Methods and compositions for treating bleeding events in subjects with hemophilia

Abstract

To provide alternative methods of treatment for subjects with bleeding disorders such as hemophilia.SOLUTION: The present invention provides iRNA, e.g., double-stranded ribonucleic acid (dsRNA) compositions, that target the Serpinc1 gene, and methods of using such iRNA, e.g., dsRNA compositions, to treat bleeding events in subjects with hemophilia (e.g., with or without inhibitors).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 530,518, filed July 10, 2017, U.S. Provisional Patent Application No. 62 / 599,223, filed December 15, 2017, U.S. Provisional Patent Application No. 62 / 614,111, filed January 5, 2018, and U.S. Provisional Patent Application No. 62 / 673,424, filed May 18, 2018. The entire contents of each of the foregoing patent applications are incorporated herein by reference.

[0002] This application is also related to International Application No. PCT / US2016 / 065245, filed December 7, 2016, U.S. Provisional Patent Application No. 62 / 264,013, filed December 7, 2015, U.S. Provisional Patent Application No. 62 / 315,228, filed March 30, 2016, U.S. Provisional Patent Application No. 62 / 366,304, filed July 25, 2016, and U.S. Provisional Patent Application No. 62 / 429,241, filed December 2, 2016. The entire contents of each of the foregoing patent applications are incorporated herein by reference.

[0003] Additionally, this application is related to U.S. Provisional Patent Application No. 61 / 992,057, filed May 12, 2014, U.S. Provisional Patent Application No. 62 / 089,018, filed December 8, 2014, U.S. Provisional Patent Application No. 62 / 102,281, filed January 12, 2015, and International Application No. PCT / US2015 / 030337, filed May 12, 2015. The entire contents of each of the foregoing patent applications are incorporated herein by reference.

[0004] This application is also related to U.S. Provisional Patent Application No. 61 / 638,952, filed April 26, 2012, U.S. Provisional Patent Application No. 61 / 669,249, filed July 9, 2012, U.S. Provisional Patent Application No. 61 / 734,573, filed December 7, 2012, U.S. Provisional Patent Application No. 13 / 837,129, filed March 15, 2013, now U.S. Patent No. 9,127,274, U.S. Patent Application No. 14 / 806,084, filed July 22, 2015, now U.S. Patent No. 9,376,680, U.S. Patent Application No. 15 / 070,358, filed March 15, 2016, and International Application No. PCT / US2013 / 038218, filed April 25, 2013. This application is also related to International Application No. PCT / US2012 / 065601, filed November 16, 2012. The entire contents of each of the aforementioned patent applications are incorporated herein by reference.

[0005] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 2, 2018, is named 117811-02720_SL.TXT and is 21,147 bytes in size. [Background technology]

[0006] Serpinc1 is a member of the serine proteinase inhibitor (serpin) superfamily. Serpinc1 is a plasma protease inhibitor that inhibits thrombin and other active serine proteases of the coagulation system, such as factors X, IX, XI, XII, and VII, thereby regulating the blood coagulation cascade. The anticoagulant activity of Serpinc1 is enhanced by the presence of heparin and other related glycosaminoglycans, which catalyze the formation of the thrombin:antithrombin (TAT) complex.

[0007] Bleeding disorders, either inherited or acquired, are conditions in which blood clots insufficiently. For example, hemophilia is a group of inherited, hereditary bleeding disorders that impair the body's ability to control blood clotting, or coagulation. Hemophilia A is a recessive, X-linked genetic disorder involving a lack of functional clotting factor VIII and accounts for 80% of hemophilia cases. Hemophilia B is a recessive, X-linked genetic disorder involving a lack of functional clotting factor IX. Hemophilia B accounts for approximately 20% of hemophilia cases. Hemophilia C is an autosomal inherited disorder involving a lack of functional clotting factor XI. Hemophilia C is not fully recessive, as heterozygous individuals also exhibit increased bleeding.

[0008] Currently, there is no cure for hemophilia, but it can be controlled by regular infusion of deficient clotting factors, e.g., factor VIII in hemophilia A. However, some hemophilia patients develop antibodies (inhibitors) against the replacement factors given to them, thereby becoming resistant to the replacement clotting factors. Therefore, bleeding in such patients cannot be adequately controlled. Summary of the Invention [Problem to be solved by the invention]

[0009] For example, the development of high-titer inhibitors against factor VIII and other coagulation factors is the most serious problem in the treatment of hemophilia, making the treatment of bleeding extremely difficult.Currently, the only way to stop bleeding in such subjects is to use "bypass drugs" such as factor VIII inhibitor bypassing agent (fiba) and recombinant activated factor VII (rFVIIa), plasma exchange therapy, continuous factor replacement, and immune tolerance therapy, but none of them are completely effective.Therefore, there is a need in the art for alternative treatments for subjects with bleeding disorders such as hemophilia. [Means for solving the problem]

[0010] The present invention provides, at least in part, a therapeutic effect of an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the Serpincl gene in a subject with hemophilia who is free of an inhibitor and who is administered a therapeutically effective amount of the iRNA composition, e.g., as described by the World Federation of Hemophilia (e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6, July 2012; DOI:10.1111 / j.1365-2516.2012.02909.x) and / or the Food and Drug Administration, and in a subject with hemophilia who has an inhibitor and has been administered a therapeutically effective amount of an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the Serpincl gene, the bleeding event can be treated with a therapeutically effective amount of a replacement factor, such as factor VIII or factor XI, that is less than the recommended effective amount of the replacement factor as recommended by the Food and Drug Administration; and in a subject with hemophilia who has an inhibitor and has been administered a therapeutically effective amount of an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the Serpincl gene, e.g., by the World Federation of Hemophilia (see, e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6, July 2012; DOI:10.1111 / j.1365-2516.2012.02909.x) and / or the Food and Drug Administration (FDA).

[0011] Thus, in one aspect, the present invention provides a method for treating a bleeding event in a subject with a bleeding disorder, such as hemophilia, without an inhibitor, comprising administering to the subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15). the sense strand comprises a region complementary to an mRNA encoding Serminc1, the region comprising a nucleotide corresponding to a nucleotide selected from the group consisting of a nucleotide sequence identical to that of Serminc1, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a replacement factor, the effective amount of which is reduced compared to a recommended effective amount of the replacement factor, thereby treating a bleeding event in a subject with hemophilia without an inhibitor.

[0012] In another aspect, the present invention provides a method for treating a bleeding event in a subject with a bleeding disorder, such as hemophilia with an inhibitor, comprising: administering to the subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a bypassing agent, wherein the effective amount of the bypassing agent is reduced compared to the recommended effective amount of the bypassing agent, thereby treating a bleeding event in a subject with hemophilia with an inhibitor.

[0013] In one embodiment, the present invention provides a method for treating a bleeding event in a subject with a bleeding disorder, such as inhibitor-free hemophilia, by administering to the subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a replacement factor, wherein the effective amount of the replacement factor is reduced compared to the recommended effective amount of the replacement factor, thereby treating a bleeding event in a subject with inhibitor-free hemophilia.

[0014] In another aspect, the present invention provides a method for treating a bleeding event in a subject with a bleeding disorder, such as hemophilia with an inhibitor, comprising: administering to the subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a bypassing agent, wherein the effective amount of the bypassing agent is reduced compared to the recommended effective amount of the bypassing agent, thereby treating a bleeding event in a subject with hemophilia with an inhibitor.

[0015] The double-stranded RNAi agent can be administered to a subject in two or more doses.

[0016] In some embodiments, the double-stranded RNAi agent is administered to a subject once a month, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 2 months, once quarterly, or as needed. can be.

[0017] In one embodiment, double-stranded RNAi agent is administered to the subject once a month.In another embodiment, double-stranded RNAi agent is administered to the subject once every 6 weeks.In one embodiment, double-stranded RNAi agent is administered to the subject once every 2 months.In yet another embodiment, double-stranded RNAi agent is administered to the subject once a quarter.

[0018] The double-stranded RNAi agent may be, for example, between about 25 mg and about 100 mg, e.g., between about 25 mg and about 95 mg, between about 25 mg and about 90 mg, between about 25 mg and about 85 mg, between about 25 mg and about 80 mg, between about 25 mg and about 75 mg, between about 25 mg and about 70 mg, between about 25 mg and about 65 mg, between about 25 mg and about 60 mg, between about 25 mg and about 50 mg, between about 50 mg and about 100 mg, between about 50 mg and about 95 mg, between about 50 mg and about 90 mg, between about 50 mg and about 85 mg, between about 50 mg and about 80 mg, between about 30 mg and about 100 mg, between about 30 mg and about 90 mg, between about 30 mg and about 80 mg, between about 40 mg and about 5 ... The compound can be administered to a subject as a fixed dose of between 0 mg and about 100 mg, between about 40 mg and about 90 mg, between about 40 mg and about 80 mg, between about 60 mg and about 100 mg, between about 60 mg and about 90 mg, between about 25 mg and about 55 mg, between about 25 mg and about 65 mg, between about 30 mg and about 95 mg, between about 30 mg and about 85 mg, between about 30 mg and about 75 mg, between about 30 mg and about 65 mg, between about 30 mg and about 55 mg, between about 40 mg and about 95 mg, between about 40 mg and about 85 mg, between about 40 mg and about 75 mg, between about 40 mg and about 65 mg, between about 40 mg and about 55 mg, or between about 45 mg and about 95 mg.

[0019] In some embodiments, the double-stranded RNAi agent can be administered at a fixed dose of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg.

[0020] In some embodiments, the double-stranded RNAi agent is administered to the subject at a fixed dose of about 25 mg; or at a fixed dose of about 50 mg; or at a fixed dose of about 80 mg; or at a fixed dose of about 100 mg.

[0021] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0022] In one embodiment, the subject is a human.

[0023] Hemophilia can be hemophilia A, hemophilia B, or hemophilia C.

[0024] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0025] In one embodiment, the modified nucleotides are independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

[0026] The region of complementarity may be at least 17 or 19 nucleotides in length.

[0027] In one embodiment, the region of complementarity is between 19 and 21 nucleotides in length. In another embodiment, the region of complementarity is between 21 and 23 nucleotides in length.

[0028] In one embodiment, each strand is no more than 30 nucleotides in length.

[0029] At least one strand of the double-stranded RNAi agent may comprise a 3' overhang of at least 1 nucleotide or at least 2 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 another embodiment, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0030] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may be one or more GalNAc linked to the RNAi agent via a monovalent, bivalent, or trivalent branched linker. The ligand may be conjugated to the 3' end of the sense strand of the double-stranded RNAi agent, the 5' end of the sense strand of the double-stranded RNAi agent, the 3' end of the antisense strand of the double-stranded RNAi agent, or the 5' end of the antisense strand of the double-stranded RNAi agent.

[0031] In some embodiments, a double-stranded RNAi agent of the invention comprises multiple, e.g., 2, 3, 4, 5, or 6, GalNAc, each independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0032] In certain embodiments, the ligand is: [ka]

[0033] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0034] In one embodiment, X is O.

[0035] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0036] In one embodiment, the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence of 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0037] In one embodiment, the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

[0038] In one embodiment, the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and the sense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), [ka] (wherein X is O or S). It is conjugated to a ligand as shown in FIG.

[0039] In one embodiment, the agent is administered as a pharmaceutical composition. In one embodiment, the RNAi agent is administered in an unbuffered solution, such as saline or water.

[0040] In another embodiment, the siRNA is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0041] In one embodiment, administration of the dsRNA agent to a subject reduces Serpinc1 activity by about 75% or more.

[0042] In one embodiment, the replacement factor is factor VIII.The therapeutically effective amount of factor VIII administered to a subject can be less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, or less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg. In one embodiment, the therapeutically effective amount of Factor VIII administered to a subject is about two-thirds to about one-fifth of the recommended effective amount of Factor VIII, for example, a dose of about 5 IU / kg to 20 IU / kg, or about 10 IU / kg to about 20 IU / kg, for example, about 5, 10, 15, or 20 IU / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0043] In another embodiment, the replacement factor is Factor IX. A therapeutically effective amount of Factor IX is less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, or less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg. In one embodiment, the therapeutically effective amount of Factor IX administered to a subject is about half to about one-sixth of the recommended effective amount of Factor IX, e.g., a dose of about 10 IU / kg to about 30 IU / kg, or about 20 to about 30 IU / kg, e.g., a dose of about 10, 15, 20, 25, or about 30 IU / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0044] In one embodiment, the bypassing agent is activated prothrombin complex concentrate (aPCC). The therapeutically effective amount of aPCC can be less than about 100 U / kg, or less than about 90 U / kg, or less than about 80 U / kg, or less than about 70 U / kg, or less than about 60 U / kg, or less than about 50 U / kg, or less than about 40 U / kg, or less than about 30 U / kg, or less than about 20 U / kg, or less than about 10 U / kg. In one embodiment, the therapeutically effective amount of aPCC administered to a subject is about one-half to one-third of the recommended effective amount of aPCC, e.g., a dose of about 30 to about 50 U / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0045] In another embodiment, the bypassing agent is recombinant Factor VIIa (rFVIIa). The therapeutically effective amount of the bypassing agent can be less than about 120 μg / kg, or less than about 110 μg / kg, or less than about 100 μg / kg, or less than about 90 μg / kg, or less than about 80 μg / kg, or less than about 70 μg / kg, or less than about 60 μg / kg, or less than about 50 μg / kg, or less than about 40 μg / kg, or less than about 30 μg / kg, or less than about 20 μg / kg. In one embodiment, the therapeutically effective amount of rFVIIa administered to the subject is about half the recommended effective amount of rFVIIa, for example, a dose of about 45 μg / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0046] In one embodiment, the invention provides a method of treating a bleeding event in a subject with hemophilia without inhibitors, eg, hemophilia A, hemophilia B, or hemophilia C. The method includes administering to a subject, e.g., subcutaneously administering, about 80 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and the 3'-end of the sense strand is linked to a nucleotide sequence as shown in the following schematic diagram: [ka] (wherein X is O or S). and administering to the subject a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced compared to the recommended effective amount of the replacement factor, thereby treating a bleeding event in a subject with hemophilia without inhibitors.

[0047] In another embodiment, the invention provides a method of treating a bleeding event in a subject with hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C, who has an inhibitor. The method includes administering to a subject, e.g., subcutaneously administering, about 80 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and the 3'-end of the sense strand is linked to a nucleotide sequence as shown in the following schematic diagram: [ka] (wherein X is O or S). and administering to the subject a therapeutically effective amount of a bypassing agent, wherein the 'effective amount of bypassing agent' is reduced compared to the recommended effective amount of the bypassing agent, thereby treating a bleeding event in a subject with hemophilia who has an inhibitor.

[0048] In one embodiment, a fixed dose of RNAi is administered subcutaneously to a subject.

[0049] In one embodiment, a step dose of RNAi is administered to a subject once a month.

[0050] Hemophilia is referred to as hemophilia A, hemophilia B, or hemophilia C. [Brief explanation of the drawings]

