Methods and compositions for treating SERPINC1-associated disorders

Abstract

To provide iRNA, e.g., double stranded ribonucleic acid (dsRNA), targeting the Serpinc1 gene, compositions, and methods of using such iRNA, e.g., dsRNA, and compositions to inhibit expression of Serpinc1 and to treat subjects having a Serpinc1-associated disease, e.g., a bleeding disorder, such as hemophilia.SOLUTION: A method of preventing at least one symptom in a subject having a disorder that should benefit from reduction in Serpinc1 expression comprises administering to the subject a fixed dose of about 25 mg to about 100 mg of a double stranded ribonucleic acid (RNAi) agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to 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.

[0002] 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 PCT Patent Application No. PCT / US2015 / 030337, filed May 12, 2015. 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 / 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. 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 PCT Patent Application No. PCT / US2013 / 038218, filed April 25, 2013. This application is also related to PCT Patent Application No. PCT / US2012 / 065601, filed November 16, 2012. The entire contents of each of the foregoing patent applications are incorporated herein by reference.

[0004] 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 December 6, 2016, is named 121301-05220_SL.TXT and is 21,140 bytes in size. [Background technology]

[0005] 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.

[0006] 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.

[0007] 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]

[0008] 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 "bypassing agents" 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]

[0009] The present invention provides a method for treating a subject having a disorder that would benefit from inhibiting or reducing expression of the Serpinc1 gene, e.g., a bleeding disorder such as hemophilia, using an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the Serpinc1 gene to inhibit expression of the Serpinc1 gene.

[0010] The present invention is based at least in part on the surprising discovery that GalNAc-linked double-stranded RNAi agents containing specific chemical modifications at very low doses (for example, at least about 30 times lower than the doses taught in the art) show exceptional potency in inhibiting the expression of Serpincl, and exceptional duration of inhibition of Serpincl expression.Specifically, low doses of RNAi agents containing GalNAc ligands and sense and antisense strands in which substantially all of the nucleotides are modified, for example, RNAi agents containing one or more motifs of three identical modifications at three consecutive nucleotides, including one motif at or near the cleavage site of the agent, six phosphorothioate linkages, and GalNAc ligands, are shown herein to be exceptionally effective and long-lasting in silencing the activity of Serpincl gene.

[0011] Therefore, in one aspect, the present invention provides a method for preventing at least one symptom of a subject with a disorder that will benefit from reducing Serpincl expression.The method comprises administering to the subject a double-stranded RNAi agent at a dose of about 0.200mg / kg to about 1.825mg / kg, the double-stranded RNAi agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that are different from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that are different from the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides, and substantially all the nucleotides of the sense strand and substantially all the nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with the ligand, for example, the ligand is bound to the 3 ' end of the sense strand.

[0012] In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from reduced Serpincl expression. The method includes administering to the subject a double-stranded RNAi agent at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi The agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, 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 double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that is attached at the 3' end of the sense strand.

[0013] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0014] In another embodiment, the sense and antisense strands comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one sequence listed in any one of Tables 2 and 3.

[0015] In some embodiments, the modified nucleotides are independently selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In yet other embodiments, the modified nucleotides are selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-hydroxyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

[0016] In another embodiment of the double-stranded RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0017] 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.

[0018] 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.

[0019] In certain embodiments, the ligand is: [ka]

[0020] In another aspect, the present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced expression of Serpincl, the method comprising administering to the subject a double-stranded RNAi agent at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding Serpincl, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) is represented by n p ' is a 2 nucleotide overhang and n peach nucleotide in the ' is linked to an adjacent nucleotide via a phosphorothioate linkage; N respectively a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications at three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; the sense and antisense strands each independently contain two phosphorothioate linkages at their 5' ends; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker; Thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0021] In another aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced expression of Serpincl, the method comprising administering to the subject a double-stranded RNAi agent at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding Serpincl, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) Represented by: n p ' is a 2 nucleotide overhang and n peach nucleotide in the ' is linked to an adjacent nucleotide via a phosphorothioate linkage; N respectively a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications at three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; the sense and antisense strands each independently contain two phosphorothioate linkages at their 5' ends; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker; Thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0022] In one embodiment, the double-stranded RNAi agent is administered to the subject in a single dose or in two or more doses, for example, three, four, five, or six doses.

[0023] In one embodiment, the double-stranded RNAi agent is administered to the subject once a month, once every six weeks, once every two months, once a quarter, or as needed.

