasma iron levels as a percentage of pre-dose levels in cynomolgus monkeys (n=3 per group) administered a single 3 mg / kg or 10 mg / kg dose of the indicated dsRNA duplex subcutaneously on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 post-dose. [Figure 4] FIG. 4 is a graph showing percent transferrin saturation levels in cynomolgus monkeys (n=3 per group) administered a single 3 mg / kg or 10 mg / kg dose of the indicated dsRNA duplex subcutaneously on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 post-treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] The present invention provides iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the transmembrane protease, serine 6 (TMPRSS6) gene. The gene can be in a cell, for example, in a subject, such as a human. The use of these iRNAs allows for targeted degradation of the mRNA of the corresponding gene (TMPRSS6) in a mammal.

[0106] The iRNAs of the invention were designed to target the human transmembrane protease, serine 6 (TMPRSS6) gene, including portions of the gene that are conserved in TMPRSS6 orthologs of other mammalian species. Without intending to be limited by theory, it is believed that combinations or subcombinations of the aforementioned features and specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of the iRNAs of the invention.

[0107] Thus, the present invention provides methods for treating and preventing transmembrane protease, serine 6 (TMPRSS6) associated disorders, such as iron overload associated disorders and / or ineffective erythropoiesis disorders, such as hereditary hemochromatosis, beta thalassemia (e.g., beta thalassemia major and beta thalassemia intermedia), polycythemia vera, myelodysplastic syndromes, congenital dyserythroplastic anemia, pyruvate kinase deficiency, using iRNA compositions that undergo RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the TMPRSS6 gene.

[0108] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region that is up to about 30 nucleotides in length or less, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the TMPRSS6 gene.

[0109] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that are substantially complementary to at least a portion of an mRNA transcript of the TMPRSS6 gene. In some embodiments, such an iRNA agent with a longer antisense strand can include, for example, a second RNA strand (sense strand) that is 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0110] The use of iRNA of the present invention allows the targeted degradation of mRNA of corresponding gene (TMPRSS6 gene) in mammals.The inventors have demonstrated by in vitro assay that iRNA targeting TMPRSS6 gene can strongly mediate RNAi, resulting in significant inhibition of the expression of TMPRSS6 gene.Therefore, the method and composition comprising these iRNA are useful for treating subjects with TMPRSS6-related disorders, such as iron overload-related disorders and / or ineffective erythropoiesis disorders, such as hereditary hemochromatosis, β-thalassemia (e.g., β-thalassemia major and β-thalassemia intermedia), polycythemia vera, myelodysplastic syndrome, congenital dyserythrocytic anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.

[0111] Thus, the present invention provides methods and combination therapies for treating subjects having disorders that would benefit from inhibiting or reducing expression of the TMPRSS6 gene, e.g., transmembrane protease, serine 6 (TMPRSS6) associated disorders, e.g., iron overload associated disorders and / or ineffective erythropoiesis disorders, e.g., hereditary hemochromatosis, beta thalassemia (e.g., beta thalassemia major and beta thalassemia intermedia), polycythemia vera, myelodysplastic syndromes, congenital dyserythroplastic anemia, pyruvate kinase deficiency, using iRNA compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the TMPRSS6 gene.

[0112] The present invention also provides methods for preventing at least one symptom in a subject having a disorder that would benefit from inhibiting or reducing expression of the TMPRSS6 gene, such as an iron overload-related disorder and / or an ineffective erythropoiesis disorder, such as hereditary hemochromatosis, beta thalassemia (e.g., beta thalassemia major and beta thalassemia intermedia), polycythemia vera, myelodysplastic syndrome, congenital dyserythroid anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.

[0113] The following detailed description of the invention discloses methods of making and using compositions containing iRNA that inhibit expression of the TMPRSS6 gene, as well as compositions, uses, and methods of treating subjects who would benefit from inhibition and / or reduction of expression of the TMPRSS6 gene, e.g., subjects susceptible to or diagnosed with a TMPRSS6-associated disorder.

[0114] I. Definition So that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that 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 present invention.

[0115] 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 to more than one element, e.g., a plurality of elements.

[0116] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0117] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand."

[0118] The term "about" is used herein to mean within a typical tolerance 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 before a series of numbers or ranges, it is understood that "about" can modify each of the series of numbers or ranges.

[0119] The term "at least", "more than" or "or more" before a number or sequence of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included, if the context is clear. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides of a 21 nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the indicated property. When at least is before a sequence of numbers or ranges, it is understood that "at least" can modify each of the sequence of numbers or ranges.

[0120] As used herein, "less than" or "or less than" is understood as the value adjacent to the phrase and the logically smaller value or integer of that value up to zero if logical from the context. For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides. When "less than" precedes a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges. As used herein, a range includes both upper and lower limits.

[0121] As used herein, a method of detection can include a determination that the amount of analyte present is below the detection level of the method.

[0122] In case of a conflict between a shown target site and the nucleotide sequence for either the sense or antisense strand, the shown sequence controls.

[0123] In the event of a discrepancy between a sequence on the transcript or other sequence and its indicated site, the nucleotide sequence listed herein takes precedence.

[0124] As used herein, "transmembrane protease, serine 6", which is used interchangeably with the term "TMPRSS6", refers to a type II plasma membrane serine protease (TTSP) gene or protein. TMPRSS6 is also known as matriptase-2, IRIDA (iron-refractory iron deficiency anemia), transmembrane protease serine 6, type II transmembrane serine protease 6, and membrane-bound mosaic serine proteinase matriptase-2. TMPRSS6 is a serine protease type II transmembrane protein approximately 899 amino acids in length. TMPRSS6 contains multiple domains, including a short endodomain, a transmembrane domain, a sea urchin sperm protein / enteropeptidase domain / agrin (SEA) domain, two complement factor / sea urchin embryonic growth factor / BMP domains (CUBs), three LDL-R class a domains (LDLa), and a trypsin-like serine protease domain with a conserved His-Asp-Ser triad (HDS).

[0125] The sequence of human TMPRSS6 mRNA transcript can be found, for example, in GenBank Accession No. GI:1755203660 (NM_153609.4, SEQ ID NO:1, reverse complement, SEQ ID NO:2). The sequence of mouse TMPRSS6 mRNA can be found, for example, in GenBank Accession No. GI:125656151 (NM_027902.2, SEQ ID NO:3, reverse complement, SEQ ID NO:4). The sequence of rat TMPRSS6 mRNA can be found, for example, in GenBank Accession No. GI:194474097 (NM_001130556.1, SEQ ID NO:5, reverse complement, SEQ ID NO:6). The sequence of cynomolgus monkey TMPRSS6 mRNA can be found, for example, in GenBank Accession No. GI:982272225 (XM_005567384.2, SEQ ID NO:7, reverse complement, SEQ ID NO:8). The sequence of rhesus TMPRSS6 mRNA can be found, for example, in GenBank Accession No. GI:1622838152 (XM_015150283.2, SEQ ID NO:9, reverse complement, SEQ ID NO:10).

[0126] Further examples of TMPRSS6 mRNA sequences are readily available through public databases such as, for example, GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0127] Further information regarding TMPRSS6 can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=TMPRSS6.

[0128] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are hereby incorporated by reference as of the filing date of this application.

[0129] The term TMPRSS6 as used herein also refers to the variation of the TMPRSS6 gene, including the variants provided in SNP databases.A large number of sequence variations within the TMPRSS6 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=TMPRSS6, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0130] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TMPRSS6 gene, such as an mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-dependent cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TMPRSS6 gene.

[0131] The target sequence can be about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides in length, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0132] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0133] Generally, each of "G", "C", "A", "T" and "U" represents a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moiety. For example, but not limited to, a nucleotide that contains inosine as its base can base pair with a nucleotide that contains adenine, cytosine or uracil. Thus, a nucleotide that contains uracil, guanine or adenine can be replaced with, for example, a nucleotide that contains inosine in the nucleotide sequence of the dsRNA featured in the present invention. In another embodiment, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form GU wobble bases that pair with the target mRNA. Sequences containing such replacements are suitable for the compositions and methods featured herein.

[0134] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent" as used interchangeably herein refer to agents that contain RNA as the term is defined herein and mediate the cleavage of targets of RNA transcription via the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNAs through a process known as RNA interference (RNAi). iRNAs regulate, e.g., inhibit, the expression of the TMPRSS6 gene in cells, e.g., in cells in a subject, e.g., a mammalian subject.

[0135] 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 TMPRSS6 target mRNA sequence, and mediates cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into a cell is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes the dsRNA into 19-23 base pair small interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases can unravel the siRNA duplex, thereby inducing target recognition to the complementary antisense strand (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). That is, in one aspect, the present invention relates to a single-stranded RNA (siRNA) that is generated in cells and promotes the formation of a RISC complex to cause the silencing of a target gene, i.e., the TMPRSS6 gene. Thus, the term "siRNA" is also used herein to refer to the iRNA described above.

[0136] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded siRNAs is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs as described herein or as single-stranded siRNAs that are chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0137] In certain embodiments, the "iRNA" used in the compositions, uses and methods of the present invention is double-stranded RNA, and is referred to herein as "double-stranded RNA agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure, comprising two antiparallel substantially complementary nucleic acid strands, which are referred to as having "sense" or "antisense" orientation with respect to target RNA, i.e., TMPRSS6 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of target RNA, for example, mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0138] Generally, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides.In addition, as used herein, "iRNA" may include ribonucleotides with chemical modifications, and iRNA may include substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase, or any combination thereof.Thus, the term modified nucleotide includes the substitution, addition, or removal of, for example, a functional group or atom, to the internucleoside linkage, sugar moiety, or nucleobase.Modifications suitable for use in the agent of the present invention include all types of modifications disclosed herein or known in the art.When used in siRNA type molecules, any of these modifications are encompassed by "iRNA" or "RNAi agent" for the purposes of this specification and claims.

[0139] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0140] The duplex region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may be about 19-36 base pairs in length, e.g., about 19-30 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 19-30, 19 The length of the duplex region may range from 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In certain embodiments, the duplex region is 19 to 21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of the present disclosure.

[0141] The two strands forming the duplex structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are parts of one larger molecule and are connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand that forms the duplex structure, the connected RNA strands are called "hairpin loops." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may be 10 or fewer nucleotides. In some embodiments, a hairpin loop may be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may be 4-10 unpaired nucleotides. In some embodiments, a hairpin loop may be 4-8 nucleotides.

[0142] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, they can, but do not necessarily, be covalently linked. When the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand that forms a duplex structure, the connecting structure is called a "linker". The RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi can include one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0143] In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, eg, the TMPRSS6 gene, and mediates cleavage of the target RNA.

[0144] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a TMPRSS6 target mRNA sequence, and mediates cleavage of the target RNA.

[0145] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain at least one nucleotide overhang, or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. An overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, a given overhang nucleotide may be present on the 5' end, the 3' end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0146] In one embodiment, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0147] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' or 5' end, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' or 5' end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0148] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3'-end or 5'-end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang on the sense strand or on the antisense strand or on both strands can include an extended length of more than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on 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 on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on 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. In certain embodiments, the overhang comprises a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0149] "Blunt" or "blunt end" means that there is no unpaired nucleotide at the end of the double-stranded RNA agent, i.e., there is no nucleotide overhang.A "blunt-ended" double-stranded RNA agent is double-stranded over its entire length, i.e., there is no nucleotide overhang at either end of the molecule.The RNAi agent of the present invention includes an RNAi agent that has no nucleotide overhang at one end (i.e., an agent that has one overhang and one blunt end) or an RNAi agent that has no nucleotide overhang at either end.In most cases, such a molecule will be double-stranded over its entire length.