[0051] [Figure 1A] FIG. 1A is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg of AT3SC-001 on plasma thrombin formation levels in one healthy human subject. [Figure 1B] FIG. 1B is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg of AT3SC-001 on plasma thrombin formation levels in one healthy human subject. [Figure 1C] FIG. 1C is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg of AT3SC-001 on plasma thrombin formation levels in one healthy human subject. [Figure 1D] FIG. 1D is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg of AT3SC-001 on plasma thrombin formation levels in one healthy human subject. [Figure 2A] FIG. 2A is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg of AT3SC-001 on plasma AT (Serpincl) protein levels in one healthy human subject. [Figure 2B] FIG. 2B is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg of AT3SC-001 on plasma AT (Serpincl) protein levels in one healthy human subject. [Figure 3] FIG. 3 is a graph showing the association between the percentage of AT (Serpinc1) knockdown and the percentage increase in peak thrombin formation in healthy subjects receiving a single subcutaneous dose of 0.03 mg / kg AT3SC-001. [Figure 4] FIG. 4 is a graph showing the effect of multiple doses of 0.015 mg / kg, 0.045 mg / kg, or 0.075 mg / kg AT3SC-001 on plasma AT (Serpincl) protein levels in human subjects with hemophilia A or B. [Figure 5A]Figure 5A is a graph showing the effect of multiple doses of 0.225 mg / kg, 0.450 mg / kg, 0.900 mg / kg, 1.800 mg / kg, or 80 mg of AT3SC-001 on plasma AT (Serpincl) protein levels in human subjects with hemophilia A or B. [Figure 5B] FIG. 5B is a graph showing the dose-dependent effect of AT3SC-001 on plasma AT (Serpincl) protein levels in human subjects. [Figure 6A] FIG. 6A is a graph showing the effect of multiple doses of 0.015 mg / kg or 0.045 mg / kg AT3SC-001 on peak thrombin levels in human subjects with hemophilia A or B. [Figure 6B] FIG. 6B is a graph depicting the effect of multiple doses of 0.015 mg / kg or 0.045 mg / kg AT3SC-001 on thrombin generation in human subjects with hemophilia A or B as percent change relative to the baseline group. [Figure 7] FIG. 7 is a graph showing the effect of multiple doses of 0.045 mg / kg AT3SC-001 on clot formation time and clotting time in one subject with hemophilia A (subject 101-009). [Figure 8] FIG. 8 is a graph showing mean maximum AT reduction with monthly equivalent doses. [Figure 9] FIG. 9 is a graph showing the effect of multiple doses of AT3SC-001 on thrombin generation by quartile decline in AT. [Figure 10A] FIG. 10A is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by Factor VIII as measured in subjects administered 225 mcg / kg qM AT3SC-001. [Figure 10B] FIG. 10B is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by Factor VIII measured in subjects administered 1800 mcg / kg qM AT3SC-001. [Figure 10C]FIG. 10C is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, as measured in subjects administered 80 mg qM AT3SC-001. [Figure 11] FIG. 11 is a graph showing the effect of multiple doses of AT3SC-001 on bleeding events by quartile decline in AT. [Figure 12] FIG. 12 is a table showing bleeding event data for subjects enrolled in Part C of the Phase I clinical trial of AT3SC-001. [Figure 13A] FIG. 13A is a graph showing the median annualized bleeding rate (ABR) for all dosing cohorts in Part C of a Phase I clinical trial of AT3SC-001 before the start of the study, during the initiation portion of the study, and during the observational portion of the study. [Figure 13B] Figure 13B is a graph showing the median annualized bleeding rate (ABR) before the start of the study, during the initiation portion of the study, and during the observational portion of the study for the 80 mg once monthly (80 mg qM x 3) cohort of Part C of a Phase I clinical trial of AT3SC-001. [Figure 14A] FIG. 14A is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects receiving a fixed dose of 50 mg AT3SC-001 once monthly. [Figure 14B] FIG. 14B is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, as measured in inhibitor subjects receiving a fixed dose of 50 mg AT3SC-001 once monthly. [Figure 14C] FIG. 14C is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects receiving a fixed dose of 50 mg AT3SC-001 once monthly. [Figure 14D]FIG. 14D is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects receiving a fixed dose of 50 mg AT3SC-001 once monthly. [Figure 14E] FIG. 14E is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects receiving a fixed dose of 50 mg AT3SC-001 once monthly. [Figure 14F] FIG. 14F is a graph showing the relative AT activity associated with the percentage of peak thrombin formation achieved by factor VIII, as measured in inhibitor subjects receiving a fixed dose of 50 mg AT3SC-001 once monthly. [Figure 15] FIG. 15 is a graph showing the effect of multiple doses of 50 mg or 80 mg of AT3SC-001 on mean AT (Serpincl) activity relative to baseline in human subjects with hemophilia A or B with inhibitors. [Figure 16] FIG. 16 is a graph showing that the AT-lowering effect of multiple doses of 50 mg AT3SC-001 correlates with increased thrombin formation in hemophilia A subjects. [Figure 17A] FIG. 17A is a table showing bleeding event data for subjects enrolled in Part D of a Phase I clinical trial of AT3SC-001. [Figure 17B] FIG. 17B is a graph showing the median annualized bleeding rate (ABR) for all subjects in Part D of a Phase I clinical trial of AT3SC-001 before the start of the study, during the initiation portion of the study, and during the observational portion of the study. [Figure 18] Figure 18 is a graph showing the effect of multiple doses of 80 mg of AT3SC-001 on mean AT (Serpinc1) activity relative to baseline in human subjects with hemophilia without inhibitors in a Phase II open-label extension (OLE) study of AT3SC-001. [Figure 19A]Figure 19A is a graph showing the effect of multiple doses of 50 mg or 80 mg of AT3SC-001 on mean AT (Serpinc1) activity relative to baseline in human subjects with hemophilia A or B with or without inhibitors in a Phase II open-label extension (OLE) study of AT3SC-001. [Figure 19B] 19B is a graph showing the effect of multiple doses of 50 mg or 80 mg of AT3SC-001 on peak thrombin formation in human subjects with hemophilia A or B, with or without inhibitors, in a Phase II, open-label extension (OLE) study of AT3SC-001. The shaded area of ​​the graph represents the range of peak thrombin levels observed in healthy human volunteers (HV) administered AT3SC-001, indicating less than 25% AT knockdown in the Phase I study of AT3SC-001 described in Example 1. The dotted line within the HV range represents the median peak thrombin level observed in healthy human volunteers (HV), indicating less than 25% AT knockdown with administration of AT3SC-001 in the Phase I study of AT3SC-001 described in Example 1. [Figure 20A] 1 is a graph showing the median annualized bleeding rate (ABR) before study initiation, at the start of the study, and during the observation portion of the study for subjects with hemophilia A or B without inhibitors in a Phase II OLE clinical trial of AT3SC-001. [Figure 20B] 1 is a graph showing the median annualized bleeding rate (ABR) before study initiation and during the observational portion of the study for subjects with hemophilia A or B with inhibitors in a Phase II OLE clinical trial of AT3SC-001. [Figure 21] 1 is a table showing the characteristics of bleeding events experienced by patients with hemophilia A or B, with or without inhibitors, in the Phase II OLE clinical trial of AT3SC-001. [Figure 22] 1 is a table showing the management of bleeding events experienced by patients with hemophilia A or B without inhibitors in the Phase II OLE clinical trial of AT3SC-001. [Figure 23] 1 is a table showing the characteristics of bleeding events experienced by patients with hemophilia A or B and inhibitors in the Phase II OLE clinical trial of AT3SC-001. [Figure 24A] 1 is a graph showing the effect of a monthly fixed dose of AT3SC-001 50 mg or 80 mg on the amount of factor VIII needed to control bleeding in subjects with hemophilia A without inhibitors. [Figure 24B] 1 is a graph showing the effect of a monthly fixed dose of AT3SC-001 50 mg or 80 mg on the amount of factor IX needed to control bleeding in subjects with hemophilia B without inhibitors. [Figure 24C] 1 is a graph showing the effect of a monthly fixed dose of AT3SC-001 50 mg or 80 mg on the amount of rFVIIa needed to control bleeding in subjects with hemophilia A or B with inhibitors. [Figure 24D] 1 is a graph showing the effect of a monthly fixed dose of AT3SC-001 50 mg or 80 mg on the amount of aPCC needed to control bleeding in subjects with hemophilia A or B with inhibitors. [Figure 25A] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A with inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 25B] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A with inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26A] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26B]1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26C] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26D] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26E] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26F] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26G] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26H] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26I] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26J] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26K] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26L] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26M] 1 is a graph showing the effect of adding aPCC on thrombin formation in plasma samples from subjects with hemophilia A who do not have inhibitors, before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 26N] 1 is a graph showing the effect of adding rFVIIa on thrombin formation in plasma samples from subjects with hemophilia A without inhibitors before (bottom line) and after (top line) the subjects were administered AT3SC-001. [Figure 27A] 1 is a graph showing the effect of multiple doses of AT3SC-001 on thrombin generation by quartile decline of AT in Phase I / II open-label extension (OLE) clinical samples. [Figure 27B] 1 is a graph showing the simulated effect of multiple doses of AT3SC-001 on thrombin generation by quartile decline in AT. [Figure 27C] 27B is a scatter plot showing a strong correlation between simulated TG (FIG. 27B) and measured TG (FIG. 27A). [Figure 28A] FIG. 1 shows simulated in silico thrombin generation curves for various AT levels and 0.1% factor FVIII (simulating severe hemophilia A). [Figure 28B] 1 is a heat map representation of peak thrombin at various factor VIII doses (single dose) and AT levels for severe hemophilia A. [Figure 28C] 1 is a heat map representation of peak thrombin at various FIX doses (single dose) and AT levels for severe hemophilia B. [Figure 29A] 1 is a graph showing simulated peak thrombin potential (nM) as a function of time for 5, 10, 20, and 50 IU / kg of FVIII with AT at 100%. [Figure 29B] 1 is a graph showing simulated peak thrombin generation capacity (nM) as a function of time for 5, 10, and 20 IU / kg of FVIII with AT at 20% of baseline. DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention relates, at least in part, to the prevention of hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C) in a subject who is free of an inhibitor and who is administered a therapeutically effective amount of an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the Serpincl gene, as defined by, for example, the World Federation of Hemophilia (see, e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6, July 2012; DOI: 10.1111 / j.1365-2516.2012.02909.x; the contents of which are incorporated herein by reference in their entirety) and / or by the Food and Drug Administration (e.g., ADVATE (Antihemophilic Factor (Recombinant)) product insert; 11 / 2016; BeneFIX (Coagulation Factor IX (Recombinant ... (See insert; 11 / 2011; the contents of each of which are incorporated herein by reference in their entirety.) The present invention is based on the unexpected discovery that bleeding events can be treated with a therapeutically effective amount of a replacement factor, such as factor VIII or factor XI, that is less than the recommended effective amount of the replacement factor. Accordingly, the present invention provides methods for treating bleeding events in subjects with hemophilia who do not have inhibitors.The method includes administering to a subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a replacement factor, wherein the effective amount of the replacement factor is reduced compared to the recommended effective amount of the replacement factor, thereby treating a bleeding event in a subject with hemophilia without an inhibitor.

[0053] In another aspect, the present invention provides a method for treating a bleeding event in a subject with hemophilia without inhibitors. The method includes administering to the subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a replacement factor, wherein the effective amount of the replacement factor is reduced compared to the recommended effective amount of the replacement factor, thereby treating a bleeding event in a subject with hemophilia without inhibitors.

[0054] The present invention provides, at least in part, the prevention of hemophilia as defined by, for example, the World Federation of Hemophilia (see, e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6, July 2012; DOI: 10.1111 / j.1365-2516.2012.02909.x) and / or the Food and Drug Administration (see, e.g., NovoSeven RT, Coagulation Factor VIIA (Recombinant) product insert; 07 / 2014; FEIBA, Anti-Inhibitor ... (See, e.g., 11 / 2013, Complex product insert; the contents of each of which are incorporated herein by reference in their entireties.) The present invention is also based on the discovery that bleeding events can be treated with therapeutically effective amounts of bypassing agents, such as activated prothrombin complex concentrates (aPCC) or recombinant factor VIIa (rFVIIa), that are lower than the recommended effective amounts of the bypassing agents.

[0055] Therefore, in another aspect, the present invention provides a method for treating a bleeding event in a subject with hemophilia and an inhibitor, the method comprising: administering to the subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits the expression of Serpinc1, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a bypassing agent, the effective amount of which is reduced compared to the recommended effective amount of the bypassing agent, for example, approved by the Food and Drug Administration, thereby treating a bleeding event in a subject with hemophilia and an inhibitor.

[0056] In another aspect, the present invention provides a method for treating a bleeding event in a subject with hemophilia and an inhibitor, the method comprising: administering to the subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits the expression of Serpinc1, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; and administering to the subject a therapeutically effective amount of a bypassing agent, the effective amount of which is reduced compared to the recommended effective amount of the bypassing agent, for example, approved by the Food and Drug Administration, thereby treating a bleeding event in a subject with hemophilia and an inhibitor.

[0057] iRNA agents for use in the methods of the invention generally are about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, The RNA strand (antisense strand) comprises an RNA strand having a region of 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 Serpinc1 gene.

[0058] In other 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, or 27-53 nucleotides in length, and have a region of at least 19 contiguous nucleotides that are substantially complementary to at least a portion of an mRNA transcript of the Serpincl gene. In some embodiments, the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0059] In some embodiments, iRNA agents for use in the methods of the invention include an RNA strand (antisense strand) that can be up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides in length, and that has a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the Serpincl gene. In some embodiments, such iRNA agents with longer antisense strand lengths can also include 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.

[0060] The detailed description below discloses how to make and use compositions comprising iRNA to inhibit expression of the Serpincl gene, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from inhibiting and / or reducing expression of this gene.

[0061] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a parameter is recited, it is intended that values ​​and ranges intermediate to the recited values ​​are also part of the invention.

[0062] 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. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0063] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0064] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0065] As used herein, "Serpinc1" refers to a specific polypeptide expressed in cells. Serpinc1 is also known as serpin peptidase inhibitor, clade C (antithrombin; AT), member 1; antithrombin III; AT3; antithrombin; and heparin cofactor 1. The sequence of human Serpinc1 mRNA transcript can be found, for example, in GenBank Accession No. GI:254588059 (NM_000488; SEQ ID NO: 1). Rhesus monkey Serpinc1 m The sequence of the RNA can be found, for example, in GenBank accession number GI:157167169 (NM_001104583; SEQ ID NO:2). The sequence of mouse Serpinc1 mRNA can be found, for example, in GenBank accession number GI:237874216 (NM_080844; SEQ ID NO:3). The sequence of rat Serpinc1 mRNA can be found, for example, in GenBank accession number GI:58865629 (NM_001012027; SEQ ID NO:4).

[0066] The term "Serpincl" as used herein also refers to the specific polypeptide expressed in cells due to the naturally occurring DNA sequence variation of the Serpincl gene, such as the single nucleotide polymorphism of the Serpincl gene.Several SNPs in the Serpincl gene have been identified, and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).Non-limiting examples of SNPs in the Serpincl gene can be found under NCBI dbSNP accession numbers rs677;rs5877;rs5878;rs5879;rs941988;rs941989;rs1799876;rs19637711;rs2008946;and rs2227586.

[0067] As used herein, a "subject" is an animal such as a mammal, including a primate (such as a human or a non-human primate, e.g., a monkey or chimpanzee), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, and whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition described herein that would benefit from reduced Serpincl expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced Serpincl expression; a human being with a disease, disorder, or condition that would benefit from reduced Serpincl expression; and / or a human being treated for a disease, disorder, or condition that would benefit from reduced Serpincl expression.

[0068] As used herein, the terms "treating" or "treatment" refer to beneficial or desired results, including, but not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the severity of bleeding, stabilization of bleeding (i.e., not worsening), improvement or reduction of bleeding, or cessation of bleeding. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. In the methods of the present invention, treatment includes on-demand treatment and control, perioperative management of bleeding, and routine prophylaxis to reduce the frequency of bleeding episodes.

[0069] The term "reducing" in relation to the level of Serpincl or a disease marker or symptom in a subject refers to a statistically significant decrease in such level, which may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, preferably to a level that is accepted as being within the normal range for individuals without such disorder.

[0070] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of the Serpincl gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with the disease, disorder, or condition, such as bleeding, or other symptoms. For example, reducing one or more risk factors for bleeding. The likelihood of bleeding is reduced when a child has no bleeding or experiences bleeding with less severity compared to a population of children with the same risk factors but not treated as described herein. Effective prevention is considered to be a failure to develop a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a disease, disorder, or condition (e.g., by at least about 10% on a clinically recognized scale for the disease or disorder), or a delay in symptoms (e.g., by days, weeks, months, or years).

[0071] As used herein, the term "bleeding disorder" refers to a disease or disorder that results in poor blood clotting and / or excessive bleeding. A bleeding disorder may be an inherited disorder, such as hemophilia or von Willebrand's disease, or an acquired disorder, such as disseminated intravascular coagulation, pregnancy-associated eclampsia, vitamin K deficiency, autoimmune disorders, inflammatory bowel disease, ulcerative colitis, skin disorders (e.g., psoriasis, pemphigus), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease), allergic drug reactions (e.g., as a result of medications such as aspirin, heparin, and warfarin), diabetes, acute hepatitis B infection, acute hepatitis C infection, malignant tumors or solid tumors (e.g., prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervical, breast, melanoma, kidney, and / or hematologic malignancies). In one embodiment, the inherited bleeding disorder is hemophilia, e.g., hemophilia A, B, or C. In one embodiment, a subject with an inherited bleeding disorder, e.g., hemophilia, becomes afflicted with an inhibitor, e.g., an alloantibody inhibitor, to replacement coagulation therapy and is referred to herein as an "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.

[0072] In one embodiment, the bleeding disorder is a rare bleeding disorder (RBD). RBD may be acquired or inherited. Genetic RBDs include disorders involving deficiencies of the clotting factors fibrinogen, FII, FV, combined FV and FVIII, FVII, FX, FXI, and FXIII, as well as congenital deficiency of vitamin K-dependent factors (VKCFD). They are generally transmitted as autosomal recessive conditions, but in some cases, such as FXI and dysfibrinogenemia, they can be autosomal dominant. RBDs are reported in most populations, with homozygous or double heterozygous incidence ranging from 1 in 500,000 for FVII deficiency to 1 in 2 to 3 million for prothrombin and FXIII deficiencies. Relative frequencies vary across populations and are higher when consanguinity or intramarriage is common, increasing the frequency of specific mutant alleles.

[0073] Exemplary RBDs include afibrinogenemia (fibrinogen; factor I deficiency); hypofibrinogenemia (fibrinogen; factor I deficiency); dysfibrinogenemia (fibrinogen; factor I deficiency); dysfunctional fibrinogenemia (fibrinogen; factor I deficiency); hypoprothrombinemia (prothrombin; factor II deficiency); prothrombin deficiency (prothrombin; factor II deficiency); thrombophilia (prothrombin; factor II deficiency); congenital antithrombin III deficiency (thromboplastin; factor III; tissue factor); parahemophilia (proaccelerin; factor V; labile factor); O'Hall disease ( Activated protein C resistance (proaccelerin; factor V; labile factor); Alexander disease (stable factor proconvertin; factor VII); congenital proconvertin / factor VII deficiency (stable factor proconvertin; factor VII); Stuart-Prower deficiency (Stuart-Prower factor; factor X); congenital factor XIIIa / b deficiency (fibrin stabilizing factor; factor XIII); hereditary factor XIII deficiency (fibrin stabilizing factor; factor XIII); and fibrin stabilizing factor deficiency (fibrin stabilizing factor; factor XIII).

[0074] A "therapeutically effective amount," as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject with a bleeding disorder and bleeding, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0075] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject with a bleeding disorder but who is not bleeding, e.g., a subject with a bleeding disorder and who is planned for surgery (e.g., perioperative treatment). Ameliorating a disease includes slowing the course of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the iRNA, how the agent is administered, the degree of risk for the disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other individual characteristics of the patient being treated.

[0076] A "therapeutically effective amount" or "prophylactically effective amount" also includes the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio for any treatment. The iRNAs utilized in the methods of the invention can be administered in amounts sufficient to produce a reasonable benefit / risk ratio appropriate for such treatment.

[0077] The "Recommended Therapeutically Effective Amounts of Replacement Factors" and "Recommended Therapeutic Effective Amounts of Bypassing Agents" are recommended by the World Federation of Hemophilia (e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6, July 2012; DOI: 10.1111 / j.1365-2516.2012.02909.x; ADVATE (Antihemophilic and BeneFIX (Coagulation Factor IX (Recombinant) product insert; 11 / 2016; see BeneFIX; and BeneFIX (Coagulation Factor IX (Recombinant) product insert; 11 / 2011) are doses of replacement factor or bypassing agent, respectively, sufficient to form thrombin and eliminate bleeding and / or achieve peak plasma factor levels in a bleeding subject. Each of the foregoing applications is incorporated herein by reference in its entirety.

[0078] For example, for subjects with minor bleeding, the recommended dose of a replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor VIII level of about 10-40 IU / dL; for subjects with moderate bleeding, the recommended dose of a replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor VIII level of about 30-60 IU / dL; for subjects with major bleeding, the recommended dose of a replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor VIII level of about 60-100 IU / dL; and for subjects undergoing surgery, the recommended dose of a replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor VIII level of about 30-60 IU / dL (see, e.g., Tables 1 and 2 of the ADVATE (Antihemophilic Factor (Recombinant)) product insert; 11 / 2016).