[0024] The double-stranded RNAi agent may be administered at a concentration of, for example, 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, 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, g / 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 0.2 0.250 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 1.50 0 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 0.42. 5 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 0.4 75 to about 1.700 mg / kg, about 0.475 to about 1.600 mg / 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 0.50 0 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 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 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 0.92 The subject can be administered a monthly dose of 5 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, e.g., once a month for 1, 2, 3, 4, 5, 6, 7, 8 or more months.

[0025] The subject can be a human, such as a human with an acquired or inherited bleeding disorder, eg, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0026] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0027] In one embodiment, administration of the double-stranded RNAi agent to a subject results in increased blood clotting and / or decreased Serpinc1 protein accumulation.

[0028] In one embodiment, the method further comprises measuring the thrombin level in the subject.

[0029] The double-stranded RNAi agent can be administered subcutaneously or intravenously.

[0030] In one embodiment, substantially all nucleotides of the antisense strand and substantially all nucleotides of the sense strand of the RNAi agent comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification. In one embodiment, every nucleotide of the sense strand and every nucleotide of the antisense strand of the RNAi agent is a modified nucleotide.

[0031] In one embodiment, the YYY motif occurs at or near the site of cleavage in the sense strand.

[0032] In one embodiment, the Y'Y'Y' motif occurs at positions 11, 12, and 13 from the 5' end of the antisense strand.

[0033] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0034] Each strand may have 15 to 30 nucleotides, or 19 to 30 nucleotides.

[0035] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

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

[0037] In one embodiment, the ligand is attached to the 3' end of the sense strand.

[0038] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0039] In one embodiment, the base pair at the first position of the 5' end of the antisense strand of the duplex is an AU base pair.

[0040] In one embodiment, the RNAi agent is AD-57213 ((sense (5'→3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf (SEQ ID NO: 13); antisense (5'→3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; 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)).

[0041] 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.

[0042] 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).

[0043] In one aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpincl expression. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated to a ligand that binds at the 3' end of the sense strand.

[0044] In another aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpincl expression. The method includes administering to the subject a double-stranded ribonucleic acid (RNAi) agent at a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, and the antisense strand comprises the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15). The double-stranded RNAi agent comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ from the sequence by no more than 3 nucleotides, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that is attached at the 3' end of the sense strand.

[0045] The double-stranded RNAi agent can be administered to a subject in two or more doses.

[0046] 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.

[0047] The double-stranded RNAi agent may be administered at a concentration of, for example, 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, 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, g / 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 0.2 0.250 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 1.50 0 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 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 0.42 5 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, about 0.475 to about 1.500 mg / kg, about 0.475 to about 1.400 mg / kg, about 0.475 to about 1.200 mg / kg 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 to about 1.500 mg / kg, about 0.900 to about 1.400 mg / kg, about 0.900 to about 1.40 0 mg / kg, about 0.900 to about 1.200 mg / kg, about 0.900 to about 1.100 mg / kg, about 0.900 to about 1.000 mg / kg, about 0.925 to 1.825 mg / kg, about 0.925 to 1.800 mg / kg, about 0.925 to about 1.700 mg / kg, about 0.925 to 1.600 mg / kg, about 0.925 to about 1.500 mg / kg, about 0.925 to 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.It can be administered to subjects as a monthly dose of 925 to 1,000 mg / kg.

[0048] In some embodiments, the dose of the double-stranded RNAi agent is administered to the subject as a monthly dose of about 0.200 mg / kg to about 0.250 mg / kg; or as a monthly dose of about 0.425 mg / kg to about 0.475 mg / kg; or as a monthly dose of about 0.875 mg / kg to about 0.925 mg / kg; or as a monthly dose of about 1.775 mg / kg to about 1.825 mg / kg.

[0049] In one embodiment, the double-stranded RNAi agent is administered to the subject as a monthly dose of 0.225 mg / kg.

[0050] In another embodiment, the double-stranded RNAi agent is administered to the subject as a monthly dose of 0.450 mg / kg.

[0051] In yet another embodiment, the double-stranded RNAi agent is administered to the subject as a monthly dose of 0.900 mg / kg.

[0052] In one embodiment, the double-stranded RNAi agent is administered to the subject as a monthly dose of 1.800 mg / kg.

[0053] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0054] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by about 70% to about 95%.

[0055] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0056] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0057] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%.

[0058] In yet another embodiment, administration of the dose of the double-stranded RNAi agent to the subject reduces the subject's on-demand annualized bleeding rate (ABR) by about 80 to about 95% when compared to the median historical ABR of subjects not administered the double-stranded RNAi agent who have a disorder that would benefit from reduced Serpinc1 expression.