[0150] 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., TMPRSS6 mRNA.

[0151] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a TMPRSS6 nucleotide sequence, as defined herein. If the complementary region is not completely complementary to the target sequence, the mismatch may be in the internal or terminal region of the molecule. In general, the most tolerable mismatch is in the terminal region, e.g., within 5, 4, or 3 nucleotides of the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNA agent of the present invention comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the target mRNA, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the sense strand, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention comprises a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the antisense strand, for example, the sense strand comprises 4, 3, 2, 1 or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within, for example, 5, 4, 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is within, for example, the 3' end nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0152] Thus, the RNAi agent described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agent described herein contains 3 or less mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agent described herein contains 2 or less mismatches. In one embodiment, the RNAi agent described herein contains 1 or less mismatches. In one embodiment, the RNAi agent described herein contains 0 mismatches. In certain embodiments, when the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last 5 nucleotides from either the 5' or 3' end of the complementary region. For example, in such an embodiment, for a 23 nucleotide RNAi agent, the strand complementary to the region of the TMPRSS6 gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether the RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the TMPRSS6 gene. In particular, if a particular complementary region in the TMPRSS6 gene is known to have polymorphic sequence variation within the population, it is important to consider the efficacy of a mismatched RNAi agent to inhibit expression of the TMPRSS6 gene.

[0153] 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.

[0154] As used herein, "substantially all of the nucleotides are modified" means broadly but not entirely modified and can include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0155] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0156] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence in the context of a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex structure under specific conditions with an oligonucleotide or polynucleotide comprising a second nucleotide sequence, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. Those skilled in the art may determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0157] A complementary sequence in an iRNA, for example, in a dsRNA as described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its final application, for example, in vitro or in vivo, inhibition of gene expression. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered as mismatches for the purpose of 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, may still be referred to as "fully complementary" for purposes described herein.

[0158] "Complementary" sequences, as used herein, may also include or be formed entirely from non-Watson-Crick base pairs, or base pairs formed from non-natural modified nucleotides, so long as they satisfy the above requirements regarding their ability to hybridize, including, but not limited to, G:U wobble base pairs or Hoogsteen base pairs.

[0159] The terms "complementary," "fully complementary," and "substantially complementary," as used herein, may be used in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or the antisense strand of a double-stranded RNA agent and a target sequence, as will be understood from the context of their use.

[0160] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding the TMPRSS6 gene). For example, a polynucleotide is complementary to at least a portion of a TMPRSS6 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the TMPRSS6 gene.

[0161] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target TMPRSS6 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target TMPRSS6 sequence and include a contiguous nucleotide sequence that is at least 80% complementary, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of any one of the nucleotide sequences of SEQ ID NO:1, 3, 5, 7, or 9, or to a fragment of any one of SEQ ID NO:1, 3, 5, 7, or 9, over its entire length.

[0162] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target TMPRSS6 sequence, and include nucleotides 187-210; 227-254; 322-363; 362-390; 398-420; 404-429; 410-435; 439-461; 443-467; 448-474; 460-483; 466-488; 496-519; 519-542; 526-530; 532-543; 536-537; 538-541; 538-542; 538-543; 538-544; 538-545; 538-546; 538-547; 538-549; 540-544; 540-545; 540-546; 540-547 ... 548;557~593;641~671;652~676;687~713;725~762;757~794;886~908;921~951;956~987;1051~1082;1233~1269;1279~1313;1313~1341;1327~1351;1415~1439;1447~1480;1464~1486;1486~1509;1559~1589;1571~1595;1579~ 1609;1707~1735;1738~1764;1806~1828;1864~1886;1934~1966;1967~1991;2008~2031;2015~2043;2042~2072;2287~2311;2297~2354;2336~2361;2360~2384;2416~2438;2481~2510;2496~2527;2526~2558;2665~2693;2693~2 719; 2707-2729; 2799-2821; 2851-2874; 2971-2999; 2981-3006; and 3155-3195.

[0163] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target TMPRSS6 sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to a fragment of SEQ ID NO:1 selected from the group consisting of nucleotides 230-252, 324-346, 560-578, 560-582, 2338-2360, 3163-3185, 3169-3191, and 3172-3194 of SEQ ID NO:1.

[0164] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target TMPRSS6 sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to a fragment of SEQ ID NO:1 selected from the group of nucleotides 560-578, 2338-2360, and 3169-3191 of SEQ ID NO:1.

[0165] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target TMPRSS6 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to any one of the sense strand nucleotide sequences in any one of Tables 2-7 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-7, over its entire length.

[0166] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to the antisense polynucleotide and thus identical to the target TMPRSS6 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to a nucleotide sequence equivalent region of SEQ ID NO:2, 4, 6, 8, or 10, or to a fragment of any one of SEQ ID NO:2, 4, 6, 8, or 10, over its entire length.

[0167] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target TMPRSS6 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-7, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-7.

[0168] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1556360, AD-1571158, AD-1571033, AD-1554875, AD-1571160, AD-1555117, AD-1554911, and AD-1556915.

[0169] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1556360, AD-1571158, and AD-1571033.

[0170] Generally, "iRNA" includes ribonucleotides that have chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any of these modifications, when used in dsRNA molecules, are encompassed by "iRNA" for the purposes of this specification and claims.

[0171] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0172] In one aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can stoichiometrically inhibit translation by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule can be about 14 to about 30 nucleotides in length and can have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can include a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0173] The phrase "contacting a cell with an iRNA", such as dsRNA, as used herein includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contacting can be performed directly or indirectly. Thus, for example, an iRNA can be physically contacted with a cell by performing a method separately, or the iRNA can be placed in a situation that allows or will subsequently contact a cell.

[0174] The contacting of cells in vitro can be achieved, for example, by incubating cells with iRNA.The contacting of cells in vivo can be achieved, for example, by injecting iRNA into or near the tissue in which the cells are present, or by injecting iRNA into another area, for example, into the bloodstream or subcutaneous cavity, so that the agent subsequently reaches the tissue in which the cells to be contacted are present.For example, the iRNA can contain or be bound to a ligand, such as GalNAc, that guides the iRNA to a target site, such as the liver.A combination of in vitro and in vivo contact methods is also possible.For example, the cell can be contacted with iRNA in vitro and then transplanted into a subject.

[0175] In certain embodiments, contacting a cell with an iRNA includes "introducing" or "delivering an iRNA into a cell" by promoting or causing uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through spontaneous diffusion or active intracellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. Introduction into a cell in vitro includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0176] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharma- ceutically active molecule, such as a nucleic acid molecule, such as an iRNA or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0177] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (such as a human, a monkey, and a non-human primate, such as a chimpanzee), a non-primate (such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses a target gene either endogenously or heterologously. In an embodiment, the subject is a human, such as a human being that is treated or evaluated for a disease or disorder that would benefit from a decrease in TMPRSS6 expression, a human being at risk for a disease or disorder that would benefit from a decrease in TMPRSS6 expression, a human being that has a disease or disorder that would benefit from a decrease in TMPRSS6 expression, or a human being that is treated for a disease or disorder that would benefit from a decrease in TMPRSS6 expression as described herein. In some embodiments, the subject is a human being that is female. In other embodiments, the subject is a human being that is male. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0178] As used herein, the term "treat" or "treatment" refers to a beneficial or desired outcome, such as reducing at least one sign or symptom of a TMPRSS6-associated disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesired TMPRSS6 expression, reducing the degree of undesired TMPRSS6 activation or stabilization, improving or alleviating undesired TMPRSS6 activation or stabilization. "Treatment" can also mean extending survival time compared to the expected survival time in the absence of treatment. The term "lower" in the context of TMPRSS6 levels or disease markers or symptoms in a subject refers to a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the reduction is at least 50% in disease marker, for example, in protein level or gene expression level.In the context of the level of TMPRSS6 in a subject, "reduce" refers to reducing to a level that is accepted as being within the normal range of an individual without the disorder.In certain embodiments, "reduce" refers to the reduction in the difference between the level of marker or symptom in a subject suffering from a disease and the level that an individual is accepted within the normal range, for example, the level of weight reduction between an individual who is obese and an individual whose weight is accepted within the normal range.

[0179] As used herein, "prevention" or "preventing" refers to a reduction in the likelihood that a subject will develop a disease, disorder, or condition that may be treated or improved by reducing the expression of the TMPRSS6 gene, such as undesirable or excessive TMPRSS6 expression, such as symptoms of elevated iron levels or iron dysregulation. The likelihood of developing elevated iron levels or iron dysregulation is reduced, for example, when an individual with one or more risk factors for elevated iron levels or iron dysregulation does not develop elevated iron levels or iron dysregulation, or develops elevated iron levels or iron dysregulation that is less severe than a population with the same risk factors and not treated as described herein. Not developing a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a disease, disorder, or condition (e.g., a reduction of at least about 10% of the clinically acceptable magnitude for the disease or disorder), or a delay in the onset of delayed symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0180] As used herein, the term "transmembrane protease, serine 6-associated disease" or "TMPRSS6-associated disease" refers to a disease or disorder caused by or associated with TMPRSS6 gene expression or TMPRSS6 protein production. The term "TMPRSS6-associated disease" includes diseases, disorders, or conditions that would benefit from a decrease in TMPRSS6 gene expression, replication, or protein activity.

[0181] In some embodiments, the TMPRSS6-related disease is iron overload-related disorder, condition characterized by elevated iron level, or iron dysregulation.Iron overload can be caused by, for example, hereditary condition, increased dietary iron intake, or excess iron administered parenterally, including intravenous injection of excess iron and transfusion iron overload.

[0182] In some embodiments, the TMPRSS6-related disease is ineffective erythropoiesis disorder. Ineffective erythropoiesis disorder is an abnormal proliferation of red blood cell precursors with non-productive synthesis of enucleated red blood cells, resulting in anemia and hypoxia. In particular, the increase in red blood cells does not cause a corresponding increase in red blood cells. As a result, iron absorption still increases in response to stress, but iron is deposited in organs instead of being used to generate more red blood cells.

[0183] In some embodiments, TMPRSS6-associated disorders include, but are not limited to, hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis, sideroblastic anemia, hemolytic anemia, dyserythroid anemia, sickle cell anemia, hemoglobinopathies, thalassemia (e.g., beta thalassemia and alpha thalassemia), polycythemia vera, myelodysplastic syndromes, congenital dyserythroid anemia, pyruvate kinase deficiency, chronic liver disease, porphyria cutanea tarda, erythropoietic porphyria, atransferrinemia, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, renal hemosiderosis.

[0184] In some embodiments, TMPRSS6-associated disorders include disorders associated with oral administration of excess iron, transfusional iron overload, and intravenous administration of excess iron.

[0185] In other embodiments, TMPRSS6-related disorders also include disorders with symptoms associated with or that may be caused by iron overload. Such symptoms include liver disease (cirrhosis, cancer), heart attack or heart failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and in some cases, increased risk of early death. In one embodiment, TMPRSS6-related disorders include neurodegenerative disorders associated with iron overload and / or iron dysregulation, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedreich's ataxia, epilepsy, and multiple sclerosis. Administration of an iRNA targeting TMPRSS6, such as an iRNA listed in any one of Tables 2-7, can treat one or more of these symptoms or prevent the onset or progression of a disease or disorder that is exacerbated by increased iron levels.