[0079] For subjects with minor bleeding, the recommended dose of replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor IX level of about 10-30 IU / dL; for subjects with moderate bleeding, the recommended dose of replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor IX level of about 25-50 IU / dL; for subjects with major bleeding, the recommended dose of replacement factor or bypassing agent is a dose sufficient to achieve a peak plasma factor IX level of about 25-50 IU / dL. The recommended dose of replacement factor or bypassing agent for a subject is a dose sufficient to achieve a peak plasma factor IX level of about 50-100 IU / dL.

[0080] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human and animal subjects without undue toxicity, irritation, allergic response, or other problem or impairment, commensurate with a reasonable benefit / risk ratio.

[0081] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc, or stearic acid), or solvent that contains materials that participate in the transport or transportation of a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol, propylene glycol, propylene glycol stearate ... (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (24) other non-toxic, compatible substances utilized in pharmaceutical formulations.

[0082] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpincl gene, including mRNA that is the product of RNA processing of the primary transcription product. In one embodiment, the target portion of the sequence will be long enough to serve as a substrate for iRNA-induced cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the Serpincl gene.

[0083] The target sequence can be about 9 to 36 nucleotides in length, e.g., about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 30 ... The length can be 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. Ranges and lengths intermediate to the above-listed ranges and lengths are also part of the invention. Something is intended to be.

[0084] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence referenced using standard nucleotide nomenclature.

[0085] "G," "C," "A," "T," and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine, and uracil, respectively, as bases. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, as further detailed 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 substituted with other moieties without substantially changing the base pairing properties of an oligonucleotide containing a nucleotide with such a replacement moiety. For example, without limitation, a nucleotide containing inosine as a base can form base pairs with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine in the nucleotide sequence of a dsRNA featured in the present invention can be substituted with, for example, a nucleotide containing inosine. In another example, any adenine and cytosine in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with a target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the present invention.

[0086] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," as used interchangeably herein, refer to agents containing RNA that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as those terms are defined herein. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, the expression of Serpincl in cells, e.g., cells within a subject, such as a mammalian subject.

[0087] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a Serpincl target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, a RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs 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 unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). When it binds to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir et al., (2001) Genes Dev.15:188). Thus, in one aspect, the present invention relates to the single-stranded RNA (siRNA) formed in cells, which promotes the formation of the RISC complex, causing the silencing of the target gene, i.e., the Serpincl gene. Therefore, the term "siRNA" is used herein to also refer to RNAi as described above.

[0088] In another embodiment, the RNAi agent is administered to a cell or organism to inhibit a target mRNA. The single-stranded siRNA may be introduced. The single-stranded RNAi agent binds to 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 siRNA 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 antisense nucleotide sequence described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA chemically modified by the method described in Lima et al., (2012) Cell 150;:883-894.

[0089] In another embodiment, the "iRNA" for use in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "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" and "antisense" orientations relative to the target RNA, i.e., the Serpincl gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of target RNA, for example, mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0090] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, "RNAi agent" can also include ribonucleotides with chemical modifications; RNAi agents can also contain significant modifications in multiple nucleotides.

[0091] As used herein, the term "modified nucleotide" refers to a nucleotide that has independently modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.Therefore, the term modified nucleotide includes, for example, the substitution, addition, or removal of functional group or atom to internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present invention include any type of modification disclosed herein or known in the art.For the purpose of this specification and claims, any such modification used in siRNA type molecules is included in "RNAi agent".

[0092] The duplex region can be of any length that allows for specific degradation of the desired target RNA by the RISC pathway, and can be about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 2 The length may be in the range of 0-30, 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. Intermediate ranges and lengths within the above-listed ranges and lengths are also contemplated by the present invention. It is intended to be part of the light.

[0093] The two strands forming a duplex structure may be different parts of a larger single RNA molecule, or they may be separate RNA molecules. When the two strands are part of a larger single molecule and are therefore linked by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand that form the duplex structure, the linked RNA strands are referred to as a "hairpin loop." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides.

[0094] When the two substantially complementary strands of dsRNA are contained by separate RNA molecules, these molecules do not need to be covalently linked, but can be.When the two strands are covalently linked by anything other than a continuous chain of nucleotides between the 3'-end of one strand and the 5'-end of the other strand that forms a double-stranded structure, the linking structure is called a "linker".The RNA strands can have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure.In addition to the double-stranded structure, RNAi can also contain one or more nucleotide overhangs.

[0095] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a Serpincl target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs 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 unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0096] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. 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 can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang can be present at the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0097] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end and / or 5'-end. In another embodiment, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0098] In certain embodiments, the overhang on the sense strand or the antisense strand, or on both strands, can have an extended length of more than 10 nucleotides, e.g., 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is at the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the extended overhang are substituted with a nucleoside thiophosphate.

[0099] " Blunt" or " blunt end " means that there is no unpaired nucleotide at the end of double-stranded RNAi agent, that is, there is no nucleotide overhang. " Blunt-ended " RNAi agent is double-stranded throughout its entire length, that is, there is no nucleotide overhang at either end of the molecule. The RNAi agent of the present invention includes the RNAi agent that has nucleotide overhang at one end (that is, the agent that has one overhang and one blunt end) or the RNAi agent that has nucleotide overhang at both ends.

[0100] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that contains a region that is substantially complementary to a target sequence, e.g., Serpinc1 mRNA. As used herein, the term "region of complementarity" refers to a region in the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a Serpinc1 nucleotide sequence as defined herein. If the region of complementarity is not completely complementary to the target sequence, there may be mismatches in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the iRNA.

[0101] 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 that term is defined herein.

[0102] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site of the target where cleavage occurs. In some embodiments, the cleavage region includes either end of the cleavage site and the three bases immediately adjacent thereto. In some embodiments, the cleavage region includes either end of the cleavage site and the two bases immediately adjacent thereto. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region includes nucleotides 11, 12, and 13.

[0103] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence with respect to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under specific conditions, as would be understood by one of skill in the art. Such conditions can be, for example, stringent conditions, which can include the following: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by a wash (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may apply, such as physiologically relevant conditions that may be encountered inside an organism. One of skill in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0104] A complementary sequence within an iRNA, such as a dsRNA described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both of the nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or 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 retaining the ability to hybridize under conditions most relevant to their end use, such as inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for purposes described herein.

[0105] As used herein, a "complementary" sequence may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0106] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein to refer to base matches between the sense and antisense strands of a dsRNA or between the antisense strand of an iRNA agent and a target sequence, as will be understood in the context of their use.

[0107] As used herein, a polynucleotide "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 Serpinc1). For example, if a sequence is substantially complementary to a contiguous portion of an mRNA encoding Serpinc1, then the polynucleotide is complementary to at least a portion of the Serpinc1 mRNA. be.

[0108] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target Serpincl sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target Serpincl sequence, and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the corresponding region of the nucleotide sequence of SEQ ID NO: 1, or a fragment of SEQ ID NO: 1, over its entire length.

[0109] In one embodiment, the RNAi agent of the present invention comprises a sense strand that is substantially complementary to the antisense polynucleotide and thus complementary to the target Serpincl sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the corresponding region of the nucleotide sequence of SEQ ID NO: 5, or any one fragment of SEQ ID NO: 5, over its entire length.

[0110] In one embodiment of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense RNA molecule that inhibits target mRNA by an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner 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 RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from any one of the antisense sequences described herein.

[0111] The term "inhibiting," as used herein, is used synonymously with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes all levels of inhibition.

[0112] The phrase "inhibiting the expression of Serpinc1," as used herein, includes the inhibition of the expression of any Serpinc1 gene encoding a Serpinc1 protein (e.g., mouse Serpinc1 gene, rat Serpinc1 gene, monkey Serpinc1 gene, or human Serpinc1 gene, etc.), as well as Serpinc1 gene variants or mutants.

[0113] "Inhibiting expression of the Serpinc1 gene" includes any level of inhibition of the Serpinc1 gene, such as at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition, e.g., at least partial suppression of expression of the Serpinc1 gene.

[0114] Serpinc1 gene expression can be assessed based on the level of any variable associated with Serpinc1 gene expression, such as Serpinc1 mRNA level, Serpinc1 protein level, or thrombin:antithrombin complex level, for example, as a measure of thrombin formation capacity, bleeding time, prothrombin time (PT), platelet count, and / or activated partial thromboplastin time (aPTT). Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as a baseline level before administration, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive drug control).

[0115] In one embodiment, at least partial suppression of Serpincl gene expression is assessed by a decrease in the amount of Serpincl mRNA that can be isolated from or detected in a first cell or group of cells that transcribes the Serpincl gene and that has been treated to inhibit Serpincl gene expression, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been treated to inhibit Serpincl gene expression (control cells). The degree of inhibition can be expressed in terms of:

[0116]

number

[0117] The phrase "contacting cells with RNAi agents" such as dsRNA as used herein includes contacting cells by any possible means.Contacting cells with RNAi agents includes contacting cells with iRNA in vitro or contacting cells with iRNA in vivo.Contacting can be performed directly or indirectly.Therefore, for example, RNAi agents can be physically contacted with cells by performing individual methods, or RNAi agents can be placed in a situation that allows or will allow them to contact cells later.

[0118] Contacting cells in vitro can be carried out, for example, by incubating cells with RNAi agent.Contacting cells in vivo can be carried out, for example, by injecting RNAi agent into the tissue where cells are or nearby, or by injecting RNAi agent into another area, for example, bloodstream or subcutaneous space, so that RNAi agent will then reach the tissue where cells are contacted.For example, RNAi agent can comprise and / or be bound to a ligand, for example, GalNAc3, that guides RNAi agent to target site, for example, liver.Combined contacting methods in vitro and in vivo are also possible.For example, cell can be contacted with RNAi agent in vitro, and then transplanted into subject.

[0119] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA to a cell" by facilitating or causing uptake or absorption into the cell. Absorption or uptake of the iRNA can occur by unassisted diffusion processes, active intracellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or can be administered systemically. In vivo delivery is described herein by reference in its entirety. Beta-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Patent Application Publication No. 2005 / 0281781, are incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below and / or known in the art.

[0120] II. The Methods of the Invention The invention provides for the combination of an iRNA agent, or a pharmaceutical composition comprising an iRNA agent of the invention, in an amount that reduces Serpincl activity in a subject, e.g., by about 75% or more, and a medicament as approved by, e.g., the World Federation of Hemophilia (see, e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6, July 2012; DOI: 10.1111 / j.1365-2516.2012.02909.x) and / or the Food and Drug Administration (see, e.g., ADVATE (Antihemophilic Factor (Recombinant)) product insert; 11 / 2016; BeneFIX (Coagulation Factor IX (Recombinant ...

[0010] Provided is a therapeutic method for treating a bleeding event in a subject with hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C), comprising administering to the subject a therapeutically effective amount of a replacement factor or bypassing agent that is reduced compared to the recommended therapeutically effective amount of the replacement factor or bypassing agent (see insert; 11 / 2011). The foregoing is incorporated herein by reference in its entirety.

[0121] As described in the Examples below, it has been surprisingly discovered that in subjects with hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C, with or without inhibitors, administration of an RNAi agent that inhibits expression of Serpinc1 in an amount that reduces Serpinc1 activity in the subject by about 75% or more reduces the median annualized bleeding rate and spontaneous annualized bleeding rate, and the bleeding can be managed (thrombin is formed and bleeding stops) with a therapeutically effective amount of a replacement factor or bypassing agent that is reduced compared to the recommended therapeutically effective amount of the replacement factor or bypassing agent.

[0122] Suitable replacement factors for use in the methods of the invention include factor VIII, such as Advate, Eloctate, Haemate, Helixate, Immunate, Octanate, Recombinate, and Refacto, or factor IX, such as Aimafix, Benefix, Immunine, and Refacto. Suitable bypassing agents for use in the methods of the invention include activated prothrombin complex concentrates (aPCC), such as FEIBA and Prothromplex, and recombinant factor VIIa (rFVIIa), such as NovoSeven.

[0123] The replacement factor can be factor VIII, and a therapeutically effective amount of the replacement factor administered to a subject in the methods of the invention is a dose sufficient to achieve a peak plasma factor VIII level of about 10-100 IU / dL, e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 IU / dL.

[0124] For example, a therapeutically effective amount of replacement factor VIII administered to a subject may be less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg. The therapeutically effective amount of factor VIII administered to a subject may be less than about 80 IU / kg, less than about 70 IU / kg, less than about 60 IU / kg, less than about 50 IU / kg, less than about 40 IU / kg, less than about 30 IU / kg, less than about 20 IU / kg, or less than about 10 IU / kg. In one embodiment, the therapeutically effective amount of factor VIII administered to a subject is about two-thirds to about one-fifth of the recommended effective amount of replacement factor, for example, a dose of about 5 to about 20 IU / kg, or about 10 to about 20 IU / kg, for example, 5, 10, 15, or 20 IU / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0125] The replacement factor can be factor IX, and a therapeutically effective amount of the replacement factor administered to a subject in the methods of the invention is a dose sufficient to achieve a peak plasma factor IX level of about 10-100 IU / dL, e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 IU / dL.

[0126] For example, a therapeutically effective amount of replacement factor IX can be less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, or less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg. In some embodiments, the therapeutically effective amount of Factor IX administered to a subject is about half to about one-sixth of the recommended therapeutically effective amount of replacement factor, for example, about 10 to about 30 IU / kg or about 20 to about 30 IU / kg, for example, about 10, 15, 20, 25, or 30 IU / kg. In some embodiments, the bleeding event is moderate bleeding. In other embodiments, the bleeding event is severe bleeding.

[0127] The bypassing agent can be an aPCC, and the therapeutically effective amount of the bypassing agent administered to a subject in the methods of the invention is a dose sufficient to form thrombin and stop bleeding.

[0128] For example, the therapeutically effective amount of the bypassing agent aPCC can be less than about 100 U / kg, or less than about 90 U / kg, or less than about 80 U / kg, or less than about 70 U / kg, or less than about 60 U / kg, or less than about 50 U / kg, or less than about 40 U / kg, or less than about 30 U / kg, or less than about 20 U / kg, or less than about 10 U / kg. In one embodiment, the therapeutically effective amount of aPCC administered to a subject is about half to about one third of the recommended effective amount of replacement factor, for example, a dose of about 30 to about 50 U / kg, for example, about 30, 35, 40, 45, or 50 U / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0129] The bypassing agent can be rFVIIa, and the therapeutically effective amount of the bypassing agent administered to a subject in the methods of the invention is a dose sufficient to form thrombin and stop bleeding.

[0130] For example, the therapeutically effective amount of the bypassing agent rFVIIa can be less than about 120 μg / kg, or less than about 110 μg / kg, or less than about 100 μg / kg, or less than about 90 μg / kg, or less than about 80 μg / kg, or less than about 70 μg / kg, or less than about 60 μg / kg, or less than about 50 μg / kg, or less than about 40 μg / kg, or less than about 30 μg / kg, or less than about 20 μg / kg. In one embodiment, the therapeutically effective amount of rFVIIa administered to a subject is about half the recommended effective amount of the replacement factor. , for example, a dose of about 45 μg / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a major bleeding event.

[0131] In some embodiments, the RNAi agent is between about 25 mg and about 100 mg, e.g., between about 25 mg and about 95 mg, between about 25 mg and about 90 mg, between about 25 mg and about 85 mg, between about 25 mg and about 80 mg, between about 25 mg and about 75 mg, between about 25 mg and about 70 mg, between about 25 mg and about 65 mg, between about 25 mg and about 60 mg, between about 25 mg and about 50 mg, between about 50 mg and about 100 mg, between about 50 mg and about 95 mg, between about 50 mg and about 90 mg, between about 50 mg and about 85 mg, between about 50 mg and about 80 mg, between about 30 mg and about 100 mg, between about 30 mg and about 90 mg, between about 30 mg and about 8 and about 40 mg to about 100 mg, about 40 mg to about 90 mg, about 40 mg to about 80 mg, about 60 mg to about 100 mg, about 60 mg to about 90 mg, about 25 mg to about 55 mg, about 25 mg to about 65 mg, about 30 mg to about 95 mg, about 30 mg to about 85 mg, about 30 mg to about 75 mg, about 30 mg to about 65 mg, about 30 mg to about 55 mg, about 40 mg to about 95 mg, about 40 mg to about 85 mg, about 40 mg to about 75 mg, about 40 mg to about 65 mg, about 40 mg to about 55 mg, or about 45 mg to about 95 mg.

[0132] In some embodiments, the RNAi agent is administered at a fixed dose of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg.

[0133] In one embodiment, the RNAi agent is administered to the subject at a dose that reduces Serpinc1 activity by about 75% or more.

[0134] Thus, in one embodiment, the present invention provides a method for treating a bleeding event in a subject with hemophilia who has an inhibitor, comprising administering to the subject about 30 mg to about 90 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1 (e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand comprises a 3'- and administering to the subject a therapeutically effective amount of the replacement factor, the effective amount of which is reduced compared to the recommended effective amount of the replacement factor (e.g., an amount sufficient to achieve a peak plasma factor VIII level of about 10-100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor VIII, e.g., a dose of about 5 to about 20 IU / kg of factor VIII); or an amount sufficient to achieve a peak plasma factor IX level of about 10-100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor IX, e.g., a dose of about 10 to about 30 IU / kg of factor IX)), thereby treating a bleeding event in a subject with hemophilia who does not have an inhibitor.

[0135] In another aspect, the present invention provides a method for treating a bleeding event in a subject with hemophilia who has an inhibitor, comprising administering to the subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl (e.g., an amount that reduces Serpincl activity in the subject by about 75% or more). The NAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpincl comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-terminus. and administering to the subject a therapeutically effective amount of a bypassing agent, wherein the effective amount of the bypassing agent is reduced compared to the recommended effective amount of the bypassing agent (e.g., an amount sufficient to form thrombin and stop bleeding, e.g., a dose of less than about 100 U / kg of aPCC (e.g., a dose of about 30 to 50 U / kg of aPCC); a dose of less than about 120 μg / kg of rFVIIa (e.g., a dose of about 45 μg / kg of rFVIIa)), thereby treating bleeding events in a subject with hemophilia who has an inhibitor.

[0136] In another aspect, the present invention provides a method for treating a bleeding event in a subject with hemophilia without an inhibitor, comprising administering to the subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1 (e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1 that comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand comprises a 3'- and administering to the subject a therapeutically effective amount of the replacement factor, the effective amount of which is reduced compared to the recommended effective amount of the replacement factor (e.g., an amount sufficient to achieve a peak plasma factor VIII level of about 10-100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor VIII, e.g., a dose of about 5 to about 20 IU / kg of factor VIII); or an amount sufficient to achieve a peak plasma factor IX level of about 10-100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor IX, e.g., a dose of about 10 to about 30 IU / kg of factor IX)), thereby treating a bleeding event in a subject with hemophilia who does not have an inhibitor.