[0059] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0060] In one embodiment, the subject is a human.

[0061] The disorder may be an acquired or inherited bleeding disorder, for example, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0062] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0063] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0064] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0065] 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.

[0066] The region of complementarity may be at least 17 or 19 nucleotides in length.

[0067] 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.

[0068] In one embodiment, each strand is no more than 30 nucleotides in length.

[0069] At least one strand of the double-stranded RNAi agent has a 3' overhang of at least 1 nucleotide or at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10 In some embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0070] 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.

[0071] 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.

[0072] In certain embodiments, the ligand is: [ka]

[0073] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0074] In one embodiment, X is O.

[0075] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0076] 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).

[0077] 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 ribose A, C, G, or U; 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.

[0078] In one aspect, the present invention provides a method for preventing at least one symptom of a subject with a disorder that would benefit from reducing Serpincl expression.The method comprises administering to the subject a fixed dose of about 25mg to about 100mg of double-stranded RNAi agent, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides, and substantially all the nucleotides of the sense strand and substantially all the nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated with a ligand that binds to the 3' end of the sense strand.

[0079] In another aspect, the present invention provides a method for treating a subject with a disorder that would benefit from reduced Serpincl expression. The method comprises administering to the subject a fixed dose of about 25 mg to about 100 mg of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, and the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides. the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that is attached at the 3' end of the sense strand.

[0080] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0081] In another embodiment, the sense and antisense strands comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one sequence listed in any one of Tables 2 and 3.

[0082] In some embodiments, the modified nucleotides are independently selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In yet other embodiments, the modified nucleotides are selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-hydroxyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

[0083] In another embodiment of the double-stranded RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0084] In another aspect, the present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced expression of Serpincl. The method comprises administering to the subject a fixed dose of about 25 mg to about 100 mg of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding Serpincl, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) Represented by: n p ' is a 2 nucleotide overhang and n p each nucleotide in the ' is linked to an adjacent nucleotide via a phosphorothioate linkage; N respectively a and N a' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro. It is a b-modification; the sense and antisense strands each independently contain two phosphorothioate linkages at their 5' ends; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker; Thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0085] In another aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpincl expression. The method comprises administering to the subject a fixed dose of about 25 mg to about 100 mg of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding Serpincl, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (IIIe): Sense: 5'-N a -YYY-N a -3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-5'(IIIe) Represented by: n p ' is a 2 nucleotide overhang and n p each nucleotide in the ' is linked to an adjacent nucleotide via a phosphorothioate linkage; N respectively a and Na ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications at three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; the sense and antisense strands each independently contain two phosphorothioate linkages at their 5' ends; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker; Thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0086] In one embodiment, the double-stranded RNAi agent is administered to the subject in a single dose or in two or more doses, for example, three, four, five, or six doses.

[0087] In one embodiment, 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.

[0088] 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 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, It can be administered to a subject as a fixed dose of 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, e.g., as a fixed dose for 1, 2, 3, 4, 5, 6, 7, 8 months or more.

[0089] In some embodiments, the double-stranded RNAi agent can be administered to a subject as 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.

[0090] The subject can be a human, such as a human with an acquired or inherited bleeding disorder, eg, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0091] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0092] In one embodiment, administration of the double-stranded RNAi agent to a subject results in increased blood clotting and / or decreased Serpinc1 protein accumulation.

[0093] In one embodiment, the method further comprises measuring the thrombin level in the subject.

[0094] The double-stranded RNAi agent can be administered subcutaneously or intravenously.

[0095] In one embodiment, substantially all nucleotides of the antisense strand and substantially all nucleotides of the sense strand of the RNAi agent comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification. In one embodiment, every nucleotide of the sense strand and every nucleotide of the antisense strand of the RNAi agent is a modified nucleotide.

[0096] In one embodiment, the YYY motif occurs at or near the site of cleavage in the sense strand.

[0097] In one embodiment, the Y'Y'Y' motif occurs at positions 11, 12, and 13 from the 5' end of the antisense strand.

[0098] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0099] Each strand may have 15 to 30 nucleotides, or 19 to 30 nucleotides.

[0100] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0101] In one embodiment, the ligand is: [ka]

[0102] In one embodiment, the ligand is attached to the 3' end of the sense strand.

[0103] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0104] In one embodiment, the base pair at the first position of the 5' end of the antisense strand of the duplex is an AU base pair.