[0186] In one embodiment, the TMPRSS6-related disorder is beta thalassemia. Beta thalassemia is any one of a group of genetic disorders characterized by a genetic deficiency in the synthesis of beta globin chains. In the homozygous state, beta thalassemia (thalassemia major) causes severe transfusion-dependent anemia. In the heterozygous state, beta thalassemia trait (thalassemia minor) causes mild to moderate microcytic anemia. "Thalassemia intermedia" is beta thalassemia in which the clinical severity of the disease in a subject lies between the mild symptoms of beta thalassemia minor and beta thalassemia major. Several laboratory tests, such as complete blood count to determine red blood cell and hemoglobin counts, blood smear test, hemoglobin electrophoresis, gene sequencing, or iron tests to check iron, ferritin, unsaturated iron binding capacity, total iron binding capacity, or transferrin saturation levels, can be used to help detect and diagnose thalassemia. The type and relative amount of hemoglobin present in red blood cells is another indicator of thalassemia. Beta thalassemia causes an imbalance in the formation of beta and alpha hemoglobin chains, leading to an increase in minor hemoglobin components. Thus, patients with beta thalassemia major usually have a greater proportion of Hb F. Patients with beta thalassemia minor usually have an elevated proportion of Hb A2.

[0187] In one embodiment, the β thalassemia is thalassemia major. In another embodiment, the β thalassemia is thalassemia intermedia.

[0188] In some embodiments, the TMPRSS6-related disorder is polycythemia vera.Polycythemia vera is a type of blood cancer that causes bone marrow to produce excess red blood cells.These excess cells usually thicken blood vessels, which makes patients more susceptible to blood clots and other complications, such as stroke or heart attack.To help detect and diagnose polycythemia vera, some tests can be performed, such as complete blood count, blood smear test, erythropoietin level test, bone marrow aspiration or biopsy, or gene sequencing.

[0189] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a subject with a TMPRSS6-related disease, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0190] "Prophylactically effective amount" as used herein is intended to include an amount of an RNAi agent that is sufficient to prevent or ameliorate disease or one or more symptoms of a disease when administered to a subject with a TMPRSS6-associated disorder. Ameliorating a disease includes slowing down the progression of the disease or reducing the severity of disease that will develop. A "prophylactically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment in some cases, and other personal characteristics of the patient to be treated.

[0191] A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired 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.

[0192] The phrase "pharmacologically acceptable" is used herein to refer to those compounds, materials (including salts), compositions 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.

[0193] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transportation of the subject compound from one organ or body part to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material. 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. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0194] The term "sample" as used herein includes similar fluids, cells, or tissues isolated from a subject, and 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 from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples can be from the liver (e.g., the entire liver or a specific segment of the liver, or a specific type of cell within the liver, such as stem cells). In some embodiments, a "sample from a subject" refers to urine obtained from a subject. A "sample from a subject" can also refer to blood from a subject, or serum or plasma from blood.

[0195] II. iRNAs of the Invention The present invention provides iRNA that inhibits the expression of TMPRSS6 gene.In certain embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of TMPRSS6 gene in cells, such as cells in mammals, such as subjects, such as humans, susceptible to TMPRSS6-related disorders, such as iron overload-related disorders and / or ineffective erythropoiesis disorders, such as hereditary hemochromatosis, β-thalassemia (e.g., β-thalassemia major and β-thalassemia intermedia), polycythemia vera, myelodysplastic syndromes, congenital dyserythropoietic anemia, pyruvate kinase deficiency, erythropoietin porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of TMPRSS6 gene. The region of complementarity is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0196] When the iRNA is contacted with a cell expressing the TMPRSS6 gene, it inhibits the expression of the TMPRSS6 gene (e.g., human, primate, non-primate, or rat TMPRSS6 gene) by at least about 50%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence, for example, using Western blot or flow cytometry techniques. In certain embodiments, the inhibition of expression is determined by the qPCR method provided in the Examples herein, using siRNA at a concentration of, for example, 10 nM, in a suitable biological cell line provided therein. In certain embodiments, the inhibition of expression in vivo is determined by knocking down the human gene in a rodent expressing the human gene, for example, a mouse expressing a human target gene or an AAV-infected mouse, when administered, for example, as a single dose, at a minimum of 3 mg / kg of RNA expression.

[0197] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary and generally completely complementary to the target sequence. The target sequence can be obtained from the sequence of the mRNA formed during the expression of the TMPRSS6 gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that the two strands hybridize to form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be comprised as a self-complementary region of a single nucleic acid molecule, such that it is opposite on separate oligonucleotides.

[0198] 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, 19-32, 19-34, 19-36, 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, 19-66, 19-67, 19-68, 19-69, 20-7 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 18-25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, e.g., 19-21 base pairs in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of the present disclosure.

[0199] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 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, e.g., 19-23 nucleotides in length, or 21-23 nucleotides in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of the disclosure.

[0200] In some embodiments, the duplex structure is 19-30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19-30 nucleotides in length.

[0201] In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. In general, the dsRNA is long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can function as substrates for Dicer. As those skilled in the art will recognize, the region of an RNA targeted for cleavage is most often a portion of a longer RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to be a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0202] Those skilled in the art will recognize that the duplex region is the primary functional portion of the dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 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 base pairs. That is, in one embodiment, an RNA molecule or a complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, so long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, one skilled in the art will recognize that, in one embodiment, 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 TMPRSS6 gene expression is not generated in a target cell by cleavage of a larger dsRNA.

[0203] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Moreover, the overhang nucleotide may be present on the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the dsRNA.

[0204] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi compounds of the present invention can be prepared using a two-step approach. 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 offers the advantage that the oligonucleotide strands containing non-natural nucleotides or modified nucleotides can be easily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0205] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the sequences provided in any one of Tables 2-7, and the corresponding antisense strand of the sense strand is selected from the sequences provided in any one of Tables 2-7. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of the mRNA generated upon expression of the TMPRSS6 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand in any one of Tables 2-7, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-7.

[0206] In certain embodiments, the substantially complementary sequences of the dsRNA are contained on each oligonucleotide, while in other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0207] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-7.

[0208] For example, the sequences of Tables 3 or 5 are not described as modified or conjugated sequences, but the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, can include any one of the sequences set forth in any one of Tables 2-7, which are unmodified, unconjugated, or modified or conjugated differently than those set forth therein. In other words, the invention encompasses the dsRNA of Tables 2-7, which are unmodified, unconjugated, modified, or conjugated, as described herein.

[0209] Those skilled in the art are well aware that dsRNAs with duplex structures of about 20 to 23 base pairs, e.g., 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 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 embodiments, due to the nature of the oligonucleotide sequences provided in any one of Tables 2-7, the dsRNAs described herein can include at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-7, minus only a few nucleotides on one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides that are derived from any one of the sequences in Tables 2-7 and that differ from dsRNAs containing the entire sequence in their ability to inhibit expression of the TMPRSS6 gene by an inhibition rate of about 5, 10, 15, 20, 25, or 30% or less are contemplated to be within the scope of the present invention.

[0210] Additionally, the RNAs provided in Tables 2-7 identify sites in the TMPRSS6 transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within the specific site. Such iRNAs will generally comprise at least about 19 contiguous nucleotides from any one of the sequences provided in any one of Tables 2-7 linked to additional nucleotide sequences taken from regions adjacent to the selected sequence within the TMPRSS6 gene.

[0211] III. Modified iRNAs of the Invention In certain embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain any chemical modifications or conjugations, e.g., known in the art and described herein. In other embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all of the nucleotides of the iRNA or substantially all of the nucleotides of the iRNA are modified, i.e., no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 unmodified nucleotide is present in the strand of the iRNA.

[0212] Nucleic acids featured in the present invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SLet al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted ligation) or 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), base modifications, such as substitutions with stabilizing bases, destabilizing bases, or bases that base pair with partners in the extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including modifications or substitutions of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or containing non-natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have phosphorus atom in backbones are included.For the purpose of this specification, as sometimes referred to in the art, modified RNAs that do not have phosphorus atom in their internucleoside backbones can also be considered as oligonucleosides.In some embodiments, modified iRNAs will have phosphorus atom in their internucleoside backbones.

[0213] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' and 5'-3' or 2'-5' and 5'-2'. Also included are various salts, mixed salts, and free acid forms. In some embodiments of the invention, the dsRNA agent of the invention is in free acid form. In other embodiments of the invention, the dsRNA agent of the 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 in the agent as counterion to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0214] Representative U.S. patents which teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,71 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. 5,47 No. 6,925, 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 , 587,361, 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, Nos. 6,639, 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. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0215] Modified RNA backbones that do not contain phosphorus atoms in the backbone include 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, including those with 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 N, O, S and CH 2 Others have mixed component parts.

[0216] Representative United States patents which teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the contents of each of which are incorporated herein by reference in their entirety.

[0217] RNA mimics suitable for use in the iRNA provided herein are considered in which both the sugar and the internucleoside linkage of the nucleotide unit, i.e., its backbone, are replaced with novel groups. The base unit is maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, which is an RNA mimic 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. Nucleic acid bases are retained and directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331 and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0218] Some embodiments featured in the present invention include RNA with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly those with the --CH 2 --NH--CH 2 -, --CH 2 --N(CH 3 )--O--CH 2 -- [known as the methylene (methylimino) or MMI backbone], --CH 2 --O--N(CH 3 )--CH 2 --, --CH 2 --N(CH 3 )--N(CH 3 )--CH 2 -- and --N(CH 3 )--CH 2 --CH 2-- and the amide backbone of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

[0219] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein may include one of the following at the 2' position: OH, F, O-, S- or N-alkyl, O-, S- or N-alkenyl, O-, S- or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: 1 ~C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, interfering agents, groups for improving the pharmacokinetic properties of iRNA or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification is 2'-methoxyethoxy (2'-O--CH 2 CH 2 OCH 3 , 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 includes 2'-dimethylaminooxyethoxy, i.e., O(CH 2 ) 2 ON(CH 3 ) 2 groups, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH 2 --O--CH 2 --N(CH 3 ) 2 Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides, (both R and S isomers within these three families), 2'-alkoxyalkyls, and 2'-NMA (N-methylacetamide).

[0220] Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in a 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. An iRNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include U.S. Patent Application Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, Nos. 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, some of which are commonly owned with this application, the entire contents of each of the foregoing are incorporated herein by reference.

[0221] iRNAs may also include modifications or substitutions of nucleobases (often referred to in the art simply as "bases"). 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 deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine ... and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some 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 0-6 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 exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0222] Representative United States patents which teach the preparation of certain of the above modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-referenced 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.

[0223] In some embodiments, the RNAi agents of the present disclosure may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a ring formed by bridging two carbons, whether adjacent or non-adjacent. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a ring formed by bridging two carbon atoms of the sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, a bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally through a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide having a 4'-CH 2It is a nucleotide that contains a bicyclic sugar moiety that contains an -O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural 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 that contain a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, an antisense polynucleotide agent of the invention includes one or more bicyclic nucleosides containing a 4' to 2' bridge.