[0137] In another aspect, the present invention provides a method for treating a bleeding event in a subject with hemophilia who has an inhibitor, comprising administering to the subject about 40 mg to about 90 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1 (e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to an mRNA encoding Serpinc1 that comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), and wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand differ by no more than 3 nucleotides. The method includes the steps of: all nucleotides are modified nucleotides, and the sense strand is conjugated to a ligand attached at the 3'-end; and administering to the subject a therapeutically effective amount of a bypassing agent, wherein the effective amount of the bypassing agent is reduced compared to the recommended effective amount of the bypassing agent (e.g., an amount sufficient to form thrombin and stop bleeding, e.g., a dose of less than about 100 U / kg of aPCC (e.g., a dose of about 30 to 50 U / kg of aPCC); a dose of less than about 120 μg / kg of rFVIIa (e.g., a dose of about 45 μg / kg of rFVIIa)), thereby treating bleeding events in a subject with hemophilia who has an inhibitor.

[0138] In one aspect, the present invention provides a method for treating hemophilia without inhibitors, such as hemophilia A, hemophilia B, or a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1 suitable for administration to a subject at a fixed dose of about 30 mg to 90 mg (e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more), for use in a method for treating a bleeding event in a subject with hemophilia C; and a replacement factor suitable for administration to a subject at a reduced dose compared to the recommended effective amount of the replacement factor (e.g., an amount sufficient to achieve a peak plasma factor VIII level of about 10 to 100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor VIII, e.g., a dose of about 5 to about 20 IU / kg of factor VIII); or an amount sufficient to achieve a peak plasma factor IX level of about 10 to 100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor IX, e.g., a dose of about 10 to about 30 IU / kg of factor IX)). The RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end.

[0139] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl suitable for administration to a subject at a fixed dose of about 30 mg to 90 mg (e.g., an amount that reduces Serpincl activity in the subject by about 75% or more) for use in a method of treating a bleeding event in a subject with hemophilia who has an inhibitor, e.g., hemophilia A, hemophilia B, or hemophilia C; and a bypassing agent suitable for administration to a subject at a reduced dose compared to the recommended effective amount of the bypassing agent (e.g., an amount sufficient to form thrombin and stop bleeding, e.g., a dose of less than about 100 IU / kg of aPCC (e.g., a dose of about 30 to 50 U / kg of aPCC); or a dose of less than about 120 μg / kg of rFVIIa (e.g., a dose of about 45 μg / kg of rFVIIa)). The RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end.

[0140] In one aspect, the invention provides a method for treating a bleeding event in a subject with hemophilia without an inhibitor, e.g., hemophilia A, hemophilia B, or hemophilia C, comprising administering to the subject a fixed dose of about 40 mg to 90 mg (e.g., an amount that reduces Serpincl activity in the subject by about 75% or more); and a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl suitable for administration to the subject at a reduced dose (e.g., about 10-100 mg) relative to the recommended effective amount of a replacement factor. and a replacement factor suitable for administration to a subject in an amount sufficient to achieve a peak plasma factor VIII level of about 10 to 100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor VIII, e.g., a dose of about 5 to about 20 IU / kg of factor VIII); or in an amount sufficient to achieve a peak plasma factor IX level of about 10 to 100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor IX, e.g., a dose of about 10 to about 30 IU / kg of factor IX). The RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpincl, the region comprising at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-terminus.

[0141] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl suitable for administration to a subject at a fixed dose of about 40 mg to 90 mg (e.g., an amount that reduces the subject's Serpincl activity by about 75% or more) for use in a method of treating a bleeding event in a subject with hemophilia who has an inhibitor, e.g., hemophilia A, hemophilia B, or hemophilia C; and a bypassing agent suitable for administration to a subject at a reduced dose compared to the recommended effective amount of the bypassing agent (e.g., an amount sufficient to form thrombin and stop bleeding, e.g., a dose of less than about 100 IU / kg of aPCC (e.g., a dose of about 30 to 50 U / kg of aPCC); or a dose of less than about 120 μg / kg of rFVIIa (e.g., a dose of about 45 μg / kg of rFVIIa)). The RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end.

[0142] In the above methods and uses, in one embodiment the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0143] In one embodiment, the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence of 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0144] In one embodiment, the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

[0145] In one embodiment, the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and the sense strand is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0146] In one embodiment, the agent is administered as a pharmaceutical composition, hi one embodiment, the RNAi agent is administered in a non-buffered solution, such as saline or water.

[0147] In another embodiment, the RNAi agent is administered with a buffer, such as a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[0148] In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl suitable for administration to a subject at a fixed dose of about 80 mg (e.g., an amount that reduces Serpincl activity in the subject by about 75% or more), for use in a method of treating a bleeding event in a subject with hemophilia without an inhibitor, e.g., hemophilia A, hemophilia B, or hemophilia C; and a replacement factor suitable for administration to a subject at a reduced dose relative to the recommended effective amount of the replacement factor (e.g., an amount sufficient to achieve a peak plasma factor VIII level of about 10-100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor VIII, e.g., a dose of about 5 to about 20 IU / kg of factor VIII); or an amount sufficient to achieve a peak plasma factor IX level of about 10-100 IU / dL (e.g., a dose of less than about 200 IU / kg of factor IX, e.g., a dose of about 10 to about 30 IU / kg of factor IX)). The RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and the 3'-end of the sense strand is linked to the following schematic diagram: [ka] (wherein X is O or S). It is conjugated to a ligand as shown in FIG.

[0149] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpinc1 suitable for administration to a subject at a fixed dose of about 80 mg (e.g., an amount that reduces the subject's Serpinc1 activity by about 75% or more), for use in a method of treating a bleeding event in a subject with hemophilia who has an inhibitor, e.g., hemophilia A, hemophilia B, or hemophilia C; and a bypassing agent suitable for administration to a subject at a reduced dose compared to the recommended effective amount of the bypassing agent (e.g., an amount sufficient to form thrombin and stop bleeding, e.g., a dose of less than about 100 U / kg of aPCC (e.g., a dose of about 30 to 50 U / kg of aPCC); or a dose of less than about 120 μg / kg of rFVIIa (e.g., a dose of about 45 μg / kg of rFVIIa)). The RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and the 3'-end of the sense strand is linked to the following schematic diagram: [ka] (wherein X is O or S). It is conjugated to a ligand as shown in FIG.

[0150] In one embodiment, the fixed dose of the RNAi agent is suitable for subcutaneous administration.

[0151] In one embodiment, the fixed dose of the RNAi agent is suitable for administration to a subject once a month.

[0152] The present invention also provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression, e.g., a bleeding disorder, e.g., hemophilia. The method includes administering to the subject a prophylactically effective amount of an iRNA agent, e.g., a dsRNA (e.g., a pharmaceutical composition comprising a dsRNA of the present invention), e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg, thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the method includes administering to the subject a prophylactically effective dose, e.g., a fixed dose of about 50 mg, of an iRNA agent, e.g., a dsRNA (e.g., a pharmaceutical composition comprising a dsRNA of the present invention), thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced Serpinc1 expression. In another embodiment, the method includes administering to a subject a prophylactically effective dose, e.g., a fixed dose of about 80 mg, of an iRNA agent, e.g., a dsRNA, of the invention (e.g., a pharmaceutical composition comprising a dsRNA of the invention), thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0153] In another aspect, the invention provides a method of treating a subject with a disorder that would benefit from reduced Serpinc1 expression, e.g., a bleeding disorder, e.g., hemophilia, comprising administering to a subject, e.g., a human, a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg, of an iRNA agent targeting the Serpinc1 gene or a pharmaceutical composition comprising an iRNA agent targeting the Serpinc1 gene, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the method comprises administering to the subject a therapeutically effective dose, e.g., a fixed dose of about 50 mg, of an iRNA agent, e.g., a dsRNA of the invention (e.g., a pharmaceutical composition comprising a dsRNA of the invention), thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression. In another embodiment, the method includes administering a therapeutically effective dose, e.g., a fixed dose of about 80 mg, of an iRNA agent, e.g., a dsRNA, of the invention (e.g., a pharmaceutical composition comprising a dsRNA of the invention) to the subject, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0154] In another aspect, the present invention provides the use of an iRNA, e.g., a dsRNA, of the present invention at a prophylactically effective dose, e.g., an amount that reduces Serpincl activity in a subject by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg, to prevent at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibited Serpincl expression, such as hemophilia. In one embodiment, the present invention provides the use of an iRNA, e.g., a dsRNA, of the present invention at a prophylactically effective dose, e.g., a fixed dose of about 50 mg, to prevent at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibited Serpincl expression, such as hemophilia. In another embodiment, the present invention provides the use of an iRNA, e.g., a dsRNA, of the present invention at a prophylactically effective dose, e.g., a fixed dose of about 80 mg, to prevent at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibited Serpincl expression, such as hemophilia.

[0155] In a further aspect, the present invention provides a prophylactically effective dose, e.g., a therapeutically effective amount of Serpincl in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibited Serpincl expression, such as hemophilia. The present invention provides for the use of an iRNA agent of the present invention in an amount that reduces inc1 activity by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg. In one embodiment, the present invention provides for the use of an iRNA agent of the present invention in a prophylactically effective dose, e.g., a fixed dose of about 50 mg, in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a bleeding disorder, e.g., hemophilia, that would benefit from reduced and / or inhibited Serpinc1 expression. In another embodiment, the present invention provides for the use of an iRNA agent of the present invention in a prophylactically effective dose, e.g., a fixed dose of about 80 mg, in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a bleeding disorder, e.g., hemophilia, that would benefit from reduced and / or inhibited Serpinc1 expression.

[0156] In another aspect, the invention provides for the use of an iRNA agent of the invention at a therapeutically effective dose, e.g., an amount that reduces Serpincl activity in a subject by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg, to treat a subject, e.g., a subject that would benefit from reduced and / or inhibited Serpincl expression. In one embodiment, the invention provides for the use of an iRNA agent of the invention at a therapeutically effective dose, e.g., a fixed dose of about 50 mg, to treat a subject, e.g., a subject that would benefit from reduced and / or inhibited Serpincl expression. In another embodiment, the invention provides for the use of an iRNA agent of the invention at a therapeutically effective dose, e.g., a fixed dose of about 80 mg, to treat a subject, e.g., a subject that would benefit from reduced and / or inhibited Serpincl expression.

[0157] In yet another aspect, the invention provides use of an iRNA agent of the invention targeting the Serpinc1 gene, e.g., a dsRNA, or a pharmaceutical composition comprising an iRNA agent targeting the Serpinc1 gene in a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in a subject by about 75% or more, e.g., about 25 mg to about 100 mg, in the manufacture of a medicament for treating a subject who would benefit from reduced and / or inhibited Serpinc1 expression, such as a subject with a bleeding disorder, e.g., hemophilia. In one embodiment, the invention provides use of an iRNA agent of the invention targeting the Serpinc1 gene, e.g., a dsRNA, or a pharmaceutical composition comprising an iRNA agent targeting the Serpinc1 gene in a therapeutically effective dose, e.g., a fixed dose of about 50 mg, in the manufacture of a medicament for treating a subject who would benefit from reduced and / or inhibited Serpinc1 expression, such as a subject with a bleeding disorder, e.g., hemophilia. In another embodiment, the invention provides the use of an iRNA agent of the invention targeting the Serpinc1 gene, e.g., a dsRNA, or a pharmaceutical composition comprising a therapeutically effective dose, e.g., a fixed dose of about 80 mg, of an iRNA agent targeting the Serpinc1 gene, in the manufacture of a medicament for treating a subject who would benefit from reduced and / or inhibited Serpinc1 expression, such as a subject with a bleeding disorder, e.g., hemophilia.

[0158] In some embodiments of the present invention, for example, when double-stranded RNAi agent comprises a sense strand and an antisense strand, and the antisense strand comprises a region complementary to the mRNA encoding Serpinc1, comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) by no more than 3 nucleotides, and substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end, such agent is administered at a fixed dose of about 25mg to about 100mg, for example, at a fixed dose of about 25mg; or at a fixed dose of about 50mg; or at a fixed dose of about 80mg; or at a fixed dose of about 100mg.In one embodiment, the fixed dose is 50mg.In another embodiment, the fixed dose is 80mg.

[0159] Thus, in one aspect, the present invention provides a method for predicting at least one symptom of a subject having a disorder, e.g., a bleeding disorder, e.g., hemophilia, that would benefit from reduced Serpincl expression. The present invention provides a method for preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression. The method includes administering to a subject a double-stranded ribonucleic acid (RNAi) agent (e.g., a pharmaceutical composition comprising the RNAi agent) at a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in a subject by about 75% or more, e.g., about 25 mg to about 100 mg, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand that binds at its 3' end, thereby preventing at least one symptom of a disorder that would benefit from reduced Serpinc1 expression. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0160] In another aspect, the invention provides a method of treating a subject having a disorder that would benefit from reduced Serpincl expression, e.g., a bleeding disorder, e.g., hemophilia, comprising administering a therapeutically effective dose, e.g., an amount that reduces Serpincl activity in the subject by about 75% or more, e.g., about 25 mg to about 100 mg, of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, comprising a sense strand and an antisense strand, wherein the antisense strand differs by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15). The present invention includes administering to a subject (e.g., a human) a pharmaceutical composition comprising a double-stranded ribonucleic acid (RNAi) agent or an iRNA agent targeting the Serpinc1 gene, the pharmaceutical composition comprising a region complementary to the mRNA encoding Serpinc1, comprising at least 15 consecutive nucleotides, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end, thereby treating a subject with a disorder that would benefit from reducing Serpinc1 expression. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0161] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for use in preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced and / or inhibited Serpinc1 expression, such as a bleeding disorder, e.g., hemophilia, at a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in the subject by about 75% or more, e.g., about 25 mg to about 100 mg, fixed dose, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0162] In a further aspect, the present invention provides a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), and wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced and / or inhibited Serpinc1 expression, such as bleeding disorders, e.g., hemophilia. The present invention provides a double-stranded ribonucleic acid (RNAi) agent, wherein all nucleotides are modified nucleotides, and the sense strand is conjugated to a ligand attached at the 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0163] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for use in treating a subject, for example, a subject who would benefit from reduced and / or inhibited Serpinc1 expression, at a therapeutically effective dose, for example, an amount that reduces Serpinc1 activity in the subject by about 75% or more, for example, about 25 mg to about 100 mg, in a fixed dose. The double-stranded ribonucleic acid (RNAi) agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0164] In yet another aspect, the invention provides use of an iRNA agent, e.g., a dsRNA, or pharmaceutical composition comprising a double-stranded ribonucleic acid (RNAi) agent of the invention targeting the Serpinc1 gene in the manufacture of a medicament for treating a subject who would benefit from reduced and / or inhibited Serpinc1 expression, such as a subject with a bleeding disorder, e.g., hemophilia, in a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in a subject by about 75% or more, e.g., about 25 mg to about 100 mg, fixed dose, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand that binds at its 3' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0165] The methods and uses of the present invention may be used in combination with other methods, such as those described above, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80 days. In one embodiment, the expression of the target Serpincl gene is reduced for a long duration, e.g., at least about 7 days or more, e.g., about 1 week, 2 weeks, 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, about a quarter, or more.

[0166] The reduction of gene expression can be evaluated by any method known in the art.For example, the reduction of Serpinc1 expression can be measured by using a method that is common to those skilled in the art, such as Northern blotting, qRT-PCR to determine the mRNA expression level of Serpinc1; by using a method that is common to those skilled in the art, such as Western blotting, immunological method, etc. to determine the protein level of Serpinc1; and / or can be determined, for example, by determining the biological activity of Serpincl, which affects one or more molecules associated with the cellular blood clotting mechanism (or, in an in vivo environment, blood clotting itself). In one embodiment, to assess Serpincl expression, for example, thrombin formation time, clot formation time, and / or clotting time are determined using ROTEM® Thromboelastometry analysis of whole blood.

[0167] The administration of dsRNA by the method and use of the present invention can reduce the severity, signs, symptoms and / or markers of the patients with Serpincl-related disease of this disease or disorder.In this context, " reduction " means the statistically significant reduction of this level.For example, reduction can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100%.

[0168] The effectiveness of disease treatment or prevention can be evaluated, for example, by measuring the levels of disease progression, disease remission, symptom severity, bleeding frequency, pain relief, quality of life, the dose of drug required to maintain the effect of treatment, disease markers, or any other measurable parameter appropriate for the given disease being treated or targeted for prevention. Monitoring the effectiveness of treatment or prevention by measuring any one or any combination of such parameters is well within the capabilities of one skilled in the art. For example, the effectiveness of treatment of bleeding disorders can be evaluated by, for example, periodically monitoring thrombin:antithrombin levels. Comparison of later measurements with earlier measurements provides the physician with an indication of whether the treatment is effective. Monitoring the effectiveness of treatment or prevention by measuring any one or any combination of such parameters is well within the capabilities of one skilled in the art. In the context of administering an iRNA targeting Serpinc1 or a pharmaceutical composition thereof, "effective against" a bleeding disorder indicates that administration in a clinically relevant manner results in a beneficial effect, such as improvement in symptoms, amelioration, disease remission, prolongation of life, improved quality of life, or other effect generally recognized as positive by a physician knowledgeable in the treatment of bleeding disorders and their associated causes, for at least a statistically significant proportion of patients.

[0169] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when symptoms do not worsen or develop as would otherwise be expected. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease indicates an effective treatment. The effectiveness of a given iRNA drug or formulation of the drug can also be determined using an experimental animal model for a given disease known in the art. When using an experimental animal model, the effectiveness of the treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0170] Alternatively, efficacy can be assessed by a reduction in disease severity as determined by a person skilled in the art of diagnosis based on a clinically accepted disease severity assessment scale. For example, any positive change resulting from a reduction in disease severity as determined using an appropriate scale indicates successful treatment with the iRNA or iRNA formulation described herein.

[0171] The iRNA (or pharmaceutical composition comprising the iRNA) can be administered to a subject approximately once a week, approximately twice a month, approximately once every six weeks, approximately once every two months, or once a quarter.

[0172] Double-stranded iRNA agent can be administered to the subject in one or more doses.For example, double-stranded iRNA agent can be administered to the subject in a monthly dose of about 0.200mg / kg to about 1.825mg / kg.Alternatively, double-stranded iRNA agent can be administered to the subject in a fixed dose of about 25mg to about 100mg.

[0173] In one embodiment, the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to the mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) by no more than 3 nucleotides, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated with a ligand that binds at its 3' end. The double-stranded RNAi agent is administered to a subject at a fixed dose of about 25 to about 100 mg, for example, about 25 mg, 50 mg, 80 mg, or 100 mg. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0174] Administration can be repeated on a regular basis, for example, once a month for one, two, three, four or more months.After the initial treatment regimen, treatment can also be administered less frequently.For example, administration can be repeated once a month for three months, followed by administration once a quarter for one year or more.

[0175] Thus, in some embodiments, the RNAi agent is administered in a regimen that includes a "loading phase" of closely spaced administrations, followed by a "maintenance phase" in which the RNAi agent is administered at longer intervals.

[0176] The loading dosing schedule and / or maintenance dosing schedule may optionally be repeated one or more times, the number of repetitions depending on achieving a desired effect, e.g., suppression of the Serpincl gene, and / or achieving a therapeutic or prophylactic effect, e.g., increased blood clotting, decreased clot formation time, and / or decreased clotting time.

[0177] Administration of iRNA may, for example, increase Serpincl levels in the patient's cells, tissues, blood, urine, or other compartments by at least about 5%, 6%, 7%, 8%, 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%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or , 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0178] The iRNA can be administered by intravenous infusion over a period of time, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 minutes.

[0179] Prior to administration of the full dose of iRNA, the patient can be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as allergic reactions. In another example, undesirable side effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels, can be monitored. Patients can be monitored for any adverse immunostimulatory effects.

[0180] Due to the inhibitory effect on Serpinc1 expression, the composition according to the present invention or a pharmaceutical composition prepared therefrom can improve quality of life.

[0181] The iRNA of the present invention can be administered in a "naked" form, or as a "free iRNA." Naked iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA can also be in a suitable buffer. The buffer can include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS). The pH and osmolality of the buffer containing the iRNA can be adjusted to suit administration to a subject.

[0182] Alternatively, the iRNA of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0183] Subjects who would benefit from reduced and / or inhibited Serpincl gene expression are subjects with a bleeding disorder described herein, e.g., a genetic bleeding disorder or an acquired bleeding disorder. In one embodiment, the subject with a genetic bleeding disorder has hemophilia, e.g., hemophilia A, B, or C. In one embodiment, the subject with a genetic bleeding disorder, e.g., hemophilia, is an inhibitor subject (a subject who has become resistant to replacement clotting factors). In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C. Treatments for subjects who would benefit from reduced and / or inhibited Serpincl gene expression include therapeutic treatments (e.g., on-demand, e.g., when the subject is bleeding (spontaneously or as a result of trauma) and not clotting) and prophylactic treatments (e.g., when the subject is not bleeding and / or undergoing surgery).

[0184] The present invention further provides methods and uses for the use of iRNAs or pharmaceutical compositions thereof to treat subjects who would benefit from reduced and / or inhibited Serpincl expression, e.g., subjects with bleeding disorders, e.g., in combination with other pharmaceutical agents and / or other therapeutic methods, e.g., known pharmaceutical agents and / or known therapeutic methods, such as those currently utilized to treat these disorders.

[0185] For example, in certain embodiments, an iRNA targeting Serpinc1 is administered in combination with an agent useful in treating bleeding disorders, for example, as described elsewhere herein. For example, additional therapeutic agents and methods suitable for treating subjects who would benefit from reduced Serpinc1 expression, e.g., subjects with bleeding disorders, include fresh frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrate; virally inactivated vWF-containing FVIII concentrate; desensitization therapy, which may include large doses of FVIII or FIX along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasma exchange therapy in conjunction with immunosuppression and infusion of FVIII or FIX, with or without antifibrinolytic therapy; immune tolerance induction (ITI), with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and / or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytic drugs such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilic drugs; corticosteroids; immunosuppressants; and estrogen.

[0186] The iRNA and additional therapeutic agents and / or treatments can be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agents can be administered as part of separate compositions or at different times and / or in a manner known in the art. Alternatively, it can be administered by another method described herein.

[0187] In one embodiment, the present invention provides a method for administering compound AT3SC-001 (AD-57213 - sense strand: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and antisense strand: 5'-u

[0013] Provided is a method for treating a subject suffering from a bleeding disorder, e.g., hemophilia, by subcutaneously administering to the subject sUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0188] III. iRNAs for Use in the Methods of the Invention Described herein are methods for the use of improved double-stranded RNAi agents to inhibit expression of the Serpinc1 gene in cells, such as cells within a subject, e.g., a mammal, such as a human, with a Serpinc1-associated disorder, e.g., a bleeding disorder, e.g., hemophilia.

[0189] Thus, the present invention provides double-stranded RNAi agents having chemical modifications capable of inhibiting the expression of a target gene (i.e., the Serpincl gene) in vivo. In certain aspects of the present invention, substantially all nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all nucleotides of the iRNA of the present invention are modified. An iRNA of the present invention in which "substantially all nucleotides are modified" is largely, but not completely, modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0190] RNAi agents include a sense strand and an antisense strand. Each strand of an RNAi agent can be in the range of 12 to 30 nucleotides in length. For example, each strand can be between 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 19 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0191] The sense strand and antisense strand typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent can be 12 to 30 nucleotide pairs in length. For example, the duplex region can be 14 to 30 nucleotide pairs, 17 to 30 nucleotide pairs, 27 to 30 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the duplex region is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0192] In one embodiment, the RNAi agent can contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, or 3 to 4 nucleotides in length. The overhang can be 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being offset. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be another sequence. The first and second strands can also be joined by additional bases, for example, to form a hairpin, or other non-basic linker.

[0193] In one embodiment, each nucleotide in the overhang region of the RNAi agent can be independently modified or unmodified nucleotide, including but not limited to 2'-sugar modifications such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.For example, TT can be the overhang sequence at either end of either strand.The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0194] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent is 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 one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands.In one embodiment, the 3'-overhang is present in the antisense strand.In one embodiment, the 3'-overhang is present in the sense strand.

[0195] RNAi agents can contain only a single overhang, which can enhance the buffering activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand, or alternatively at the 3'-end of the antisense strand.RNAi can also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa.Usually, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without wishing to be bound by theory, the asymmetric blunt ends at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand are advantageous for the introduction of guide strands into the RISC process.

[0196] Any nucleic acid featured in the present invention may be synthesized and / or modified by methods established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotides, reverse linkage, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base that base-pairs with a wide range of partners, removal of a base (abasic nucleotide), or a conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and / or backbone modifications, including modification or substitution 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 lacking natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have phosphorus atoms in backbones include those that do not have phosphorus atoms in backbones.For the purpose of this specification and as sometimes referred to in the art, the modified RNA that does not have phosphorus atoms in its internucleoside backbone is also considered to be oligonucleoside.In some embodiments, modified iRNA has phosphorus atoms in its internucleoside backbone.

[0197] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, including 3'-alkylenephosphonates, chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0198] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,717. ;No. 5,321,131;No. 5,399,676;No. 5,405,939;No. 5,453,496;No. 5,455,233;No. 5,466,677;No. 5,476,9 No. 25; 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. 5,58 No. 7,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6 ,239,265;No.6,277,603;No.6,326,199;No.6,346,614;No.6,444,423;No.6,531,590;No.6,534,639 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0199] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include 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, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonic acid and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S, and CH2 moieties.

[0200] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; ,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0201] In other embodiments, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further 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.

[0202] Some embodiments featured herein include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) of the aforementioned U.S. Patent No. 5,489,677, as well as the amide backbones of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, RNAs featured herein have the morpholino backbone structures of the aforementioned U.S. Patent No. 5,034,506.

[0203] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein, can 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 C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsDNA comprises at the 2' position one of the following: C1 to C 10The modified iRNA may be one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacokinetic properties of iRNA, or group that improves the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the Examples herein below.

[0204] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications are also made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar of the 3'-terminal nucleotide or 2'-5'-linked dsRNA, and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; and 5,700,920, several of which are co-owned with the present application. The entire contents of each of the foregoing are incorporated herein by reference.

[0205] iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (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 ( and other synthetic and natural nucleobases such as 4-isopropyl uracil, ...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, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, pp. 613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 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 O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making it an exemplary base substitution, especially when combined with a 2'-O-methoxyethyl sugar modification. Furthermore.

[0206] Representative United States patents that teach the preparation of the above and other modified nucleobases include, but are not limited to, 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; 5,594,12 Nos. 1, 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 contents of each of which are incorporated herein by reference in their entirety.

[0207] The RNA of an iRNA may also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, an agent of the present invention comprises an RNA of an iRNA, which is also modified to contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety, where the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural conformation. Addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O.R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0208] Examples of bicyclic nucleosides for use in polynucleotides of the invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide agents of the invention include one or more bicyclic nucleosides containing a 4'-2' bridge. Examples of such 4'-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)-O-2' (ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283). No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0209] Additional representative U.S. patents and published U.S. patent applications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,3 Nos. 99,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; and U.S. Patent Application Publication Nos. 2008 / 0039618 and 2009 / 0012281, the contents of each of which are incorporated herein by reference in their entirety.

[0210] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0211] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation, referred to herein as an "S-cEt."

[0212] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, minimizing distortion of the ribose ring.

[0213] Representative publications that teach the preparation of some of the above-described 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.

[0214] One or more of the nucleotides of the iRNA of the invention can also comprise a hydroxymethyl-substituted nucleotide, which is an acyclic 2'-3'-seco-nucleotide, also known as an "unlocked nucleic acid" ("UNA") modification.

[0215] Representative U.S. patent 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.

[0216] Potentially stable modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl 4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861. can be.

[0217] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the invention, double-stranded RNAi agents of the invention include agents having chemical modifications disclosed, for example, in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or PCT / U.S. Patent Application Publication No. 2012 / 065691, filed November 16, 2012, the contents of each of which are incorporated herein by reference in their entirety.

[0218] As shown in this specification, US Provisional Patent Application No. 61 / 561,710, and PCT / US Patent Application Publication No. 2012 / 065691, better results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand of RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of RNAi agent are otherwise completely modified.The introduction of these motifs, if present, disrupts the modification pattern of the sense strand and / or antisense strand.The RNAi agent is optionally conjugated with a GalNAc derivative ligand, for example, on the sense strand.The resulting RNAi agent exhibits better gene silencing activity.

[0219] More particularly, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded RNAi agent are 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 RNAi agent, the gene silencing activity of the RNAi agent is significantly improved.

[0220] In one embodiment, the RNAi agent is a 19 nucleotide long, double-ended The sense strand is a bluntmer, and the three consecutive nucleotides at positions 7, 8, and 9 from the 5' end The antisense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0221] In another embodiment, the RNAi agent is a 20-nucleotide long blunt-ended duplex, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 8, 9, and 10, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0222] In yet another embodiment, the RNAi agent is a 21-nucleotide long blunt-ended duplex, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 9, 10, and 11, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0223] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, while the other end comprises two nucleotide overhangs.Preferably, the two nucleotide overhangs are at the 3' end of the antisense strand.When the two nucleotide overhangs are at the 3' end of the antisense strand, the three nucleotides at the end are blunt. There are two phosphorothioate internucleotide linkages between the nucleotides, two of the three nucleotides 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 three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, all nucleotides in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of the motif, are modified nucleotides.In one embodiment, each residue is independently modified with 2'-O-methyl or 3' fluoro, for example, in the alternating motif.Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0224] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand comprising at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand being 1-4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides long, the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand such that the RNAi agent reduces expression of the target gene when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially yields siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0225] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs occurring at the cleavage site of the sense strand.

[0226] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at or near the cleavage site in the antisense strand.

[0227] In RNAi agents having a duplex region 17-23 nucleotides in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand or 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 RNAi from the 5' end.

[0228] The sense strand of RNAi agent can contain at least one motif of three identical modifications in three consecutive nucleotides at the site of strand breakage; antisense strand can have at least one motif of three identical modifications in three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned such that one motif of three nucleotides in sense strand and one motif of three nucleotides in antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0229] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications at three consecutive nucleotides. The first motif may be at or near the site of strand cleavage, and the other motifs may contain wing modifications. As used herein, the term "wing modification" refers to a motif present on another portion of the strand separated from a motif at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one or more nucleotides. When motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other; when motifs are separated by one or more nucleotides, the chemical structures may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be present at one end or on either side of the lead motif relative to the first motif at or near the cleavage site.

[0230] Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications at 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 sequence as the wing modifications that may be present in the sense strand.

[0231] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0232] In another embodiment, wing modifications in the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0233] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications may be located at the same end of the duplex region and may have an overlap of 1, 2, or 3 nucleotides.

[0234] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands are aligned such that two modifications from one strand are each located at one end of the duplex region, with an overlap of one, two, or three nucleotides; two modifications from one strand are each located at the other end of the duplex region, with an overlap of one, two, or three nucleotides; and two modifications from one strand are located on either side of the lead motif, with an overlap of one, two, or three nucleotides in the duplex region.

[0235] In one embodiment, all nucleotides in the sense strand and antisense strand of RNAi agent, including the nucleotide that is part of motif, are modified.Each nucleotide is modified with the same or different modifications, and this modification can include one or more of one or both of non-bonded phosphate oxygen and / or one or more of bonded phosphate oxygen;Modify the component of ribose sugar, for example, the 2' hydroxyl of ribose sugar;Large-scale substitution of phosphate moiety with " dephosphorylation " linker;Modify or substitute natural base;And substitute or modify ribose-phosphate backbone.

[0236] Because nucleic acids are polymers of subunits, many of the modifications, such as modifications of the base, or phosphate moiety, or non-linked O of the phosphate moiety, occur at repeated positions within the nucleic acid. In some cases, modifications are present at all of the intended positions in nucleic acid, but in many cases, this is not the case. For example, modifications may be present only at the 3' or 5' terminal position, or only at the terminal region, for example, at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications may be present in double-stranded regions, single-stranded regions, or both. Modifications may be present only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at non-binding O positions may be present only at one or both ends, or only in the terminal region, for example, at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or may be present in both double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends are phosphorylated.

[0237] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, such as the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang are modified, for example, with the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, such as modifications known in the art, such as the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribosugar of the nucleobase, and modifications of the phosphate group, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0238] In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.Strands can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0239] At least two different modifications are typically present in the sense and antisense strands, and the two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0240] In one embodiment, N a and / or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. The alternating nucleotides may refer to one at every other nucleotide or one at every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0241] The types of modifications contained in the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands is selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0242] In one embodiment, the RNAi agent of the present invention comprises an RNAi agent having an alternating motif in the antisense strand. The alternating motif in sense strand comprises a modification pattern that is shifted relative to the modification pattern.This shift can be such that the modified group of the nucleotide of sense strand corresponds to the differently modified group of the nucleotide of antisense strand, or vice versa.For example, when sense strand is paired with antisense strand in dsRNA duplex, the alternating motif in sense strand can start from "ABABAB" from 5' to 3' of the strand, and the alternating motif in antisense strand can start from "BABABA" from 5' to 3' of the strand in duplex region.As another example, the alternating motif in sense strand can start from "AABBAABB" from 5' to 3' of the strand in duplex region, and the alternating motif in antisense strand can start from "BBAABBAA" from 5' to 3' of the strand in duplex region, thereby there is a complete or partial shift in the modification pattern between sense strand and antisense strand.

[0243] In one embodiment, RNAi agent comprises the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification in sense strand, and first has the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification in antisense strand shifted, that is, the 2'-O-methyl modified nucleotide in sense strand forms base pair with the 2'-F modified nucleotide in 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.

[0244] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand interrupts the original modification pattern present in sense strand and / or antisense strand.This interruption of the modification pattern of sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand unexpectedly enhances the gene silencing activity against target gene.

[0245] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different from the modification of the motif. For example, a portion of a sequence containing a motif may be designated "...N" a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications in three consecutive nucleotides, and "N a " and "N b " represents a modification of 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, N a and / or N b may or may not be present if wing modifications are present.

[0246] RNAi agent can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.The modification of phosphorothioate or methylphosphonate internucleotide linkage can be present at any nucleotide of sense strand or antisense strand or both strands at any position of the strand.For example, the modification of internucleotide linkage can be present at all nucleotides in sense strand and / or antisense strand;The modification of each internucleotide linkage can be present in sense strand and / or antisense strand in an alternating pattern;Or sense strand or antisense strand can contain both internucleotide linkage modifications in an alternating pattern.The alternating pattern of the modification of internucleotide linkage in sense strand can be the same or different from that of antisense strand, and the alternating pattern of the modification of internucleotide linkage in sense strand can have a shift relative to the alternating pattern of the modification of internucleotide linkage in antisense strand.

[0247] In one embodiment, the RNAi comprises phosphorothioate or methylphosphonate internucleotide linkage modifications in the overhang region. For example, the overhang region comprises two It may contain two nucleotides with phosphorothioate or methylphosphonate internucleotide linkages between nucleotides.The modification of internucleotide linkages is also formed to connect overhanging nucleotides with the paired nucleotides at the ends in the double-stranded region.For example, at least 2, 3, 4 or all overhanging nucleotides are linked by phosphorothioate or methylphosphonate internucleotide linkages, and optionally there may be additional phosphorothioate or methylphosphonate internucleotide linkages that connect the overhanging nucleotides with the paired nucleotides adjacent to the overhanging nucleotides.For example, there may be at least two phosphorothioate internucleotide linkages between the three nucleotides at the ends, two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.These three nucleotides at the ends may be at the 3'-end of antisense strand, the 3'-end of sense strand, the 5'-end of antisense strand, and / or the 5'-end of antisense strand.

[0248] In one embodiment, two nucleotide overhangs are at the 3'-end of antisense strand, and there are two phosphorothioate internucleotide linkages between the three nucleotides at the end, and two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.Optionally, the RNAi agent can further have two phosphorothioate internucleotide linkages between the three nucleotides at the 5'-end of both sense strand and antisense strand.