[0224] A locked nucleoside can be represented by this structure (stereochemistry omitted): [ka] where B is a nucleobase or modified nucleobase and L is a linking group connecting the 2'-carbon and the 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH 2 )-O-2'(LNA), 4'-(CH 2 )-S-2',4'-(CH 2 ) 2 -O-2'(ENA), 4'-CH(CH 3 )-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CH 2 OCH 3 )-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845), 4'-C(CH 3 )(CH 3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,283), 4'-CH 2 -N(OCH 3 )-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH 2 -ON(CH 3 )-2' (see, for example, U.S. Patent Application Publication No. 2004 / 0171570), 4'-CH 2 -N(R)-O-2', where R is H, C1-C12 alkyl or a nitrogen protecting group (see, for example, U.S. Pat. No. 7,427,672), 4'-CH 2 -C(H)(CH 3 )-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH 2 -C(=CH 2 )-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0225] Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. 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, 7,399, and 8,413,727. Nos. 7,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, U.S. Patent Application Publication No. 2008 / 0039618 and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0226] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0227] The RNA of the iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" refers to a 4'-CH(CH 3 )-O-2' bridge (i.e., L in the preceding structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as "S-cEt."

[0228] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose, or the C3 and -C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity, resulting in less puckering of the ribose ring.

[0229] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0230] 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 their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' linkage has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' linkage of the sugar has been removed (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0231] 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 Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0232] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides such as inverted dT (idT) and inverted dA (idA), as well as inverted abasic 2'-deoxyribonucleotides (iAb) and others. Disclosure of the modifications can be found in WO 2011 / 005861.

[0233] In one example, the 3' or 5' end of the oligonucleotide is linked to an inverted 2'-deoxy modified ribonucleotide, such as an inverted dT (idT), an inverted dA (idA), or an inverted abasic 2'-deoxyribonucleotide (iAb). In one particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0234] In another embodiment, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In another embodiment, the 3' end of the sense strand is linked to an inverted dA (idA) via a 3'-3'-phosphorothioate linkage.

[0235] In one particular example, an inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of an oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0236] In another embodiment, the 3' terminal nucleotide of the sense strand is an inverted dA (idA) and is linked to the preceding nucleotide via a 3'-3'-linkage (eg, a 3'-3'-phosphorothioate linkage).

[0237] Other modifications of the nucleotides of the iRNA of the present invention include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in US Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0238] A. Modified iRNAs Containing Motifs of the Invention In certain aspects of the present invention, the double-stranded RNA agent of the present invention includes agents with chemical modifications, such as those disclosed in WO2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in WO2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense or antisense strand of dsRNAi agent, particularly at or near the cleavage site.In some embodiments, the sense and antisense strands of dsRNAi agent can be otherwise completely modified.The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present.The dsRNAi agent can be optionally conjugated with GalNAc derivative ligand, for example on the sense strand.

[0239] More specifically, gene silencing activity of a dsRNAi agent was observed when the sense and antisense strands of a double-stranded RNA agent were fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0240] Thus, the present invention provides a double-stranded RNA agent capable of inhibiting expression of a target gene (i.e., the TMPRSS6 gene) in vivo. The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0241] The sense strand and the antisense strand typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent." The duplex region of the dsRNAi agent can be, for example, a duplex region that can be 27-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0242] In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be independently 1-6 nucleotides in length, e.g., 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In certain embodiments, the overhang region may include an extended overhang region, as described above. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be another sequence. The first and second strands may also be linked, for example, by additional bases forming a hairpin, or by other non-basic linkers.

[0243] In certain embodiments, the nucleotides in the overhang region of a dsRNAi agent can each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modification, including, for example, 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.

[0244] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0245] The 5'- or 3'-overhang of the sense strand, antisense strand or both strands of dsRNAi agent can be phosphorylated.In some embodiments, the overhang region contains two nucleotides with phosphorothioate between them, and the two nucleotides can be the same or different.In some embodiments, the overhang is present at the 3' end of the sense strand, antisense strand or both strands.In some embodiments, the 3'-overhang is present in the antisense strand.In some embodiments, the 3'-overhang is present in the sense strand.

[0246] dsRNAi agent can only contain a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, single-stranded overhang can be located at the 3' end of sense strand or at the 3' end of antisense strand.RNAi can also have a blunt end, which is located at the 5' end of antisense strand (i.e., the 3' end of sense strand) or vice versa.Generally, the antisense strand of dsRNAi agent 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 antisense strand and the 3' end overhang of the antisense strand, which are asymmetric, are favorable for the insertion of guide strand into RISC process.

[0247] In certain embodiments, the dsRNAi agent is 19 nucleotides in length and double blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, 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, 13 from the 5' end.

[0248] In other embodiments, the dsRNAi agent is double blunt ended 20 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0249] In yet other embodiments, the dsRNAi agent is double blunt ended 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, 13 from the 5' end.

[0250] In certain embodiments, dsRNAi agent comprises a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at 9, 10, 11 positions from 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at 11, 12, 13 positions from 5' end, and one end of the RNAi agent is blunt, and the other end comprises a 2-nucleotide overhang.In some embodiments, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0251] In the case where a two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further has 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. In certain embodiments, all nucleotides in the sense and antisense strands of the dsRNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In certain embodiments, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the dsRNAi agent may be modified with a ligand (e.g., GalNAc 3 ).

[0252] In certain embodiments, the dsRNAi agent comprises a sense strand 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, and the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a duplex, wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand and up to 6 consecutive 3'-terminal nucleotides are unpaired 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 sense strand comprises nucleotides adjacent to the ribonucleotides of the antisense strand, thereby forming a single-stranded 5' overhang of 10-30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base pair with nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense strand and the antisense strand, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that 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 occurs 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.

[0253] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, the dsRNAi agent comprises a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer than the first strand at its 3' end, the duplex region is at least 25 nucleotides in length, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the dsRNAi agent results in an siRNA comprising the 3' end of the second strand, thereby reducing the expression of the target gene in the mammal. Optionally, the dsRNAi agent further comprises a ligand.

[0254] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the site of cleavage in the sense strand.

[0255] In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at or near the cleavage site in the antisense strand.

[0256] For dsRNAi agents having a duplex region that is 19-23 nucleotides in length, the cleavage sites in the antisense strand are typically at approximately positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, 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 can also vary depending on the length of the duplex region of the dsRNAi agent from the 5' end.

[0257] The sense strand of dsRNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the break site of the strand, and the antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the break site of the strand.When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms base pairing with at least one of the three nucleotides of the motif in the antisense strand.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0258] In some embodiments, the sense strand of the dsRNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the site of the cleavage of the strand, and the other motif may be a wing modification. As used herein, the term "wing modification" refers to a motif that occurs in another part of the strand that is separated from a motif at or near the site of the cleavage of the same strand. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature may be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each wing modification may occur at one end to the first motif at or near the cleavage site, or on either side of the lead motif.

[0259] Similar to the sense strand, the antisense strand of the dsRNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage.The antisense strand may also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0260] In some embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0261] In other embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0262] When the sense and antisense strands of a dsRNAi agent each contain at least one wing modification, the wing modifications can be at the same end of the duplex region and have an overlap of one, two, or three nucleotides.

[0263] When the sense or antisense strand of a dsRNAi agent each contains at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications from each single strand reside at one end of a duplex region with an overlap of one, two or three nucleotides, the two modifications from each single strand reside at the other end of a duplex region with an overlap of one, two or three nucleotides, and the two modifications from each single strand reside on either side of a lead motif within the duplex region with an overlap of one, two or three nucleotides.

[0264] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNAi agent, including the nucleotide that is part of the motif, can be modified.Each nucleotide can be modified with the same or different modifications, and such modifications can include one or more of the modification of one or both of non-linked phosphate oxygens, or one or more of linked phosphate oxygens, modification of the components of ribose sugar, such as modification of the 2' hydroxyl on ribose sugar, large-scale replacement of phosphate moiety with "dephosphorylation" linker, modification or replacement of naturally occurring base, and replacement or modification of ribose phosphate backbone.

[0265] Nucleic acids are polymers of subunits, so many of the modifications occur at positions that are repeated in nucleic acids, such as modifications of bases or phosphate moieties, or non-linked O of phosphate moieties. In some cases, modifications will occur at all of the target positions in nucleic acids, but in many cases they will not. For example, modifications can occur only at the 3'-end or 5'-end positions, or only at terminal regions, such as positions on the terminal nucleotides of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in the double-stranded regions of RNA, or only in the single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both termini, can occur only in terminal regions, for example, at positions on the terminal nucleotides of the strand or within the last 2, 3, 4, 5 or 10 nucleotides of the strand, or can occur in double-stranded and single-stranded regions, especially at the termini. The 5' termini can be phosphorylated.

[0266] It may be possible, for example, to enhance stability, to include specific bases in the overhang, or to include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' or 3' overhang, or in both overhangs. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, with modifications known in the art, for example, the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, modified in place of the ribosugar of the nucleobase, and modifications at the phosphate group, for example, phosphorothioate modifications. The overhang does not have to be homologous to the target sequence.

[0267] In some embodiments, each residue of the sense strand and the 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. A strand may contain two or more modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0268] At least two different modifications are typically present on the sense and antisense strands. The two modifications can be, for example, 2'-O-methyl or 2'-fluoro modifications.

[0269] In certain embodiments, N a or N b includes alternating patterns of modifications. The term "alternating motif" as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a single strand. Alternating nucleotides can refer to one every other nucleotide or one every three nucleotides or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB..." or "ABCABCABCABC...", etc.

[0270] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleotide, the alternation pattern, 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 possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0271] In some embodiments, the dsRNAi agent of the present invention comprises a modification pattern of an alternating motif on the sense strand that is shifted relative to the modification pattern of the alternating motif on the antisense strand. The shift can be such that the modified group of the nucleotide of the sense strand corresponds to the differently modified group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" from 5' to 3' of the strand in the duplex region. As another example, the alternating motif in the sense strand can start with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BBAABBAA" from 5' to 3' of the strand in the duplex region, such that there is a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0272] In some embodiments, dsRNAi agent comprises the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification on sense strand, and first has a relative shift with respect to the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification on antisense strand, i.e. comprises 2'-O-methyl modified nucleotide on base pair of sense strand and 2'-F modified nucleotide on antisense strand, and vice versa.Position 1 of sense strand can start with 2'-F modification, and position 1 of antisense strand can start with 2'-O-methyl modification.

[0273] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand or antisense strand interrupts the initial modification pattern present in the sense strand or antisense strand.The interruption of the modification pattern of the sense strand or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand or antisense strand can enhance the gene silencing activity against the target gene.

[0274] In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotides adjacent to the motif is a different modification than the modification of the motif. For example, a portion of a sequence containing a motif may be described as "...N a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification to the nucleotide adjacent 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, when wing modifications are present, N a or N b may or may not be present.

[0275] The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand, the antisense strand, or both strands at any position of the strand. For example, the internucleotide linkage modification may occur on any nucleotide on the sense strand or the antisense strand, and each internucleotide linkage modification may occur in an alternating pattern on the sense strand 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 linkage modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a relative shift to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0276] In some embodiments, the dsRNAi agent comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region can contain two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide with the terminal paired nucleotide in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and optionally there can be additional phosphorothioate or methylphosphonate internucleotide linkages that link the overhang nucleotide with 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 can be at the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 5'-end of the antisense strand.

[0277] In some embodiments, 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 adjacent to the overhanging nucleotide.Optionally, the dsRNAi 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.

[0278] In one embodiment, the dsRNAi agent comprises mismatches or combinations thereof in the duplex with the target. Mismatches can occur in overhang regions or in duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing, the most simple approach is to look at each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferred than G:C, G:U is more preferred than G:C, and I:C is more preferred than G:C (I=inosine). Mismatches, such as non-standard or non-standard pairings (described elsewhere herein), are more preferred than standard (A:T, A:U, G:C) pairings, and pairings that include universal bases are more preferred than standard pairings.

[0279] In certain embodiments, the dsRNAi agent comprises the first 1, 2, 3, 4 or 5 base pairs in the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0280] In certain embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from 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.

[0281] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT) or the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). For example, there is a short sequence of deoxythymidine nucleotides, such as two dT nucleotides on the 3' end of the sense strand, the antisense strand, or both strands.

[0282] In certain embodiments, 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) It can be expressed as: During the ceremony, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide, where Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY are all 2'-F modified nucleotides.

[0283] In some embodiments, N a or N b includes alternating pattern modifications.

[0284] In some embodiments, YYY motif occurs at or near the cleavage site of sense strand.For example, when dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of sense strand (e.g., can occur at 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), this number starting from the first nucleotide from the 5' end, or optionally this number starting from the first paired nucleotide in the double-stranded region from the 5' end.

[0285] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the 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:

[0286] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0287] 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 can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0288] 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 can independently represent an oligonucleotide sequence that contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0289] Each of X, Y and Z may be the same or different from each other.

[0290] In other embodiments, i is 0 and 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:

[0291] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0292] In one embodiment, the antisense strand sequence of the RNAi is represented by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) It can be expressed as: During the ceremony, k and l each independently represent 0 or 1; p' and q' each independently represents 0 to 6; each N a each ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; In the formula, N b ' and Y' do not have the same modification, and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0293] In some embodiments, N a ' or N b ' includes alternating pattern modifications.

[0294] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand.For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the Y'Y'Y' motif can occur at the 9th, 10th, 11th, 10th, 11th, 12th, 11th, 12th, 13th, 12th, 13th, 14th, or 13th, 14th, 15th positions of the antisense strand, the numbers starting from the first nucleotide from the 5' end, or optionally, the numbers starting from the first paired nucleotide in the duplex region from the 5' end.In some embodiments, the Y'Y'Y' motif occurs at the 11th, 12th, 13th positions.

[0295] In certain embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0296] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0297] Thus, the antisense strand has the formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(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) It can be expressed as:

[0298] 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 represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0299] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0300] 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 In some embodiments, N' independently represents an oligonucleotide sequence that contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6.

[0301] In other embodiments, k is 0 and 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:

[0302] 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.

[0303] Each of X', Y' and Z' may be the same as or different from each other.

[0304] 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.

[0305] In some embodiments, the sense strand of the dsRNAi agent may contain a YYY motif occurring at the 9th, 10th and 11th positions of the strand when the duplex region is 21nt (nucleotides), the numbers starting from the first nucleotide from the 5' end, or optionally the numbers starting from the first paired nucleotide in the duplex region from the 5' end, 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, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0306] In some embodiments, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, the number starting from the first nucleotide from the 5' end, or optionally the number starting from the first paired nucleotide in the duplex region from the 5' end, and Y' representing 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, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0307] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc) and (IId).

[0308] Thus, the dsRNAi agents used in the methods of the invention can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the iRNA duplex can have the following 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) It is expressed as During the ceremony, i, j, k and l each independently represent 0 or 1; p, p', q and q' each independently represents 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; In the formula, each n p ',n p , n q ' and n q each of which may be present or absent, 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.

[0309] 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, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0310] Exemplary combinations of sense and antisense strands that form an iRNA duplex include the following formulas: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId).

[0311] When the dsRNAi agent has the formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0312] When the dsRNAi agent has the formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0313] When the dsRNAi agent has the formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0314] When the dsRNAi agent has the formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ N independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b ’ each independently comprises an alternating pattern of modifications.

[0315] Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

[0316] When the dsRNAi 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 Y nucleotides are base-paired with a corresponding Y' nucleotide, or all three Y nucleotides are base-paired with a corresponding Y' nucleotide.

[0317] When the dsRNAi 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. Alternatively, at least two Z nucleotides are base-paired with the corresponding Z' nucleotide, or all three Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0318] When the dsRNAi 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. Alternatively, at least two of the X nucleotides are base-paired with the corresponding X' nucleotide, or all three of the X nucleotides are base-paired with the corresponding X' nucleotide.

[0319] In certain embodiments, 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, or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0320] In certain embodiments, when the dsRNAi agent has formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIid), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, and p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker (described below). a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0321] In some embodiments, when the dsRNAi agent has formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand contains at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0322] In some embodiments, dsRNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and said double strands are connected by a linker.The linker may be cleavable or non-cleavable.Optionally, said multimer further comprises a ligand.Each of said double strands can target the same gene or two different genes, or each of said double strands can target the same gene at two different target sites.

[0323] In some embodiments, dsRNAi agent is a multimer that contains three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and said double strands are connected by a linker.The linker may be cleavable or non-cleavable.Optionally, said multimer further comprises a ligand.Each of said double strands can target the same gene or two different genes, or each of said double strands can target the same gene at two different target sites.

[0324] In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at the 5' end and at one or both of the 3' ends, and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes, or each of the agents can target the same gene at two different target sites. In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modification, for example, 10 or less nucleotides with 2'-fluoro modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with 2'-fluoro modification. In certain embodiments, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modification, for example, 4 nucleotides with 2'-fluoro modification in the sense strand and 6 nucleotides with 2'-fluoro modification in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluoro modification, for example, 4 nucleotides with 2'-fluoro modification in the sense strand and 2 nucleotides with 2'-fluoro modification in the antisense strand.

[0325] In other embodiments, the RNAi agent of the present invention may contain a very small number of nucleotides that contain 2'-fluoro modification, for example, 2 or less nucleotides that contain 2'-fluoro modification.For example, the RNAi agent may contain 2, 1 or 0 nucleotides that have 2'-fluoro modification.In certain embodiments, the RNAi agent may contain 2 nucleotides that have 2'-fluoro modification, for example, 0 nucleotides that have 2-fluoro modification in the sense strand and 2 nucleotides that have 2'-fluoro modification in the antisense strand.

[0326] Various publications describe multimeric iRNAs that can be used in the methods of the invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0327] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, a 5'-vinyl phosphonate modified nucleotide of the present disclosure has the following structure: [ka] where X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH) 2 and the C5' carbon and R 5’ and the double bond between is in the E or Z configuration (e.g., E configuration); B is a nucleobase or a modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0328] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain 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.

[0329] Vinyl phosphonate modifications are also contemplated in the compositions and methods of the present disclosure. Exemplary vinyl phosphonate structures include those described above, where R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., E configuration).

[0330] As described in more detail below, iRNAs containing one or more carbohydrate moieties conjugated to the iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). 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 are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0331] The ligand may be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point", e.g., two "backbone attachment points", and (ii) at least one "tether attachment point". "Backbone attachment point", as used herein, refers to a functional group, e.g., a hydroxyl group, or generally refers to a bond that is available and suitable for incorporating the carrier into the backbone of a ribonucleic acid, e.g., a phosphate, or a modified phosphate, e.g., sulfur-containing. In some embodiments, a "tether attachment point" (TAP) refers to a constituent ring atom, e.g., a carbon atom or a heteroatom (apart from the atom that provides the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers will often contain a functional group, e.g., an amino group, or generally a bond, that provides a linkage suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0332] iRNA can be conjugated to ligand via carrier, and the carrier can be cyclic or acyclic.In one embodiment, 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.In one embodiment, acyclic group is serinol backbone or diethanolamine backbone.

[0333] i.Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermally destabilizing modifications into the seed region of the antisense strand.As used herein, "seed region" refers to positions 2-9 of the 5' end of the reference strand, or positions 2-8 of the 5' end of the reference strand.For example, thermally destabilizing modifications can be incorporated into the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

[0334] The term "thermally destabilizing modification" refers to the melting temperature (T m ) lower than the overall melting temperature (T m For example, thermally destabilizing modifications include modifications that result in a dsRNA having a T m can be decreased by 1-4 degrees Celsius, e.g., one, two, three, or four degrees Celsius. Also, the term "thermally destabilized nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0335] It has been discovered that dsRNAs having an antisense strand that includes at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand includes at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the one or more thermally destabilizing modifications of the duplex are located at positions 2-9, e.g., positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at positions 6, 7 or 8 from the 5' end of the antisense strand. In yet some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is located at positions 2, 3, 4, 5 or 9 from the 5' end of the antisense strand.

[0336] The iRNA agent includes a sense strand and an antisense strand, each strand having 14-40 nucleotides. The RNAi agent has the following formula (L): [ka] (L), which can be represented as:

[0337] 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 one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification or a 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA, 2'-O-CH2C(O)N(Me)H) modification.

[0338] C1 is a thermally destabilizing nucleotide located at a site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand, or positions 2-9 of the 5' end of the antisense strand). For example, C1 is at a position in the sense strand that pairs with a nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is at position 15 from the 5' end of the sense strand. The C1 nucleotide bears a thermally destabilizing modification that may include an abasic modification; a mismatch with the opposing nucleotide in the duplex; a sugar modification, such as, for example, a 2'-deoxy modification or an acyclic nucleotide, e.g., unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA); or a 2'-5' linked ribonucleotide (3'-RNA). In one embodiment, C1 is a thermally destabilizing modification selected from the group consisting of: i) a mismatch with the opposing nucleotide in the antisense strand; [ka] and iii) a sugar modification selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR 3 or alkyl, R 3 is H, an alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar modification that is a sugar. In one embodiment, the thermally destabilizing 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, optionally at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermally destabilizing modification in C1 is GNA or [ka] It is.

[0339] T1, T1', T2', and T3' each independently represent a nucleotide that includes a modification that provides the nucleotide with a steric bulk equal to or less than the steric bulk of the 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effect of a modification of a nucleotide are known to those skilled in the art. The modification can be at the 2' position of the ribose sugar of the nucleotide, or it can be a non-ribose nucleotide, an acyclic nucleotide, or a modification to the backbone of the nucleotide that is similar or equivalent to the 2' position of the ribose sugar, and provides the nucleotide with a steric bulk equal to or less than the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' each independently are selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

[0340] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0341] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0342] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length, or 4 is 0 nucleotides long.

[0343] q 5 are independently 0 to 10 nucleotides in length.

[0344] n 2 , and q 4 are independently 0 to 3 nucleotides in length.

[0345] Or, n 4 is 0 to 3 nucleotides in length.

[0346] In one embodiment, n 4 can be 0. In one embodiment, n 4 is 0 and q 2 and q 6 is 1. In another embodiment, n 4 is 0 and q 2 and q 6 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0347] In one embodiment, n 4 , q 2 , and q 6 are each equal to 1.

[0348] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each equal to 1.

[0349] In one embodiment, C1 is a nucleotide sequence having a sense strand length of 19 to 22 nucleotides and 4 is 1, it is at positions 14 to 17 of the 5' end of the sense strand. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0350] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand and 6 is equal to 1.

[0351] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. 2 is equal to 1.

[0352] In an 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. 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.

[0353] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0354] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. 2 is equal to 1, and non-ribose acyclic or backbone modifications at the 2' position are less sterically bulky than 2'-OMe ribose.

[0355] In one embodiment, 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 6 is equal to 1, and non-ribose acyclic or backbone modifications at the 2' position are less or equally sterically bulky than 2'-OMe-ribose.