[0249] In one embodiment, the RNAi agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. "Mismatches" can be non-canonical base pairings or other than canonical nucleotide pairings. Mismatches can occur in overhang regions or duplex regions. Base pairs are evaluated based on their tendency to promote dissociation or melting (e.g., for the free energy of binding or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With regard to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; pairings involving universal bases are preferred over canonical pairings. A "universal base" is a base that exhibits the ability to substitute for any of the four normal bases (G, C, A, and U) without significantly destabilizing nearby base pairing interactions or disrupting the expected functional bioavailability of the modified oligonucleotide. Non-limiting examples of universal bases include 2'-deoxyinosine (hypoxanthine deoxynucleotide) or its derivatives, nitroazole analogs, and hydrophobic aromatic non-hydrogen bonding bases.

[0250] In one embodiment, the RNAi agent comprises at least one of the first one, two, three, four, or five 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 a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0251] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of antisense strand is AU base pair.For example, the first base pair in the double-stranded region from the 5' end of antisense strand is AU base pair.

[0252] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, for example, two dT nucleotides, at the 3' end of the sense strand and / or antisense strand.

[0253] In one embodiment, the sense strand sequence has formula (I): 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) (In the formula: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a represents an oligonucleotide sequence containing independently 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence containing 0 to 10 modified nucleotides; p and n q independently represent overhanging nucleotides; wherein Nb and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides) Preferably, all of YYY are 2'-F modified nucleotides.

[0254] In one embodiment, N a and / or N b contains alternating patterns of modifications.

[0255] In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the duplex region from the 5' end (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13).

[0256] 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' (1b); 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).

[0257] 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 includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0258] When the sense strand is represented by formula (Ic), 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 includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0259] When the sense strand is represented as formula (Id), each N b represents an oligonucleotide sequence that independently contains 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 is independent It may refer to an oligonucleotide sequence that contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0260] Each of X, Y, and Z can be the same or different from each other.

[0261] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' n p -N a -YYY-N a -n q 3' (Ia) is expressed by

[0262] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0263] In one embodiment, the antisense strand sequence of the RNAi has the 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) (In the formula: k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' represents an oligonucleotide sequence containing independently 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides. is expressed by

[0264] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0265] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a duplex region 17 to 23 nucleotides long, the Y'Y'Y' motif can be present at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0266] In one embodiment, the Y'Y'Y' motifs are all 2'-OMe modified nucleotides.

[0267] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0268] Thus, the antisense strand has the following formula: 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (IIb); 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (IIc); or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'- N a '-n p’ 3' (IId) is expressed by

[0269] When the antisense strand is represented by formula (IIb), 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 ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0270] When the antisense strand is represented by formula (IIc), 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 ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0271] When the antisense strand is represented by formula (IId), each N b ' represents an oligonucleotide sequence that independently contains 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. b is 0, 1, 2, 3, 4, 5, or 6.

[0272] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -Na ’ -Y'Y'Y'-Na ’ -nq ’ 3' (IIa) is expressed by

[0273] When the antisense strand is represented by formula (IIa), each N a ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0274] Each of X', Y', and Z' can be the same or different from each other.

[0275] Each nucleotide of sense strand and antisense strand is independently modified with LNA, 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.

[0276] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif at positions 9, 10, and 11 of the strand, counting from the first nucleotide from the 5' end when the duplex region is 21 nucleotides; or optionally, counting from the 5' end and starting from the first paired nucleotide in the duplex region; 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; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0277] In one embodiment, the antisense strand may contain a Y'Y'Y' motif at positions 11, 12, and 13 of the strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the duplex region from the 5' end; Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0278] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0279] Thus, an RNAi agent for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex has the formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q ' 5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' represents an oligonucleotide sequence containing, independently, 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; where: Each n may or may not be present p ',n p , n q ', and n q independently represent overhanging nucleotides; 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.

[0280] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0281] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following formula: 5' n p -N a -YYY-N a -n q 3' 3' n p '-N a '-Y'Y'Y'-N a 'n q ' 5' (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) 5' -N a -YYY-N a - 3' 3' n p '-N a '-Y'Y'Y'-N a ' 5' (IIIe) Includes:

[0282] When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0283] When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence that independently contains 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0284] When the RNAi agent is represented by formula (IIIc), each N b , N b ' represents an oligonucleotide sequence that independently contains 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 represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0285] When the RNAi agent is represented by formula (IIId), each N b , N b' represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a N' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b Each of the ' independently includes an alternating pattern of modifications.

[0286] When the RNAi agent is represented by formula (IIId), each N b , N b ' represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a N' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b Each of the ' independently includes an alternating pattern of modifications.

[0287] When the RNAi agent is represented by formula (IIIe), each N a and N a ' represents an oligonucleotide sequence containing independently 0 to 25 nucleotides, which may be modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides.

[0288] Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe) can be the same as or different from one another.

[0289] When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0290] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide; or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0291] When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides. At least two of the nucleotides base pair with a corresponding X' nucleotide; or all three of the X nucleotides base pair with a corresponding X' nucleotide.

[0292] In one embodiment, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0293] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n pIn yet another embodiment, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker.

[0294] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker.

[0295] In one embodiment, the RNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe), and the double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites.

[0296] In one embodiment, the RNAi agent is a multimer that contains three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe), and the double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites.

[0297] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe) are linked to one another at one or both of the 5' and 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.

[0298] The multimeric RNAi agent that can be used in the method of the present invention is described in various publications.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of whose contents is incorporated herein by reference in its entirety.

[0299] RNAi agents containing one or more carbohydrate moieties conjugated to RNAi agents can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent is replaced with a non-carbohydrate (preferably cyclic) carrier to which another moiety, such as a carbohydrate ligand, is attached. Ribonucleotide subunits in which the ribose sugar of the subunit is replaced in this manner are referred to herein as ribose-replacement modified subunits (RRMS). The cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier may be a monocyclic ring system or may contain two or more rings, such as fused rings. The cyclic carrier may be a fully saturated ring system or may contain one or more double bonds.

[0300] The ligand is conjugated to the polynucleotide via the carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, typically containing a functional group, such as a hydroxyl group, or a backbone, such as a phosphate or modified phosphate, such as sulfur. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier often contains a functional group, such as an amino group, or generally provides a bond suitable for incorporation or tethering of another chemical moiety, such as a ligand, to the ring.

[0301] The RNAi agent may be conjugated to the ligand via a carrier, which may be a cyclic or acyclic group; preferably, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrazolin ... Preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0302] In certain embodiments, the RNAi agent for use in the methods of the invention is AT3SC-001 (AD-57213 - sense strand: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and antisense strand: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg -3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage).

[0303] These agents may further comprise a ligand.

[0304] Ligand The double-stranded RNA (dsRNA) agent of the present invention may be conjugated with one or more ligands.Ligand is bound to sense strand, antisense strand, or both strands at 3'-end, 5'-end, or both ends.For example, ligand is conjugated to sense strand.In a preferred embodiment, ligand is conjugated to 3'-end of sense strand.

[0305] In one embodiment, the ligand is a carbohydrate conjugate, such as a monosaccharide. In one embodiment, the ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to the iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to the iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to the iRNA agent of the invention via a trivalent linker.

[0306] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the invention is [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0307] In one embodiment, the GalNAc or GalNAc derivative is GalNAc3: [ka] is.

[0308] In some embodiments, a ligand, e.g., a GalNAc ligand, is attached to the 3' end of the RNAi agent. In one embodiment, the RNAi agent is represented by the following scheme: [ka] (wherein X is O or S) In one embodiment, X is O.

[0309] A variety of substances can be attached to the RNAi agents of the invention. A preferred moiety is a ligand, attached either directly or indirectly, preferably covalently, via an intervening tether.

[0310] In preferred embodiments, the ligand alters the distribution, targeting, or lifetime of the molecule into which it is incorporated. In preferred embodiments, the ligand provides improved affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, a receptor, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, compared to a species without such a ligand. Ligands that provide improved affinity for a selected target are also referred to as targeting ligands.

[0311] Some ligands may have endosomolytic properties. Endosomolytic ligands promote lysis of endosomes and / or transport of the compositions of the present invention, or components thereof, from endosomes to the cytoplasm of cells. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand is presumed to adopt its active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes lysis of endosomes and / or transport of the compositions of the present invention, or components thereof, from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586), and derivatives thereof (Turk ...). (Et al., Biochem. Biophys. Acta, 2002, 1559:56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0312] The ligands can improve the transport, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides or polymer molecules comprising any combination of the monomers and / or natural or modified ribonucleotides described herein.

[0313] Ligands generally may include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0314] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules such as synthetic polymers, e.g., synthetic polyamino acids, oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0315] Ligands can also include targeting groups, such as cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells. The targeting group can be thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD peptidomimetics, or aptamers.

[0316] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic ... 3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ tetraazamacrocycle complexes), dinitrophenyl, HRP, or AP.

[0317] The ligand may be a protein, e.g., a glycoprotein, or a peptide, e.g., a colligand. The ligand may be a molecule with specific affinity for a specific cell type, such as a ligand or antibody, e.g., an antibody that binds to a specific cell type, such as cancer cells, endothelial cells, or bone cells. Ligands may also include hormones and hormone receptors. Ligands may also include lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or non-peptide species such as aptamers. Ligands may be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0318] The ligand can be, for example, a substance, e.g., a drug, that can enhance the uptake of an iRNA agent into a cell by, for example, disrupting the cytoskeleton, e.g., by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0319] Ligands can increase cellular uptake of oligonucleotides, for example, by activating an inflammatory response. Exemplary ligands that can have such an effect include tumor necrosis factor alpha (TNF alpha), interleukin-1 beta, or gamma interferon.

[0320] In one embodiment, the ligand is lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows conjugate to be distributed to target tissue, for example, non-renal target tissue of the body.For example, target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can be used to (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, and / or (c) regulate the binding to serum protein, for example, HSA.

[0321] Lipid-based ligand can be used to regulate (for example, control) the binding of conjugate to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to target kidney, and therefore is less likely to be removed from body.The lipid or lipid-based ligand that binds less strongly to HSA can be used to make conjugate target kidney.

[0322] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that the HSA-ligand binding is not reversed.

[0323] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, so that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0324] 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, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low-density lipoprotein (LDL), and high-density lipoprotein (HDL).

[0325] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. When the agent is a peptide, modifications can be made, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide linkages, and the use of D-amino acids. Preferably, the helical agent is an alpha-helical agent, preferably having a lipophilic phase and a lipophobic phase.

[0326] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide moiety can also be a "delivery" peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the sequence from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to function as delivery peptides. Peptides or peptidomimetics, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries, are encoded by random sequences of DNA (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic linked to the iRNA agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or RGD mimic. The peptide moiety can range from about 5 amino acids to about 40 amino acids in length. The peptide moiety may have structural modifications, such as to enhance stability or direct conformational properties.Any of the following structural modifications can be utilized: RGD peptide moieties can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can promote targeting of iRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy, 8:783-787, 2001). Preferably, RGD peptides promote targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and modified, e.g., glycosylated or methylated, to promote targeting to specific tissues. For example, glycosylated RGD peptides can be α-. v iRNA agents can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers abundant on proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells that display integrins. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D-amino acids, and synthetic RGD mimetics can be used. In addition to RGD, integrins can also be used. Other moieties that target phosphoryl ligands can be used. Generally, such ligands can be used to control cell proliferation and angiogenesis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or other oncogenes, such as those described herein.

[0327] A "cell-penetrating peptide" can penetrate cells, e.g., microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells. Peptides that penetrate microbial cells can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two predominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell-penetrating peptides can be bipartite amphipathic peptides such as MPG, which is 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).

[0328] In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, Esculentinis-1, and caerin. Preferably, a number of factors are considered to maintain the integrity of helix stability. For example, a maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) are utilized, and a minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units) are utilized. Capping residues are also contemplated (e.g., Gly is an exemplary N-capping residue, and / or C-terminal amidation can be used to provide additional hydrogen bonds to stabilize the helix). Stabilization can be achieved by the formation of salt bridges between oppositely charged residues separated by positions i±3, or i±4. Cationic residues such as lysine, arginine, homo-arginine, ornithine, or histidine can form salt bridges with the anionic residues glutamic acid or aspartic acid.

[0329] Peptide and peptidomimetic ligands include natural or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides with one or more amide bonds, i.e., peptide linkages, replaced with one or more urea, thiourea, carbamate, or sulfonylurea linkages; or cyclic peptides.

[0330] The targeting ligand can be any ligand that can target a specific receptor. Examples include: folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. The ligand can also be based on nucleic acids, for example, aptamers. The aptamer can be unmodified or have any combination of modifications disclosed herein.

[0331] Endosomal release agents include imidazole, poly- or oligoimidazole, and PEI. , peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketials, orthoesters, masked or unmasked cations, or polymers with anionic charge, masked or unmasked cations, or dendrimers with anionic charge.

[0332] PK modulators refer to pharmacokinetic modulators. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also applicable as ligands (e.g., PK-modulating ligands) in the present invention.

[0333] Furthermore, aptamers that bind to serum components (eg, serum proteins) are also applicable to the present invention as PK-regulating ligands.

[0334] Other ligand conjugates applicable to the present invention are described in U.S. Patent Nos. 10 / 916,185, filed August 10, 2004; 10 / 946,873, filed September 21, 2004; 10 / 833,934, filed August 3, 2007; 11 / 115,989, filed April 27, 2005; and 11 / 944,227, filed November 21, 2007, the entire contents of which are incorporated herein by reference.

[0335] When there are two or more ligands, the ligands can all have the same properties, or all have different properties, or some ligands can have the same properties, while other ligands have different properties.For example, the ligand can have targeting properties, can have endosomolytic activity, or can have PK regulation properties.In a preferred embodiment, all ligands have different properties.

[0336] The ligand can be attached to the oligonucleotide at various positions, e.g., the 3'-terminus, the 5'-terminus, and / or an internal position. In preferred embodiments, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier as described herein. The ligand or tethered ligand can be present on the monomer when the monomer is incorporated into the growing chain. In some embodiments, the ligand is incorporated by binding to a "precursor" monomer after the "precursor" monomer is incorporated into the growing chain. For example, an amino-terminal tether (i.e., no ligand attached), e.g., TAP-(CH2) n The NH2-bearing monomer is incorporated into the growing oligonucleotide chain. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand bearing an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, is then attached to the precursor monomer by bonding between the electrophilic group of the ligand and the terminal nucleophilic group of the tether of the precursor monomer.

[0337] In another example, monomers bearing chemical groups suitable for participating in click chemistry reactions can be incorporated into, for example, azide- or alkyne-terminated tethers / linkers. In subsequent manipulations, i.e., after the precursor monomers have been incorporated into the chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be added by coupling the alkyne and azide together. can be attached to the precursor monomer.

[0338] In double-stranded oligonucleotides, the ligand can be attached to one or both strands. In some embodiments, the double-stranded iRNA agent contains a ligand conjugated to the sense strand. In other embodiments, the double-stranded iRNA agent contains a ligand conjugated to the antisense strand.

[0339] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is attached to a conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to a sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0340] Any suitable ligand in the field of RNA interference may be used, but the ligand is typically a carbohydrate, such as a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, or polysaccharide.

[0341] Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0342] In one embodiment, the dsRNA of the present invention has the formula (IV) to (VII): [ka] and conjugating the linker to a bivalent or trivalent branched linker comprising the structure shown in any one of During the ceremony, q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B , and q 5C each occurrence independently represents 0 to 20, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C each, independently at each occurrence, is absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, where one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R5A , R 5B , R 5C Each occurrence is independently absent, NH, O, S, CH2, 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., each independently at each occurrence is a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain.

[0343] The trivalent conjugate GalNAc derivative has the formula (VII): [ka] are particularly useful for use with RNAi agents to inhibit expression of target genes, such as those In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative. Examples of suitable bivalent and trivalent branched linker groups for conjugation with GalNAc derivatives include, but are not limited to, the following compounds: [ka] [ka] [ka] Includes:

[0344] 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; 5,414,077; ,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.4,789,737;No. 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. 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, and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the contents of each of which are incorporated herein by reference in their entirety.

[0345] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above modifications may be present in a single compound or even at a single nucleotide within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0346] In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., in the case of a dsRNA compound, a nucleotide. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the target nucleic acid. Additional regions of the iRNA can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For 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 significantly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, comparable results can often be obtained with shorter iRNAs compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target is typically detected by gel electrophoresis and, if necessary, by associated nucleic acid hybridization techniques known in the art.

[0347] In certain instances, the RNA of an iRNA is modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to improve the activity, cellular distribution, or cellular uptake of the iRNA, and procedures for such conjugation are available in the scientific literature.Such non-ligand moieties include cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tolyl, Lithylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above.A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using either an appropriate coupling or activation agent. The conjugation reaction can be performed with the RNA still attached to the solid support or after cleavage of the RNA in solution. The RNA conjugate can be analyzed by HPLC. Purification of the conjugate typically yields a pure conjugate.

[0348] In some embodiments, the double-stranded RNAi agent of the invention is AT3SC-001 (AD-57213).

[0349] IV. Delivery of iRNA of the Invention Delivery of an iRNA of the present invention to a cell, e.g., a cell in a subject, such as a human subject (e.g., a subject in need thereof, such as a subject with a bleeding disorder), can be achieved in several different ways. For example, delivery is achieved by contacting a cell with an iRNA of the present invention either in vitro or in vivo. In vivo delivery can also be achieved directly by administering a composition containing an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery can be achieved indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

[0350] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNAs can be minimized by local administration, e.g., by direct injection or implantation into tissue, or by administering a formulation locally. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure to systemic tissues that may be adversely affected by or degrade the agent, and can reduce the total dose of the iRNA molecule administered. Several studies have demonstrated successful knockdown of gene products when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al. (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al. (2003) Mol. Vis. 9:210-216) have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice can reduce tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ. et al. (2006) Mol. Ther. 14:343-350; Li, S. et al. (2007) Mol. Ther. 15:515-523).RNA interference can be delivered locally to the CNS by direct injection (Dorn, G. et al. (2004) Nucleic Acids 32:e49; Tan, P.H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T. et al. (2004) Neuroscience 129:521-528; Thakker, E.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602) and intranasal administration for localized delivery to the lung (Howard, K.A. et al. (2006) Mol. Ther. 14:476-484; Zhang, X. et al. (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al. (2005) Nat. Med. 11:50-55). To administer iRNA systemically for disease treatment, the RNA is modified or delivered using a drug delivery system; both methods serve to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or drug carrier allows for targeting of iRNA compositions to target tissues and can also avoid undesirable off-target effects. iRNA molecules are also modified with cholesterol to improve cellular uptake and prevent degradation. The iRNA is modified by chemical conjugation to a lipophilic group such as . For example, systemic administration of iRNA against ApoB conjugated with a lipophilic cholesterol moiety to mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al. (2004) Nature 432:173-178). Conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J. O. et al. (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA is delivered using a drug delivery system such as a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system. The positively charged cationic delivery system promotes binding of the iRNA molecule (negatively charged) and also improves interaction with the negatively charged cell membrane, allowing for efficient uptake of the iRNA by cells. Cationic lipids, dendrimers, or polymers can be attached to iRNA or can be incorporated into vesicles or micelles that encase iRNA (see, e.g., Kim SH. et al. (2008) Journal of of Controlled Release 129(2):107-116). The formation of vesicles or micelles further protects the iRNA from degradation when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, D.R. et al. (2003) J. Mol. Biol. 327:761-766; Verma, U.N. et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R. et al. (2003), supra; Verma, U.N. et al. (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S. et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y. et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E. et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A. et al. (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al. (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNAs are complexed with cyclodextrins for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins are found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0351] A. Vector-encoded iRNA of the invention iRNAs targeting the Serpincl gene are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A. et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. WO 00 / 22113; Conrad, International PCT Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (approximately hours to weeks) or sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292 pages).

[0352] The individual strands of the iRNA are transcribed from a promoter in an expression vector. Two separate expression vectors are co-introduced into a target cell (e.g., by transfection or infection) where two separate strands are expressed to produce, for example, dsRNA. Alternatively, each individual strand of the dsRNA is transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence to form a stem-loop structure.

[0353] iRNA expression vectors are generally DNA plasmids or viral vectors.Recombinant constructs for expressing iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells.Eukaryotic expression vectors are well known in the art and are available from many commercial sources.Typically, such vectors are provided containing convenient restriction sites for inserting desired nucleic acid segments.Delivery of iRNA expression vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells transplanted from a patient and then reintroduced into the patient, or any other means that allow introduction into desired target cells.

[0354] The iRNA expression plasmid is transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or more are also contemplated by the present invention. Successful introduction of the vector into the host cells is monitored using various known methods. For example, transient transfection is indicated using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, on the transfected cells.

[0355] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including, but not limited to, lentivirus vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors, or avian pox, e.g., canarypox or fowlpox, poxvirus vectors; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses can also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may optionally contain viral sequences for transfection. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in the target cell. Other aspects of vectors and constructs to consider are further described below.

[0356] Vectors useful for delivery of iRNA will contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements are selected to provide for either constitutive or regulatable / inducible expression.

[0357] iRNA expression can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0358] Viral vectors containing nucleic acid sequences encoding iRNAs are used. For example, retroviral vectors are used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitate delivery of the nucleic acid to a patient. Further details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0359] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia and cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing the iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0360] Adeno-associated virus (AVV) vectors may also be used to deliver iRNA of the present invention. (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNA is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., having either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol. 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0361] Another viral vector suitable for delivery of the iRNA of the invention is a poxvirus, such as a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or an avian pox, such as fowlpox or canarypox.

[0362] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to express different capsid protein serotypes, allowing them to target different cells. For example, see Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0363] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector is produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0364] V. Pharmaceutical Compositions of the Invention The present invention also provides pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier is also provided herein. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with the expression or activity of the Serpinc1 gene, e.g., Serpinc1-associated diseases. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral administration, e.g., subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, e.g., by injection into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the present invention are administered at a dosage sufficient to inhibit expression of the Serpinc1 gene.

[0365] In certain embodiments of the invention, for example, when a pharmaceutical composition includes a double-stranded RNAi agent containing one or more motifs of three identical modifications on three consecutive nucleotides, including such motifs at or near the cleavage site of the agent, six phosphorothioate linkages, and a GalNAc ligand, the composition may be administered at a concentration of 0.200 to about 1.825 mg / kg, 0.200 to about 1.800 mg / kg, about 0.200 to about 1.700 mg / kg, or about 0.200 to about 1.825 mg / kg. kg, about 0.200 to about 1.600 mg / kg, about 0.200 to about 1.500 mg / kg, about 0.200 to about 1.400 mg / kg, about 0.200 to about 1.400 mg / kg, about 0.200 to about 1.200 mg / kg, about 0.200 to about 1.100 mg / kg, about 0.200 to about 1.000 mg / kg, about 0.200 to about 0.900 mg / kg, about 0.200 to about 0.800 mg / kg, about 0.200 to about 0.700 mg / kg, about 0.200 to about 0.600 mg / kg, about 0.200 to about 0.500 mg / kg, about 0.200 to about 0.400 mg / kg, about 0.225 to about 1.825 mg / kg, about 0.225 to about 1.800 mg / kg, about 0.225 to about 1.700 mg / kg, about 0.225 to about 1.600 mg / kg, about 0.225 to about 1.500 mg / kg, about 0.225 to about 1.400 mg / kg, about 0.225 to about 1.400 mg / kg, about 0.225 to about 1.200 mg / kg, about 0.225 to about 1.100 mg / kg, about 0.225 to about 1.000 mg / kg, about 0.225 to about 0.900 mg / kg, about 0.225 to about 0.800 mg / kg, about 0.225 to about 0.700 mg / kg, about 0. 225 to about 0.600 mg / kg, about 0.225 to about 0.500 mg / kg, about 0.225 to about 0.400 mg / kg, about 0.250 to about 1.825 mg / kg, about 0.250 to about 1.800 mg / kg, about 0.250 to about 1.700 mg / kg, about 0.250 to about 1.600 mg / kg, about 0.250 to about 1.500 mg / kg, about 0.250 to about 1.400 mg / kg, about 0.250 to about 1.400 mg / kg, about 0.250 to about 1.200 mg / kg, about 0.250 to about 1.100 mg / kg, about 0.250 to about 1.000 mg / kg, about 0.250 to about 0.900 mg / kg, about 0.250 to about 0.800 mg / kg, about 0.250 to about 0.700 mg / kg, about 0.250 to about 0.600 mg / kg, about 0.250 to about 0.500 mg / kg, about 0.250 to about 0.400 mg / kg, about 0.425 to about 1.825 mg / kg, about 0.425 to about 1.800 mg / kg, about 0.425 to about 1.700 mg / kg, about 0.425 to about 1.600 mg / kg, about 0.425 to about 1.500 mg / kg, about 0.425 to about 1.400 mg / kg, about 0.425 to about 1.400 mg / kg, about 0.425 to about 1.200 mg / kg, about 0.425 to about 1.100 mg / kg, about 0.425 to about 1.000 mg / kg, about 0.425 to about 0.900 mg / kg, about 0.425 to about 0.800 mg / kg, about 0.425 to about 0.700 mg / kg, about 0.425 to about 0.600 mg / kg, about 0.425 to about 0.500 mg / kg, about 0.450 to about 1.825 mg / kg, about 0.450 to about 1.800 mg / kg, about 0.450 to about 1.700 mg / kg, about 0.450 to about 1.600 mg / kg, about 0.450 to about 1.500 mg / kg, about 0.450 to about 1.400 mg / kg, about 0.450 to about 1.400 mg / kg, about 0.450 to about 1.200 mg / kg, about 0.450 to about 1.100 mg / kg, about 0.450 to about 1.000 mg / kg, about 0.450 to about 0.900 mg / kg, about 0.450 to about 0.800 mg / kg, about 0.450 to about 0.700 mg / kg, about 0.450 to about 0.600 mg / kg, about 0.450 to about 0.500 mg / kg, about 0.475 to about 1.825 mg / kg, about 0.475 to about 1.800 mg / kg, about 0.475 to about 1.700 mg / kg, about 0.475 to about 1.600 mg / kg. kg, about 0.475 to about 1.500 mg / kg, about 0.475 to about 1.400 mg / kg, about 0.475 to about 1.400 mg / kg, about 0.475 to about 1.200 mg / kg, about 0.475 to about 1.100 mg / kg, about 0.475 to about 1.000 mg / kg, about 0.475 to about 0.900 mg / kg, about 0.475 to about 0.800 mg / kg, about 0.475 to about 0.700 mg / kg, about 0.475 to about 0.600 mg / kg, about 0.475 to about 0.500 mg / kg, about 0.875 to about 1.825 mg / kg, about 0.875 to about 1.800 mg / kg, about 0.875 to about 1.700 mg / kg, about 0.875 to about 1.600 mg / kg, about 0.875 to about 1.500 mg / kg, about 0.875 to about 1.400 mg / kg, about 0.875 to about 1.400 mg / kg, about 0.875 to about 1.200 mg / kg, about 0.875 to about 1.100 mg / kg, about 0.875 to about 1.000 mg / kg, about 0.875 to about 0.900 mg / kg, about 0.900 to about 1.825 mg / kg, about 0.900 to about 1.800 mg / kg, about 0.900 to about 1.700 mg / kg, about 0.900 to about 1.600 mg / kg, about 0. .900~about 1.500mg / kg, about 0.900~about 1.400mg / kg, about 0.900~about 1.400mg / kg, about 0.900~about 1.200mg / kg, about 0.9 00~about 1.100mg / kg, about 0.900~about 1.000mg / kg, about 0.925~about 1.825mg / kg, about 0.925~about 1.800mg / kg, about 0.925~ The RNAi agent is administered at a dose of about 1.700 mg / kg, about 0.925 to about 1.600 mg / kg, about 0.925 to about 1.500 mg / kg, about 0.925 to about 1.400 mg / kg, about 0.925 to about 1.400 mg / kg, about 0.925 to about 1.200 mg / kg, about 0.925 to about 1.100 mg / kg, or about 0.925 to about 1.000 mg / kg. Intermediate values ​​and ranges to the above-listed values ​​are also intended to be part of the invention; for example, the RNAi agent is administered to a subject at a dose of about 0.015 mg / kg to about 0.45 mg / kg.

[0366] For example, an RNAi agent, e.g., an RNAi agent in a pharmaceutical composition, may be about 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, about 0.5 mg / kg, 0.525 mg / kg, 0.55 mg / kg , 0.575mg / kg, about 0.6mg / kg, 0.625mg / kg, 0.65mg / kg, 0.675mg / kg, about 0.7mg / kg, 0.725mg / kg, 0.75mg / kg, 0.775mg / kg, approximately 0.8mg / kg, 0.925mg / kg, 0.95mg / kg, 0.975mg / kg, approximately 1.0mg / kg, 1.025mg / kg, 1.05mg / kg, 1.075mg / kg, approximately 1.1 mg / kg, 1.125mg / kg, 1.15mg / kg, 1.17 The compound is administered at a dose of about 5 mg / kg, about 1.2 mg / kg, 1.225 mg / kg, 1.25 mg / kg, 1.275 mg / kg, about 1.3 mg / kg, 1.325 mg / kg, 1.35 mg / kg, 1.375 mg / kg, about 1.4 mg / kg, 1.425 mg / kg, 1.45 mg / kg, 1.475 mg / kg, about 1.5 mg / kg, 1.525 mg / kg, 1.55 mg / kg, 1.575 mg / kg, about 1.6 mg / kg, 1.625 mg / kg, 1.65 mg / kg, 1.675 mg / kg, about 1.7 mg / kg, 1.725 mg / kg, 1.75 mg / kg, 1.775 mg / kg, or about 1.8 mg / kg. Values ​​intermediate to the above-listed values ​​are also intended to be part of the present invention.

[0367] In some embodiments of the invention, for example, when a double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides, which differ by no more than three nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), and substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3' end, the agent in such a pharmaceutical composition may be from about 0.200 to about 1.825 mg / kg, 0.200 to about 1.800 mg / kg, about 0.200 to about 1.700 mg / kg, about 0.200 to about 1.600 mg / kg, about 0.200 to about 1.500 mg / kg, about 0.200 to about 1.400 mg / kg, or about 0.200 to about 1.600 mg / kg. ~about 1.400mg / kg, about 0.200 to about 1.200mg / kg, about 0.200 to about 1.100mg / kg, about 0.200 to about 1.000mg / kg, about 0.200 to about 0.900mg / kg, about 0.200 to about 0.8 00mg / kg, about 0.200 to about 0.700mg / kg, about 0.200 to about 0.600mg / kg, about 0.200 to about 0.500mg / kg, about 0.200 to about 0.400mg / kg, about 0.225 to about 1.825mg / kg kg, about 0.225 to about 1.800 mg / kg, about 0.225 to about 1.700 mg / kg, about 0.225 to about 1.600 mg / kg, about 0.225 to about 1.500 mg / kg, about 0.225 to about 1.400 mg / kg, about 0 .225~about 1.400mg / kg, about 0.225~about 1.200mg / kg, about 0.225~about 1.100mg / kg, about 0.225~about 1.000mg / kg, about 0.225~about 0.900mg / kg, about 0.225~ about 0.800 mg / kg, about 0.225 to about 0.700 mg / kg, about 0.225 to about 0.600 mg / kg, about 0.225 to about 0.500 mg / kg, about 0.225 to about 0.400 mg / kg, about 0.250 to about 1.825 mg / kg, about 0.250 to about 1.800 mg / kg, about 0.250 to about 1.700 mg / kg, about 0.250 to about 1.600 mg / kg, about 0.250 to about 1.500 mg / kg, about 0.250 to about 1.400 mg / kg, about 0.250 to about 1.400 mg / kg, about 0.250 to about 1.200 mg / kg, about 0.250 to about 1.100 mg / kg, about 0.250 to about 1.000 mg / kg, about 0.250 to about 0.900 mg / kg, about 0.250 to about 0.800 mg / kg, about 0.250 to about 0.700 mg / kg, about 0.250 to about 0.600 mg / kg, about 0.250 to about 0.500 mg / kg, about 0.250 to about 0.400 mg / kg, about 0.425 to about 1.825 mg / kg, about 0.425 to about 1.800 mg / kg, about 0.425 to about 1.700 mg / kg, about 0.425 to about 1.600 mg / kg, about 0.425 to about 1.500 mg / kg, about 0.425 to about 1.400 mg / kg, about 0.425 to about 1.400 mg / kg, about 0.425 to about 1.200 mg / kg, about 0.425 to about 1.100 mg / kg, about 0.425 to about 1.000 mg / kg, about 0.425 to about 0.900 mg / kg, about 0.425 to about 0.800 mg / kg, about 0.425 to about 0.700 mg / kg, about 0.425 to about 0.600 mg / kg, about 0.425 to about 0.500 mg / kg, about 0.450 to about 1.825 mg / kg, about 0.450 to about 1.800 mg / kg, about 0.450 to about 1.700 mg / kg, about 0.450 to about 1 0.600 mg / kg, about 0.450 to about 1.500 mg / kg, about 0.450 to about 1.400 mg / kg, about 0.450 to about 1.400 mg / kg, about 0.450 to about 1.200 mg / kg, about 0.450 to about 1.100 mg / kg, about 0.450 to about 1.000 mg / kg, about 0.450 to about 0.900 mg / kg, about 0.450 to about 0.800 mg / kg, about 0.450 to about 0.700 mg / kg, about 0.450 to about 0.600 mg / kg, about 0.450 to about 0.500 mg / kg, about 0.475 to about 1.825 mg / kg, about 0.475 to about 1.800mg / kg, about 0.475 to about 1.700mg / kg, about 0.475 to about 1.600mg / kg, about 0.475 to about 1.500mg / kg, about 0.475 to about 1.400mg / kg, about 0.475 to about 1.400mg / kg, about 0. 475~about 1.200mg / kg, about 0.475~about 1.100mg / kg, about 0.475~about 1.000mg / kg, about 0.475~about 0.900mg / kg, about 0.475~about 0.800mg / kg, about 0.475~about 0.700mg / kg, about 0.475 to about 0.600 mg / kg, about 0.475 to about 0.500 mg / kg, about 0.875 to about 1.825 mg / kg, about 0.875 to about 1.800 mg / kg, about 0.875 to about 1.700 mg / kg, about 0.875 to about 1.600mg / kg, about 0.875 to about 1.500mg / kg, about 0.875 to about 1.400mg / kg, about 0.875 to about 1.400mg / kg, about 0.875 to about 1.200mg / kg, about 0.875 to about 1.100mg / kg, about 0.875~1.000mg / kg, 0.875~0.900mg / kg, 0.900~1.825mg / kg, 0.900~1.800mg / kg, 0.900~1.700mg / kg, 0.900~1.600 mg / kg, about 0.900 to about 1.500 mg / kg, about 0.900 to about 1.400 mg / kg, about 0.900 to about 1.400 mg / kg, about 0.900 to about 1.200 mg / kg, about 0.900 to about 1.100 mg / kg, about 0.900 The RNAi agent may be administered at a dose of about 0.015 mg / kg to about 1.000 mg / kg, about 0.925 to about 1.825 mg / kg, about 0.925 to about 1.800 mg / kg, about 0.925 to about 1.700 mg / kg, about 0.925 to about 1.600 mg / kg, about 0.925 to about 1.500 mg / kg, about 0.925 to about 1.400 mg / kg, about 0.925 to about 1.400 mg / kg, about 0.925 to about 1.200 mg / kg, about 0.925 to about 1.100 mg / kg, or about 0.925 to about 1.000 mg / kg. Intermediate values ​​and ranges to the above-listed values ​​are also intended to be part of the invention; for example, an RNAi agent may be administered to a subject at a dose of about 0.015 mg / kg to about 0.45 mg / kg.

[0368] For example, an RNAi agent, e.g., an RNAi agent in a pharmaceutical composition, may be administered at a concentration of about 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, about 0.5 mg / kg, 0.525 mg / kg g / kg, 0.55mg / kg, 0.575mg / kg, approximately 0.6mg / kg, 0.625mg / kg, 0.65mg / kg, 0.675mg / kg, approximately 0.7mg / kg, 0.725mg / kg, 0.75mg / kg, 0.775mg / kg, approximately 0.8mg / kg, 0.925mg / kg, 0.95mg / kg, 0.975mg / kg, approximately 1.0mg / kg, 1.025mg / kg, 1.05mg / kg, 1.075mg / kg, about 1.1mg / kg, 1.125mg / kg, 1.15mg / kg, 1.175mg / kg, about 1.2mg / kg, 1.225mg / k g, 1.25mg / kg, 1.275mg / kg, about 1.3mg / kg, 1.325mg / kg, 1.35mg / kg, 1.375mg / kg, about 1.4mg / kg, 1.425mg / kg , 1.45 mg / kg, 1.475 mg / kg, about 1.5 mg / kg, 1.525 mg / kg, 1.55 mg / kg, 1.575 mg / kg, about 1.6 mg / kg, 1.625 mg / kg, 1.65 mg / kg, 1.675 mg / kg, about 1.7 mg / kg, 1.725 mg / kg, 1.75 mg / kg, 1.775 mg / kg, or about 1.8 mg / kg. Values ​​intermediate to the above-listed values ​​are also intended to be part of this invention.