[0356] In one embodiment, T1 is at the cleavage site of the sense strand. In one embodiment, T1 is at the cleavage site of the sense strand, 2 When n is 1, it is located at position 11 from the 5' end of the sense strand. In an exemplary embodiment, T1 is located at position 11 from the 5' end of the sense strand when n is 1. 2When is 1, it is at the cleavage site of the sense strand, which is at position 11 from the 5' end of the sense strand.

[0357] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at positions 6-10 from the 5' end of the antisense strand and 4 is 1.

[0358] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., the sense strand is 19-22 nucleotides in length and 2 When is 1, it is located at the 11th position from the 5' end of the sense strand, T1' is located at the 14th position 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 of the ribose sugar or at a non-ribose acyclic or intrabackbone position that is less sterically bulky than 2'-OMe ribose, T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1, and 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 or at a non-ribose acyclic or intrabackbone position that is less sterically bulky than 2'-OMe ribose.

[0359] In one embodiment, 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 4 is 2.

[0360] In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. 4 is 1.

[0361] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0362] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0363] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0364] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0365] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0366] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0367] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0368] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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, optionally accompanied by at least 2 additional TTs at the 3' end of the antisense strand.

[0369] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 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, optionally with at least two additional TTs at the 3' end of the antisense strand, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and with two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0370] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0371] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0372] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0373] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0374] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1.

[0375] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0376] The RNAi agent may contain a phosphorus-containing group at the 5' end of the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS 2 ), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., a trans-vinyl phosphonate, [ka] 5'-Z-VP isomers (i.e., cis-vinyl phosphonates, [ka] or a mixture thereof.

[0377] In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0378] In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-P in the antisense strand.

[0379] In one embodiment, the RNAi agent comprises a 5'-PS. In one embodiment, the RNAi agent comprises a 5'-PS in the antisense strand.

[0380] In one embodiment, the RNAi agent comprises a 5'-VP. In one embodiment, the RNAi agent comprises a 5'-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5'-Z-VP in the antisense strand.

[0381] In one embodiment, the RNAi agent is a 5'-PS 2 In one embodiment, the RNAi agent comprises a 5'-PS 2 Includes.

[0382] In one embodiment, the RNAi agent is a 5'-PS 2 In one embodiment, the RNAi agent comprises 5'-deoxy-5'-C-malonyl in the antisense strand.

[0383] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0384] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0385] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0386] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 The RNAi agent also has a 5'-PS 2 Also includes.

[0387] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0388] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-P.

[0389] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The RNAi agent also includes a 5'-PS.

[0390] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, 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 with 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 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 a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0391] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 Also includes.

[0392] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0393] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0394] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 dsRNA agent also contains a 5'-PS.

[0395] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0396] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent also has a 5'-PS 2 Also includes.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0398] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-P.

[0399] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS.

[0400] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0401] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). RNAi agents also include 5'-PS 2 Also includes.

[0402] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

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

[0404] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0405] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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.

[0406] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, 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 The dsRNAi RNA agent also has a 5'-PS 2 Also includes.

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

[0408] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-P.

[0409] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The RNAi agent also includes a 5'-PS.

[0410] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 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 a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0411] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 Also includes.

[0412] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0413] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0414] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 contains a 5'-PS.

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

[0416] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent also has a 5'-PS 2 Also includes.

[0417] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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.

[0418] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-P.

[0419] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23. The RNAi agent also includes a 5'-PS.

[0420] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 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 a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0421] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-F, and q 7 is 1 with 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 Also includes.

[0422] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0423] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, 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.

[0424] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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'-OMe, and q 7 is 1 with two phosphorothioate internucleotide linkage modifications in the range of 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 and two phosphorothioate internucleotide linkage modifications in the range of 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 one embodiment, 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.

[0425] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1 to 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof), and a targeting ligand.

[0426] In one embodiment, 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.

[0427] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0428] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 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.

[0429] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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 ligand. In one embodiment, 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.

[0430] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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 ligand. In one embodiment, 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.

[0431] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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 one embodiment, 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.

[0432] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). RNAi agents also include 5'-PS 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0433] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications in the range of positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications in the range of 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 ligand. In one embodiment, 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.

[0434] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, 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.

[0435] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications in the range of 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 and two phosphorothioate internucleotide linkage modifications in the range of 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 one embodiment, 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.

[0436] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. 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 one embodiment, 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.

[0437] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0438] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 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.

[0439] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, 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.

[0440] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with two phosphorothioate internucleotide linkage modifications in the range of 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 and two phosphorothioate internucleotide linkage modifications in the range of 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 one embodiment, 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.

[0441] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. 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 one embodiment, 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.

[0442] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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 with 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0443] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, 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, with two phosphorothioate internucleotide linkage modifications in the range of 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications in the range of positions 18-23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 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.

[0444] In certain embodiments, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21 and 23, and 2'-F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20 and 22 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The dsRNA 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.

[0445] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 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) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0446] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 through 6, 8, 10, and 12 through 21, 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0447] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0448] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 9, 12 to 21, and 2'-F modifications at positions 10 and 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0449] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0450] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21, and 2'-F modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 18, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxy-nucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a four 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 particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0452] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0453] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) a length of 19 nucleotides; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5, and 7 to 9, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); And (b) an antisense strand having: (i) a length of 21 nucleotides; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end); 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.

[0454] In certain embodiments, the iRNA used in the methods of the invention is an agent selected from an agent selected from any one of Tables 2 to 7. These agents may further comprise a ligand.

[0455] III. iRNA conjugated to a ligand Another modification of the RNA of the iRNA of the invention involves chemically linking the iRNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or, for example, cellular uptake of the iRNA into cells. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556).In other embodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., a dodecanediol or undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0456] In certain embodiments, ligand changes the distribution, targeting or life span of the iRNA agent in which it is incorporated.In some embodiments, ligand brings about enhanced affinity to selected target, for example, molecule, cell or cell type, compartment, such as cell or organ compartment, tissue, organ or region of body, for example, when compared with species in which such ligand is not present.In some embodiments, ligand does not participate in double-stranded pairing in double-stranded nucleic acid.

[0457] 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 polyamino acids that are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymers, poly(L-lactide-co-glycolied) copolymers, divinyl ether-maleic anhydride copolymers, N-(2-hydroxypropyl)methacrylamide copolymers (HMPA), polyethylene glycols (PEG), polyvinyl alcohol (PVA), polyurethanes, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazines. Examples of polyamines include polyethyleneimines, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginines, amidines, protamines, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0458] The ligand may also include a targeting group, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a cell or tissue targeting agent, such as a renal cell. The targeting group may 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 mimic. In certain embodiments, the ligand is a polyvalent galactose, such as N-acetyl-galactosamine.

[0459] 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, adamantane acetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyano) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic 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, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0460] The ligand can be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, e.g., a hepatocyte. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, e.g., 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, lipopolysaccharide, activators of p38 MAP kinase, or activators of NF-κB.

[0461] The ligand can be a substance, e.g., a drug, that can increase uptake of the iRNA agent into the cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting the microtubules, microfilaments, or intermediate filaments of the cell. The drug can be, e.g., taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0462] In some embodiments, the ligand that binds 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 that contain some phosphorothioate linkages have also been found to bind to serum proteins, and therefore short oligonucleotides, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, that contain multiple phosphorothioate linkages in the backbone are also suitable for the present invention as ligands (e.g., as PK-modulating ligands). In addition, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0463] The ligand-conjugated iRNAs of the invention can be synthesized by the use of oligonucleotides bearing reactive functional pendant side chains, such as those resulting from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing a linking moiety attached thereto.

[0464] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely produced by the well-known technique of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.).Any other method for such synthesis known in the art may additionally or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, such as, for example, phosphorothioates and alkylated derivatives.

[0465] In the ligand-conjugated iRNAs and sequence-specific linked nucleosides bearing ligand molecules of the invention, oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand nucleotide or ligand nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0466] When using a nucleotide-conjugate precursor that already carries a linking moiety, typically the synthesis of the sequence-specifically linked nucleoside is completed, and then the ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to the standard and non-standard phosphoramidites that are commercially available and commonly used in oligonucleotide synthesis.

[0467] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. In one embodiment, such lipid or lipid-based molecule binds serum protein, for example human serum albumin (HSA). HSA-binding ligand allows distribution of conjugate to target tissue of the body, for example to target tissue other than the kidney. For example, the target tissue can be liver, including liver parenchymal cells. Other molecules that can bind HSA can also be used as ligand. For example, naproxen or aspirin can be used. Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) can be used to adjust binding to serum protein, for example, HSA.

[0468] Lipid-based ligand can be used to inhibit, for example, control, the binding of conjugate to target tissue.For example, lipid or lipid-based ligand that binds more strongly to HSA is less likely to target kidney, and therefore less likely to be eliminated from the body.Lipid or lipid-based ligand that does not bind too strongly to HSA can be used so that conjugate targets kidney.

[0469] In certain embodiments, the lipid-based ligand binds HSA. In one embodiment, it binds HSA with sufficient affinity such that the conjugate is distributed to non-renal tissues. However, it is preferred that the affinity is not so strong that the HSA-ligand binding is irreversible.

[0470] In other embodiments, the lipid-based ligand binds HSA weakly or not at all. In one embodiment, the conjugate is distributed to the kidney. Instead of or in addition to the lipid-based ligand, other moieties that target kidney cells can be used.

[0471] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins include vitamin B, e.g., 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).

[0472] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In some embodiments, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. In some embodiments, the helical agent is an alpha-helical agent with a lipophilic phase and a lipophobic phase.

[0473] 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 the iRNA, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0474] The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 15) can also be targeting moieties. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and cell membrane spanning proteins. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 16)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) have been found to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, e.g., from phage display libraries or one-bead-one compound (OBOC) combinatorial libraries (Lam et al., 2003). al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic tethered to a dsRNA agent via a monomeric unit incorporated for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as those that increase stability or govern conformational properties. Any of the structural modifications described below can be utilized.

[0475] 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.RGD-containing peptides and peptidiomimetics can include D-amino acids as well as synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands, such as PECAM-1 or VEGF.

[0476] A "cell-penetrating peptide" is capable of penetrating a cell, e.g., a microbial cell, e.g., a bacterial cell or a fungal cell, or a mammalian cell, e.g., a human cell. A microbial cell-penetrating peptide can be, e.g., an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). A cell-penetrating peptide can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, e.g., 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).

[0477] C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. The carbohydrate-conjugated iRNA is a composition that is advantageous for in vivo delivery of nucleic acids and suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself (which may be linear, branched or cyclic) composed of one or more monosaccharide units having at least six carbon atoms with an oxygen, nitrogen or sulfur atom bonded to each carbon atom, or a compound that has as a part thereof a carbohydrate moiety (which may be linear, branched or cyclic) composed of one or more monosaccharide units each having at least six carbon atoms with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides consisting of about 4, 5, 6, 7, 8 or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose and polysaccharide resins. 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).

[0478] In certain embodiments, the carbohydrate conjugates used in the compositions and methods of the invention are monosaccharides.

[0479] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Pat. No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, GalNAc conjugates function as ligands that target iRNA to specific cells. In some embodiments, GalNAc conjugates target iRNA to liver cells, for example, by functioning as ligands for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0480] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., via a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., via a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., via a linker as described herein.

[0481] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a tetravalent linker.