[0369] In some embodiments, a pharmaceutical composition containing an iRNA agent is administered to a subject in a fixed dose. A "fixed dose" (e.g., a dose in mg) means that a single dose of an iRNA agent is used for all subjects regardless of any particular subject-related factors, such as body weight. In a specific embodiment, the fixed dose of an iRNA agent of the present invention is based on a predetermined body weight or age.

[0370] In some embodiments, a pharmaceutical composition comprising an iRNA agent comprises between about 25 mg and about 100 mg, e.g., between about 25 mg and about 95 mg, between about 25 mg and about 90 mg, between about 25 mg and about 85 mg, between about 25 mg and about 80 mg, between about 25 mg and about 75 mg, between about 25 mg and about 70 mg, between about 25 mg and about 65 mg, between about 25 mg and about 60 mg, between about 25 mg and about 50 mg, between about 50 mg and about 100 mg, between about 50 mg and about 95 mg, between about 50 mg and about 90 mg, between about 50 mg and about 85 mg, between about 50 mg and about 80 mg, between about 30 mg and about 100 mg, or between about 30 mg and about 90 mg The dose is administered at a fixed dose of between about 30 mg and about 80 mg, between about 40 mg and about 100 mg, between about 40 mg and about 90 mg, between about 40 mg and about 80 mg, between about 60 mg and about 100 mg, between about 60 mg and about 90 mg, between about 25 mg and about 55 mg, between about 30 mg and about 95 mg, between about 30 mg and about 85 mg, between about 30 mg and about 75 mg, between about 30 mg and about 65 mg, between about 30 mg and about 55 mg, between about 40 mg and about 95 mg, between about 40 mg and about 85 mg, between about 40 mg and about 75 mg, between about 40 mg and about 65 mg, between about 40 mg and about 55 mg, or between about 45 mg and about 95 mg.

[0371] In some embodiments, a pharmaceutical composition comprising an iRNA agent is administered at a fixed dose of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg.

[0372] The pharmaceutical composition comprising the iRNA is administered to the subject about once a month, about once every five weeks, about once every six weeks, about once every two months, or once a quarter.

[0373] A pharmaceutical composition comprising an iRNA agent is administered to a subject in one or more doses. In some embodiments, a pharmaceutical composition comprising a double-stranded RNAi agent is administered in a monthly dose of about 0.200 mg / kg to about 0.250 mg / kg, a monthly dose of about 0.425 mg / kg to about 0.475 mg / kg, or a monthly dose of about 0.500 mg / kg to about 0.575 mg / kg. a monthly dose of about 5 mg / kg, a monthly dose of about 0.875 mg / kg to about 0.925 mg / kg, or a monthly dose of about 1.775 mg / kg to about 1.825 mg / kg. In some embodiments, a pharmaceutical composition comprising a double-stranded iRNA agent is administered to a subject at a fixed dose of about 25 mg to about 100 mg, e.g., about 25 mg, about 50 mg, about 80 mg, or about 100 mg.

[0374] The pharmaceutical composition is administered by intravenous infusion for a period of time such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minutes. Administration is repeated periodically, for example, weekly, every other week (i.e., every two weeks), monthly, every two months, every three months, every four months, or more. After the initial treatment regimen, the therapeutic agent is administered less frequently. For example, after three months of weekly or biweekly administration, administration is repeated once a month for six months, one year, or more.

[0375] The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two, three, or more subdoses at appropriate intervals throughout the day, or by continuous infusion or sustained-release delivery. In this case, the amount of iRNA contained in each subdose must be correspondingly small to achieve the total daily dose. The dosage unit may also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that sustains the release of the iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites and are used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0376] In other embodiments, a single dose of the pharmaceutical composition can be continued for an extended period of time, with subsequent doses administered at intervals of no more than 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a month.

[0377] Those skilled in the art will recognize that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other existing diseases, can affect the dosage and time required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective dose of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for individual iRNAs encompassed by the present invention can be estimated using conventional methodologies or based on in vivo testing using appropriate animal models as described elsewhere herein.

[0378] Advances in mouse genetics have enabled the generation of numerous mouse models for the study of various human diseases, such as bleeding disorders, that would benefit from reduced Serpincl expression. These models can be used for in vivo testing of iRNAs and for determining therapeutically effective doses. Suitable mouse models known in the art include hemophilia A and hemophilia B mouse models, e.g., mice containing knockouts of clotting factor genes, as described in Bolliger et al. (2010) Thromb Haemost 103:1233-1238, Bi L et al. (1995) Nat Genet 10:119-21, Lin et al. (1997) Blood 90:3962-6, Kundu et al. (1998) Blood 92:168-74, Wang et al. (1997) Proc Natl Acad Sci USA 94:11563-6, and Jin et al. (2004) Blood 104:1733.

[0379] The pharmaceutical compositions of the present invention may be administered in several ways depending on whether local or systemic treatment is required and on the area to be treated. Administration can be topical (e.g., via a transdermal patch), intravenous (e.g., intramuscular), or intradermal (e.g., intramuscular). For example, pulmonary administration may be by inhalation or insufflation of a powder or aerosol with a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, by implantable device; or intracranial, for example, by intraparenchymal, intrathecal, or intraventricular administration.

[0380] The iRNA is delivered to target a specific tissue, such as the liver (e.g., hepatocytes of the liver).

[0381] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be necessary or desirable. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0382] A. Further Formulations i. Emulsion The compositions of the present invention are prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and (See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) varieties. When the aqueous phase is finely divided and dispersed as minute droplets into a bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions can contain additional components in addition to the dispersed phase and active drug, which may be present in either the aqueous or oil phase as a solution, or as a separate phase. Optionally, pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion. Pharmaceutical emulsions can also be multiple emulsions consisting of more than two phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer specific advantages not offered by simple binary emulsions. Multiple emulsions, in which individual oil droplets of an o / w emulsion enclose small water droplets, constitute w / o / w emulsions. Similarly, oil enclosed within globules of water stabilized in a continuous oil phase. The droplet system provides an o / w / o emulsion.

[0383] Emulsions are characterized by little or no thermodynamic stability.In most cases, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase, and is maintained in this form by means of an emulsifier or the viscosity of the formulation.Either phase of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams.Other means of stabilizing emulsions include the use of emulsifiers incorporated into either phase of the emulsion. Emulsifiers fall into four broad categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0384] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams and Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, page 199). Surfactants are typically amphiphilic and comprise a hydrophilic portion and a hydrophobic portion. The ratio of hydrophilicity to hydrophobicity of surfactant is called hydrophilic / lipophilic balance (HLB), which is a valuable tool for classifying and selecting surfactants when preparing formulations.Surfactants are classified into different types based on the nature of their hydrophilic groups: nonionic, anionic, cationic and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 285).

[0385] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, incorporate water to form water-in-oil emulsions but possess hydrophilic properties that allow them to maintain their semisolid consistency. Finely divided solids have also been used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-expanding clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon. Glyceryl tristearate.

[0386] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions, including fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0387] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around droplets of the dispersed phase and by increasing the viscosity of the external phase.

[0388] Emulsions often contain many components, such as carbohydrates, proteins, sterols, and phosphatides, which can easily support the growth of microorganisms, so these preparations often incorporate preservatives.The preservatives commonly used in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also usually added to emulsion preparations to prevent the preparation from deteriorating.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0389] The application of emulsion formulations via dermal, oral and parenteral routes and their manufacturing methods are reviewed in the literature (e.g., Ansel's Pharmaceuticals, Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.) , New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199. Emulsion formulations for oral delivery are very widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199. Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional formulations are among the materials commonly administered orally as o / w emulsions.

[0390] ii. Microemulsions In one embodiment of the present invention, iRNA and nucleic acid compositions are formulated as microemulsions. Microemulsions are defined as a system of water, oil, and amphiphiles that is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, page 245). Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally a medium-chain alcohol, to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared using a combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is of the water-in-oil (w / o) or oil-in-water (o / w) type depends on the properties of the oil and surfactant used, as well as the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0391] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with extensive knowledge on how to formulate microemulsions (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger (See, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335.) Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that form spontaneously.

[0392] Surfactants used in preparing the microemulsions, alone or in combination with co-surfactants, include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, etc. Examples of suitable surfactants include tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), and decaglycerol decaoleate (DAO750). Cosurfactants, typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, enhance interfacial fluidity by penetrating the surfactant film and forming an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can include materials such as, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0393] Microemulsions are particularly interesting from the viewpoint of drug solubilization and drug absorption enhancement. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of drugs, including peptides (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, pp. 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, pp. 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or iRNA. Microemulsions are also effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of iRNA and nucleic acids.

[0394] The microemulsions of the present invention may also contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and enhance absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention are classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0395] iii. Particulates The RNAi agents of the present invention may be incorporated into particles, e.g., microparticles. It is produced by fog drying, but may also be produced by other methods including freeze drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0396] iv. Penetration enhancers In one embodiment, the present invention uses various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically, only lipid-soluble or lipophilic drugs can easily penetrate cell membranes. It has been discovered that even non-lipophilic drugs can penetrate cell membranes if the membrane to be penetrated is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the permeability of lipophilic drugs.

[0397] Penetration enhancers are classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above classes of penetration enhancers is described in more detail below.

[0398] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through mucous membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, p. 252).

[0399] Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C1~20 These include alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., Touitou, E. et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, pp. 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, pp. 651-654).

[0400] The physiological role of bile includes facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, N (See, e.g., New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile and any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeox...

Claims

1. 1. A method of treating a bleeding event in a subject with hemophilia without inhibitors, comprising: administering to a subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl; the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serminc1 comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; administering a therapeutically effective amount of a replacement factor to a subject, wherein the effective amount of the replacement factor is reduced compared to a recommended effective amount of the replacement factor; thereby treating a bleeding event in a subject with hemophilia without inhibitors.

2. 1. A method of treating a bleeding event in a subject with hemophilia with an inhibitor, comprising: administering to a subject a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl; the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpincl comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; administering a therapeutically effective amount of a bypassing agent to a subject, wherein the effective amount of the bypassing agent is reduced compared to a recommended effective amount of the bypassing agent; thereby treating a bleeding event in a subject with hemophilia who has an inhibitor.

3. 1. A method of treating a bleeding event in a subject with hemophilia without inhibitors, comprising: administering to a subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl; the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpincl comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; administering a therapeutically effective amount of a replacement factor to a subject, wherein the effective amount of the replacement factor is reduced compared to a recommended effective amount of the replacement factor; thereby treating a bleeding event in a subject with hemophilia without inhibitors.

4. 1. A method of treating a bleeding event in a subject with hemophilia with an inhibitor, comprising: administering to a subject a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl; the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to an mRNA encoding Serpincl comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at its 3'-end; administering a therapeutically effective amount of a bypassing agent to a subject, wherein the effective amount of the bypassing agent is reduced compared to a recommended effective amount of the bypassing agent; thereby treating a bleeding event in a subject with hemophilia who has an inhibitor.

5. 5. The method of any one of claims 1 to 4, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject once a month, once every six weeks, once every two months, or four times a year.

6. The method of any one of claims 1 to 4, wherein the double-stranded RNAi agent is administered to the subject at a fixed dose of about 50 mg.

7. The method of any one of claims 1 to 4, wherein the double-stranded RNAi agent is administered to the subject at a fixed dose of about 80 mg.

8. The method of any one of claims 1 to 7, wherein the double-stranded RNAi agent is administered subcutaneously to the subject.

9. The method of any one of claims 1 to 6, wherein the subject is a human.

10. 10. The method of claim 9, wherein the hemophilia is hemophilia A, hemophilia B, or hemophilia C.

11. The method of any one of claims 1 to 10, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

12. 12. The method of any one of claims 1 to 11, wherein the modified nucleotides are independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

13. The method of any one of claims 1 to 12, wherein the region of complementarity is at least 17 nucleotides in length.

14. The method of any one of claims 1 to 13, wherein the region of complementarity is between 19 and 21 nucleotides in length.

15. 15. The method of claim 14, wherein the region of complementarity is 19 nucleotides in length.

16. The method of any one of claims 1 to 15, wherein each strand does not exceed 30 nucleotides in length.

17. The method of any one of claims 1 to 16, wherein each of the sense and antisense strands is independently 19 to 25 nucleotides in length.

18. The method of any one of claims 1 to 17, wherein each of the sense and antisense strands is independently 21 to 23 nucleotides in length.

19. The method of any one of claims 1 to 18, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

20. The method of any one of claims 1 to 19, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

21. The method of any one of claims 1 to 19, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

22. The method of any one of claims 1 to 21, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

23. The ligand is 【Chemical 1】 23. The method of claim 22, wherein:

24. The double-stranded RNAi agent is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 2】 24. The method of claim 23, wherein X is O or S.

25. 25. The method of claim 24, wherein X is O.

26. The method of any one of claims 1 to 25, wherein the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

27. 27. The method of any one of claims 1 to 26, wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence of 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

28. the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); 28. The method of claim 27, wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

29. the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf is 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; The sense strand is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 3】 29. The method of any one of claims 1 to 28, wherein X is O or S.

30. The method of any one of claims 1 to 29, wherein the double-stranded RNAi agent is administered to the subject as a pharmaceutical composition.

31. 31. The method of any one of claims 1-30, wherein administration of the dsRNA agent to a subject reduces Serpincl activity by about 75% or more.

32. 32. The method of any one of claims 1, 3, and 5-31, wherein the replacement factor is factor VIII.

33. 33. The method of claim 32, wherein the therapeutically effective amount of Factor VIII administered to the subject is less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, or less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg.

34. 33. The method of claim 32, wherein the therapeutically effective amount of factor VIII administered to the subject is about two-thirds to about one-fifth of the recommended effective amount of factor VIII.

35. 35. The method of claim 34, wherein the therapeutically effective amount of Factor VIII administered to the subject is at a dose of about 10 to about 20 IU / kg.

36. 29. The method of any one of claims 1, 3, and 5-28, wherein the replacement factor is Factor IX.

37. 37. The method of claim 36, wherein the therapeutically effective amount of Factor IX administered to the subject is less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, or less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg.

38. 37. The method of claim 36, wherein the therapeutically effective amount of Factor IX administered to the subject is about one-half to about one-sixth the recommended effective amount of Factor IX.

39. 39. The method of claim 38, wherein the therapeutically effective amount of Factor IX administered to the subject is at a dose of about 20 to about 30 IU / kg.

40. The method of any one of claims 2 and 4 to 31, wherein the bypassing agent is an activated prothrombin complex concentrate (aPCC).

41. 41. The method of claim 40, wherein the therapeutically effective amount of aPCC administered to the subject is less than about 100 U / kg, or less than about 90 U / kg, or less than about 80 U / kg, or less than about 70 U / kg, or less than about 60 U / kg, or less than about 50 U / kg, or less than about 40 U / kg, or less than about 30 U / kg, or less than about 20 U / kg, or less than about 10 U / kg.

42. 41. The method of claim 40, wherein the therapeutically effective amount of aPCC administered to the subject is about one-half to about one-third the recommended effective amount of aPCC.

43. 43. The method of claim 42, wherein the therapeutically effective amount of aPCC administered to the subject is at a dose of about 30 to about 50 U / kg.

44. The method of any one of claims 2 and 4 to 31, wherein the bypassing agent is recombinant Factor VIIa (rFVIIa).

45. 45. The method of claim 44, wherein the therapeutically effective amount of rFVIIa administered to the subject is less than about 120 μg / kg, or less than about 110 μg / kg, or less than about 100 μg / kg, or less than about 90 μg / kg, or less than about 80 μg / kg, or less than about 70 μg / kg, or less than about 60 μg / kg, or less than about 50 μg / kg, or less than about 40 μg / kg, or less than about 30 μg / kg, or less than about 20 μg / kg.

46. 46. ​​The method of claim 45, wherein the therapeutically effective amount of rFVIIa administered to the subject is about half the recommended effective amount of rFVIIa.

47. 47. The method of claim 46, wherein the therapeutically effective amount of rFVIIa administered to the subject is a dose of about 45 μg / kg.

48. 48. The method of any one of claims 1 to 47, further comprising measuring the thrombin level in the subject.

49. 49. The method of any one of claims 1 to 48, further comprising measuring the level of a replacement factor in the subject.

50. 1. A method of treating a bleeding event in a subject with hemophilia without inhibitors, comprising: administering to the subject a fixed dose of about 80 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl; the double-stranded RNAi agent comprises a sense strand and an antisense strand; the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; The 3'-end of the sense strand is shown in the schematic diagram below. 【Chemistry 4】 (Wherein, X is O or S.) conjugating the compound with a ligand as shown in administering a therapeutically effective amount of a replacement factor to a subject, the effective amount of the supplemental factor is reduced compared to the recommended effective amount of the supplemental factor; thereby treating a bleeding event in a subject with hemophilia without inhibitors.

51. 1. A method of treating a bleeding event in a subject with hemophilia with an inhibitor, comprising: administering to the subject a fixed dose of about 80 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of Serpincl; the double-stranded RNAi agent comprises a sense strand and an antisense strand; the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; The 3'-end of the sense strand is shown in the schematic diagram below. 【Chemistry 5】 (Wherein, X is O or S.) conjugating the compound with a ligand as shown in administering a therapeutically effective amount of a bypass agent to a subject, the effective amount of the bypassing agent is reduced compared to the recommended effective amount of the bypassing agent; thereby treating a bleeding event in a subject with hemophilia who has an inhibitor.

52. 52. The method of claim 50 or 51, wherein a fixed dose of the RNAi agent is administered subcutaneously to the subject.

53. 52. The method of claim 50 or 51, wherein the fixed dose of the RNAi agent is administered to the subject once a month.

54. 52. The method of claim 50 or 51, wherein the hemophilia is hemophilia A.

55. 52. The method of claim 50 or 51, wherein the hemophilia is hemophilia B.

56. 52. The method of claim 50 or 51, wherein the hemophilia is hemophilia C.

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