[0482] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative linked to an iRNA agent. In certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0483] In some embodiments, for example, when the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted stretch of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker.The hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0484] In some embodiments, for example, when the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted stretch of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker.The hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0485] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[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. [ka] Formula II, etc.

[0487] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S. [ka]

[0488] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below. [ka]

[0489] Further exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to, [ka] (Formula XXXVI), where when one of X or Y is an oligonucleotide, the other is hydrogen.

[0490] In some embodiments, a suitable ligand is a ligand disclosed in WO 2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure: [ka]

[0491] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to the iRNA agents of the invention via a trivalent linker.

[0492] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives linked to the iRNA agent. GalNAc can be linked to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be linked to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand or the 3' end of the antisense strand. In one embodiment, GalNAc is linked to the 3' end of the sense strand, for example, via a trivalent linker.

[0493] In other embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0494] 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 stretch of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand that form 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.

[0495] 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 or a cell penetrating peptide.

[0496] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in PCT Publication Nos. WO 2014 / 179620 and WO 2014 / 179627, the contents of each of which are incorporated herein by reference in their entirety.

[0497] 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 be cleavable or non-cleavable.

[0498] The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, e.g., covalently bonds two parts of a compound. Typically, a linker is a direct bond or an atom such as oxygen or sulfur, NR8, C(O), C(O)NH, SO, SO 2 , S.O. 2A unit such as NH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylaryl alkyl, alkenylaryl alkenyl, alkenylaryl alkynyl, alkynylaryl alkyl, alkynylaryl alkenyl, alkynylaryl alkynyl, alkylheteroaryl alkyl, alkylheteroaryl alkenyl, alkylheteroaryl alkynyl, alkylheteroaryl alkynyl, alkylheteroaryl alkenyl, alkylheteroaryl alkynyl, alkylheteroaryl alkynyl, alkylheteroaryl alkyl, alkylheteroaryl alkenyl, alkylheteroaryl alkyn ... alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, etc., wherein one or more methylenes are O, S, S(O), SO 2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle, where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0499] A cleavable linking group is one that is sufficiently stable outside a cell, but once inside a target cell, is cleaved to release the two moieties that the linker is holding together. In an exemplary embodiment, the cleavable linking group is cleaved at a rate that is 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 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, for example, be selected to mimic or represent conditions found in blood or serum).

[0500] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are found to be more widespread or at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selected for a specific substrate or have no substrate specificity, such as oxidizing or reducing enzymes or reducing agents present in cells that can degrade redox-cleavable linking groups by reduction, such as mercaptans, esterases, reagents that can create endosomes or acidic environments, such as reagents that result in a pH of 5 or less, enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific), and phosphatases.

[0501] Cleavable linking groups, such as disulfide bonds, may be sensitive to pH. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from about 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 have a cleavable linking group that is cleaved at a selected pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0502] Linker can contain a cleavable linking group that can be cleaved by a specific enzyme.The type of cleavable linking group incorporated in linker can depend on the target cell.For example, liver targeting ligand can be linked to cationic lipid via a linker that contains ester group.Hepatocytes are rich in esterase, so linker will be cleaved more efficiently in hepatocyte than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testis cells.

[0503] When targeting cell types that are rich in peptidases, such as hepatocytes and synovial cells, linkers containing peptide bonds can be used.

[0504] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first and a second condition can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm by further evaluation in whole animals. In certain embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 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).

[0505] i. Redox-cleavable linking group In certain embodiments, 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 includes 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 turn to the methods described herein. For example, the candidate can be evaluated by incubating with dithiothreitol (DTT) or other reducing agent in cells, using a reagent known in the art that mimics the rate of cleavage that would be observed in, for example, a target cell. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In some conditions, the candidate compound is cleaved at up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate 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 the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0506] ii. Phosphate-based cleavable linkers In other embodiments, the cleavable linker comprises a phosphate-based cleavable linker that is cleaved by a reagent that degrades or hydrolyzes the phosphate group. An example of a reagent that cleaves the phosphate group in a cell is an enzyme such as a phosphatase in the cell. Examples of phosphate based linking groups include, -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-, where Rk in each occurrence can be independently C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0507] iii. Acid-cleavable linking groups In other embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In certain embodiments, the acid-cleavable linking group is cleaved in an acidic environment where the pH is about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower) or by a reagent such as an enzyme that can act as a general acid. In cells, certain low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). Exemplary embodiments are aryl groups, substituted alkyl groups, or tertiary alkyl groups, such as dimethylpentyl or t-butyl, when the carbon is bonded to the oxygen of the ester (alkoxy group). These candidates can be evaluated using methods similar to those described above.

[0508] iv. Ester-based linking group In other embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0509] v. Peptide-based cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved in cells by enzymes such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. The peptide-based cleaving group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and does not include all amide functional groups. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0510] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to, [ka] (formula XL), [ka] (formula XLI), [ka] (formula XLII), [ka] (Formula XLIII), and [ka] (Formula XLIV), where when one of X or Y is an oligonucleotide, the other is hydrogen.

[0511] In certain embodiments of the compositions and methods of the invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0512] In one embodiment, the dsRNA of the present invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of the following formulas (XLV) to (XLVI): Formula XXXXV Formula XLVI [ka] Formula XLVII Formula XLVIII During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C each independently represent 0 to 20 at each occurrence, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C are independent for each occurrence: absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , C.H. 2 NH, or CH 2 O, Q 2A , Q 2B , Q 3A , Q 3B , Q4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently at each occurrence, absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO 2 , N(R N ), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C is independently selected for each occurrence from absent, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, i.e., independently at each occurrence, a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents that inhibit expression of target genes, such as those of formula (XLIX): formula XLIX [ka] In the formula, L 5A , L 5B, and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0513] Examples of divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as Formulas II, VII, XI, X, and XIII.

[0514] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,07 ... No. 7, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,73 No. 7, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,082,830 No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098 No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810 Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the contents of each of which are incorporated herein by reference in their entirety.

[0515] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications can be incorporated in a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0516] A "chimeric" iRNA compound or "chimera" in the context of the present invention is an iRNA compound, such as a dsRNAi agent, that contains two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity to the target nucleic acid. Additional regions of the iRNA can serve as substrates for enzymes that are capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when using chimeric dsRNAs compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target is routinely detectable by gel electrophoresis and, optionally, associated nucleic acid hybridization techniques known in the art.

[0517] In certain instances, the RNA of an iRNA can be modified by a non-ligand group. Several non-ligand molecules have been conjugated to an iRNA to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for carrying out such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Pro...

Claims

1. 1. A double-stranded ribonucleic acid (dsRNA) agent, or a pharma- ceutically acceptable salt thereof, for inhibiting expression of transmembrane protease, serine 6 (TMPRSS6) in a cell, said dsRNA agent comprising a sense strand and an antisense strand which form a double-stranded region, wherein the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa -3' (SEQ ID NO:521); wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Gf and Uf are 2'-deoxy-2'-fluoro (2'-F) G and U, respectively; dC, dA, and dT are 2'-deoxy C, A, and T, respectively; and s is a phosphorothioate linkage; The dsRNA agent, or a pharma- ceutically acceptable salt thereof, wherein said dsRNA agent is conjugated to a ligand.

2. The dsRNA agent of claim 1, wherein the nucleotide sequence of the sense strand differs by 3 bases or less from the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395), and the nucleotide sequence of the antisense strand differs by 3 bases or less from the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa-3' (SEQ ID NO:521).

3. The dsRNA agent of claim 1, wherein the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa-3' (SEQ ID NO:521).

4. The dsRNA agent of claim 1, wherein the nucleotide sequence of the sense strand differs by one base or less from the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395), and the nucleotide sequence of the antisense strand differs by one base or less from the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa-3' (SEQ ID NO:521).

5. The dsRNA agent of claim 1, wherein the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395) and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa-3' (SEQ ID NO:521).

6. The dsRNA agent of claim 1, wherein the nucleotide sequence of the sense strand is the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395), and the nucleotide sequence of the antisense strand is the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa-3' (SEQ ID NO:521), or a pharma- ceutically acceptable salt thereof.

7. 10. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the ligand is conjugated to the 3'-end of the sense strand of the dsRNA agent.

8. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

9. 9. The dsRNA agent of claim 8, or a pharma- ceutically acceptable salt thereof, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent linker.

10. The ligand is 【Chemistry 1】 10. The dsRNA agent of claim 9, wherein:

11. A schematic diagram of the following: 【Chemistry 2】 (Wherein, X is O or S) 11. The dsRNA agent of claim 10, or a pharma- ceutically acceptable salt thereof, wherein the dsRNA agent is conjugated to a ligand as shown in:

12. 12. The dsRNA agent of claim 11, wherein X is O; or a pharma- ceutically acceptable salt thereof.

13. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, in the sodium salt form.

14. An isolated cell comprising the dsRNA agent or a pharma- ceutically acceptable salt thereof described in any one of claims 1 to 13.

15. A pharmaceutical composition for inhibiting expression of a gene encoding a transmembrane protease, serine 6 (TMPRSS6), comprising the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof.

16. The pharmaceutical composition of claim 15, wherein the dsRNA agent or a pharma- ceutically acceptable salt thereof is in a non-buffered solution.

17. 17. The pharmaceutical composition of claim 16, wherein the non-buffered solution is saline or water.

18. The pharmaceutical composition of claim 15, wherein the dsRNA agent or a pharma- ceutically acceptable salt thereof is in a buffer solution.

19. 20. The pharmaceutical composition of claim 18, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

20. 20. The pharmaceutical composition of claim 19, wherein the buffer solution is phosphate buffered saline (PBS).

21. A composition comprising a sense strand and an antisense strand, or a pharma- ceutically acceptable salt thereof, comprising: the sense strand comprises the nucleotide sequence 5'- asgscugcccUfUfUfggaauaaaguL96-3' (SEQ ID NO:395) and the antisense strand comprises the nucleotide sequence 5'- asdCsuudTadTuccadAaGfggcagcusgsa -3' (SEQ ID NO:521); wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U, respectively; Gf and Uf are 2'-deoxy-2'-fluoro (2'-F) G and U, respectively; dC, dA and dT are 2'-deoxy C, A and T, respectively; s is a phosphorothioate linkage; and L96 is represented by the following schematic diagram: 【Chemistry 3】 (Wherein, X is O) or a pharma- ceutically acceptable salt thereof, wherein the ligand is conjugated to the 3'-end of the sense strand as shown in 22. The composition of claim 21 or a pharma- ceutically acceptable salt thereof, in the sodium salt form.

23. An isolated cell comprising the composition of claim 21 or a pharma- ceutically acceptable salt thereof.

24. A pharmaceutical composition comprising the composition of claim 21 or a pharma- ceutically acceptable salt thereof.

25. The pharmaceutical composition of claim 24, wherein the composition or a pharma- ceutically acceptable salt thereof is in a non-buffered solution.

26. The pharmaceutical composition of claim 25, wherein the non-buffered solution is saline or water.

27. The pharmaceutical composition of claim 24, wherein the composition or a pharma- ceutically acceptable salt thereof is in a buffer solution.

28. The pharmaceutical composition of claim 27, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

29. The pharmaceutical composition of claim 28, wherein the buffer solution is phosphate buffered saline (PBS).

30. A composition comprising a sense strand and an antisense strand, or a pharma- ceutically acceptable salt thereof, comprising: the sense strand consists of the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaaguL96-3' (SEQ ID NO:395) and the antisense strand consists of the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa -3' (SEQ ID NO:521); wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U, respectively; Gf and Uf are 2'-deoxy-2'-fluoro (2'-F) G and U, respectively; dC, dA and dT are 2'-deoxy C, A and T, respectively; s is a phosphorothioate linkage; and L96 is represented by the following schematic diagram: 【Chemistry 4】 (Wherein, X is O) or a pharma- ceutically acceptable salt thereof, wherein the ligand is conjugated to the 3'-end of the sense strand as shown in 31. The composition of claim 30, or a pharma- ceutically acceptable salt thereof, in the sodium salt form.

32. An isolated cell comprising the composition of claim 30 or a pharma- ceutically acceptable salt thereof.

33. A pharmaceutical composition comprising the composition of claim 30 or a pharma- ceutically acceptable salt thereof.

34. The pharmaceutical composition of claim 33, wherein the composition or a pharma- ceutically acceptable salt thereof is in a non-buffered solution.

35. The pharmaceutical composition of claim 34, wherein the non-buffered solution is saline or water.

36. The pharmaceutical composition of claim 33, wherein the composition or a pharma- ceutically acceptable salt thereof is in a buffer solution.

37. The pharmaceutical composition of claim 36, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

38. The pharmaceutical composition of claim 37, wherein the buffer solution is phosphate buffered saline (PBS).

39. A double-stranded ribonucleic acid (dsRNA) agent, or a pharma- ceutically acceptable salt thereof, for inhibiting expression of a transmembrane protease, serine 6 (TMPRSS6) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand which form a double-stranded region, the sense strand comprising the nucleotide sequence 5'-asgscugcccUfUfUfggaauaaagu-3' (SEQ ID NO:395) and the antisense strand comprising the nucleotide sequence 5'-asdCsuudTadTuccadAaGfggcagcusgsa -3' (SEQ ID NO:521); wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; Gf and Uf are 2'-deoxy-2'-fluoro (2'-F) G and U, respectively; dC, dA, and dT are 2'-deoxy C, A, and T, respectively; and s is a phosphorothioate linkage; The dsRNA agent, or a pharma- ceutically acceptable salt thereof, wherein said dsRNA agent is conjugated to a ligand.

40. The dsRNA agent or a pharma- ceutically acceptable salt thereof of claim 39, in the sodium salt form.

41. A pharmaceutical composition comprising the dsRNA agent of claim 39 or a pharma- ceutically acceptable salt thereof.

42. The pharmaceutical composition of claim 41, wherein the dsRNA agent or a pharma- ceutically acceptable salt thereof is in a non-buffered solution.

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

44. The pharmaceutical composition of claim 41, wherein the dsRNA agent or a pharma- ceutically acceptable salt thereof is in a buffer solution.

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

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

47. A pharmaceutical composition comprising a dsRNA agent according to claims 1 to 13, 39 and 40 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising a composition according to any one of claims 21, 22, 30 and 31 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 15 to 20, 24 to 29, 33 to 38 and 41 to 46 for inhibiting expression of the transmembrane protease, serine 6 (TMPRSS6) in a cell.

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

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

50. The subject has a TMPRSS6-associated disorder, and optionally (i) the TMPRSS6-related disorder is an iron overload-related disorder or an ineffective erythropoiesis disorder; (ii) the TMPRSS6-associated disorder is selected from the group consisting of hereditary hemochromatosis, beta thalassemia, polycythemia vera, myelodysplastic syndromes, congenital dyserythroid anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, and Friedreich's ataxia; (iii) the TMPRSS6-related disorder is β thalassemia, optionally wherein the β thalassemia is β thalassemia major or β thalassemia intermedia; (iv) the TMPRSS6-related disorder is polycythemia vera; (v) the TMPRSS6-related disorder is hereditary hemochromatosis; (vi) the TMPRSS6-related disorder is a hemoglobinopathy; or (vii) The pharmaceutical composition of claim 49, wherein the TMPRSS6-related disorder is sickle cell anemia.

51. The pharmaceutical composition of claim 49, wherein contacting a cell with the dsRNA agent or a pharma- ceutically acceptable salt thereof, the composition or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition results in at least 50%, 60%, 70%, 80%, 90%, or 95% inhibition of expression of TMPRSS6, and optionally, the inhibition of expression of TMPRSS6 reduces TMPRSS6 protein levels in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

52. The pharmaceutical composition of claim 49, wherein contacting a cell with the dsRNA agent or a pharma- ceutically acceptable salt thereof, the composition or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition results in an increase in expression of hepcidin by at least 50%, 60%, 70%, 80%, 90%, or 95%, and optionally, the increased expression of hepcidin increases hepcidin protein levels in the serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

53. A pharmaceutical composition comprising the dsRNA agent of claims 1-13, 39 and 40 or a pharma- ceutically acceptable salt thereof, a pharmaceutical composition comprising the composition of any one of claims 21, 22, 30 and 31 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 15-20, 24-29, 33-38 and 41-46, for use in a method of treating a subject having a disorder that would benefit from a decrease in transmembrane protease, serine 6 (TMPRSS6) expression.

54. A pharmaceutical composition comprising the dsRNA agent of claims 1-13, 39 and 40 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the composition of any one of claims 21, 22, 30 and 31 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 15-20, 24-29, 33-38 and 41-46, for use in a method of preventing at least one symptom in a subject having a disorder that would benefit from a decrease in transmembrane protease, serine 6 (TMPRSS6) expression.

55. The pharmaceutical composition of claim 53, wherein the subject is a human.

56. The disorder is a TMPRSS6-associated disorder, and optionally (i) the TMPRSS6-related disorder is an iron overload-related disorder or an ineffective erythropoiesis disorder; (ii) the TMPRSS6-associated disorder is selected from the group consisting of hereditary hemochromatosis, beta thalassemia, polycythemia vera, myelodysplastic syndromes, congenital dyserythroid anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, and Friedreich's ataxia; (iii) the TMPRSS6-related disorder is β thalassemia, optionally wherein the β thalassemia is β thalassemia major or β thalassemia intermedia; (iv) the TMPRSS6-related disorder is polycythemia vera; (v) the TMPRSS6-related disorder is hereditary hemochromatosis; (vi) the TMPRSS6-related disorder is a hemoglobinopathy; or (vii) the TMPRSS6-related disorder is sickle cell anemia; 56. The pharmaceutical composition of claim 55.

57. The pharmaceutical composition of claim 55, wherein administration of the dsRNA agent, or a pharma- ceutically acceptable salt thereof, composition or a pharma- ceutically acceptable salt thereof, or pharmaceutical composition to a subject causes a decrease in iron levels, a decrease in ferritin levels, a decrease in transferrin saturation levels, an increase in hemoglobin levels, an increase in hematocrit levels, or a decrease in TMPRSS6 protein accumulation, optionally, wherein the dsRNA agent, or a pharma- ceutically acceptable salt thereof, composition or a pharma- ceutically acceptable salt thereof, or pharmaceutical composition is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg, and wherein the dsRNA agent, or a pharma- ceutically acceptable salt thereof, composition or a pharma- ceutical composition is administered subcutaneously or intravenously to the subject.

58. The pharmaceutical composition of claim 55, wherein the method further comprises determining the level of TMPRSS6 in a sample from the subject, and optionally, the level of TMPRSS6 in the subject's sample is the TMPRSS6 protein level in a blood, serum, or liver sample.

59. The pharmaceutical composition of claim 55, wherein the method further comprises determining the level of iron or hepcidin in a sample from the subject.

60. The pharmaceutical composition described in claim 55, wherein the method further comprises administering an additional therapeutic agent to the subject to treat a TMPRSS6-associated disorder.

61. 61. The pharmaceutical composition of claim 60, wherein the additional therapeutic agent is an iron chelator.

62. 62. The pharmaceutical composition of claim 61, wherein the iron chelator is selected from the group consisting of deferiprone, deferoxamine, and deferasirox.

63. The pharmaceutical composition of claim 54, wherein the subject is a human.

64. The disorder is a TMPRSS6-associated disorder, and optionally (i) the TMPRSS6-related disorder is an iron overload-related disorder or an ineffective erythropoiesis disorder; (ii) the TMPRSS6-associated disorder is selected from the group consisting of hereditary hemochromatosis, beta thalassemia, polycythemia vera, myelodysplastic syndromes, congenital dyserythroid anemia, pyruvate kinase deficiency, erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, and Friedreich's ataxia; (iii) the TMPRSS6-related disorder is β thalassemia, optionally wherein the β thalassemia is β thalassemia major or β thalassemia intermedia; (iv) the TMPRSS6-related disorder is polycythemia vera; (v) the TMPRSS6-related disorder is hereditary hemochromatosis; (vi) the TMPRSS6-related disorder is a hemoglobinopathy; or (vii) the TMPRSS6-related disorder is sickle cell anemia; 64. The pharmaceutical composition of claim 63.

65. The pharmaceutical composition of claim 63, wherein administration of the dsRNA agent, or a pharma- ceutically acceptable salt thereof, composition or a pharma- ceutically acceptable salt thereof, or pharmaceutical composition to a subject causes a decrease in iron levels, a decrease in ferritin levels, a decrease in transferrin saturation levels, an increase in hemoglobin levels, an increase in hematocrit levels, or a decrease in TMPRSS6 protein accumulation, optionally, wherein the dsRNA agent, or a pharma- ceutically acceptable salt thereof, composition or a pharma- ceutical composition is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg, and wherein the dsRNA agent, or a pharma- ceutical acceptable salt thereof, composition or a pharma- ceutical acceptable salt thereof, or pharmaceutical composition is administered subcutaneously or intravenously to the subject.

66. The pharmaceutical composition of claim 63, wherein the method further comprises determining the level of TMPRSS6 in a sample from the subject, and optionally, the level of TMPRSS6 in the subject's sample is the TMPRSS6 protein level in a blood, serum, or liver sample.

67. The pharmaceutical composition of claim 63, wherein the method further comprises determining the level of iron or hepcidin in a sample from the subject.

68. The pharmaceutical composition of claim 63, wherein the method further comprises administering an additional therapeutic agent to the subject to treat the TMPRSS6-associated disorder.

69. The pharmaceutical composition of claim 68, wherein the additional therapeutic agent is an iron chelator.

70. The pharmaceutical composition of claim 69, wherein the iron chelator is selected from the group consisting of deferiprone, deferoxamine, and deferasirox.

71. A kit comprising a dsRNA agent according to claims 1 to 13, 39 and 40, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 15 to 20, 24 to 29, 33 to 38 and 41 to 46, or a composition according to any one of claims 21, 22, 30 and 31, or a pharma- ceutically acceptable salt thereof.

72. A vial comprising a dsRNA agent according to claims 1 to 13, 39 and 40, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 15 to 20, 24 to 29, 33 to 38 and 41 to 46, or a composition according to any one of claims 21, 22, 30 and 31, or a pharma- ceutically acceptable salt thereof.

73. A syringe comprising a dsRNA agent according to claims 1 to 13, 39 and 40, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 15 to 20, 24 to 29, 33 to 38 and 41 to 46, or a composition according to any one of claims 21, 22, 30 and 31, or a pharma- ceutically acceptable salt thereof.

74. An RNA-induced silencing complex (RISC) comprising an antisense strand of a dsRNA agent or a pharma- ceutically acceptable salt thereof according to claims 1 to 13, 39 and 40, or an antisense strand of a composition or a pharma- ceutically acceptable salt thereof according to any one of claims 21, 22, 30 and 31.

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