[0105] In one embodiment, the RNAi agent is AD-57213 ((sense (5'→3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf (SEQ ID NO: 13); antisense (5'→3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; 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)).

[0106] 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.

[0107] In another embodiment, the siRNA is an siRNA selected from acetate, citrate, prolamin, carbonate, or is administered with a buffer, such as a buffer containing phosphate or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS).

[0108] In one aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpincl expression. The method includes administering a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, for example, the sense strand of the double-stranded RNAi agent is conjugated to a ligand that binds at the 3' end of the sense strand.

[0109] In another aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpincl expression. The method includes administering to the subject a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that binds at the 3' end of the sense strand.

[0110] The double-stranded RNAi agent can be administered to a subject in two or more doses.

[0111] 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.

[0112] 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.

[0113] 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 9 ... Between 0 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 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 55 mg It can be administered to a subject as a fixed dose of between about 95 mg.

[0114] In some embodiments, the double-stranded RNAi agent can be administered as 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.

[0115] In some embodiments, the double-stranded RNAi agent is administered to the subject as a fixed dose of about 25 mg; or as a fixed dose of about 50 mg; or as a fixed dose of about 80 mg; or as a fixed dose of about 100 mg.

[0116] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0117] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject reduces Serpincl activity in the subject by about 70% to about 95%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than 95%.

[0118] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0119] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0120] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%, 45%, 50%, 55%, or about 60%.

[0121] In yet another embodiment, administration of the dose of the double-stranded RNAi agent to the subject reduces the subject's on-demand annualized bleeding rate (ABR) by about 80% to about 95%, about 80% to about 85%, about 85% to about 90%, or about 90% to about 95%, when compared to the median historical ABR of subjects who have not been administered the double-stranded RNAi agent and who have a disorder that would benefit from reduced Serpinc1 expression.

[0122] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0123] In one embodiment, the subject is a human.

[0124] The disorder may be an acquired or inherited bleeding disorder, for example, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0125] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0126] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0127] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0128] 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.

[0129] The region of complementarity may be at least 17 or 19 nucleotides in length.

[0130] 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.

[0131] In one embodiment, each strand is no more than 30 nucleotides in length.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] In certain embodiments, the ligand is: [ka]

[0136] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0137] In one embodiment, X is O.

[0138] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0139] 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).

[0140] 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 ribose A, C, G, or U; 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.

[0141] 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 ribose A, C, G, or U; 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.

[0142] 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.

[0143] 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).

[0144] In a further aspect, the present invention provides kits for carrying out the methods of the present invention, which may include an RNAi agent of the present invention, instructions for use, and, optionally, means for administering the RNAi agent to a subject.

[0145] In one embodiment, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression. The method includes administering a fixed dose of about 50 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprises a region of complementarity 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 that binds at its 3' end, thereby preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0146] In another aspect, the present invention provides a method of treating a subject having a disorder that would benefit from reduced Serpincl expression, the method comprising administering a fixed dose of about 50 mg of double-stranded ribonucleic acid to a subject having a disorder that would benefit from reduced Serpincl expression. The method includes administering to a subject an (RNAi) agent, wherein a fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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' end, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0147] In one embodiment, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression. The method includes administering a fixed dose of about 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprises a region of complementarity 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 that binds at its 3' end, thereby preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0148] In another aspect, the present invention provides a method for treating a subject with a disorder that would benefit from reduced Serpinc1 expression. The method includes administering a fixed dose of about 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 to a ligand attached at its 3' end, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0149] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject reduces Serpincl activity in the subject by about 70% to about 95%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than 95%.

[0150] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0151] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0152] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%, 45%, 50%, 55%, or about 60%.

[0153] In yet another embodiment, administering the dose of the double-stranded RNAi agent to a subject is indicative of a disorder that would benefit from reduced Serpincl expression. The therapeutic effect of the present invention is to reduce a subject's on-demand annualized bleeding rate (ABR) by about 80% to about 95%, about 80% to about 85%, about 85% to about 90%, or about 90% to about 95% when compared to the median historical ABR of untreated subjects.

[0154] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0155] In one embodiment, the subject is a human.

[0156] The disorder may be an acquired or inherited bleeding disorder, for example, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0157] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0158] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0159] In one embodiment, the double-stranded RNAi agent is administered chronically to the subject.

[0160] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0161] 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.

[0162] The region of complementarity may be at least 17 or 19 nucleotides in length.

[0163] 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.

[0164] In one embodiment, each strand is no more than 30 nucleotides in length.

[0165] 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.

[0166] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand is attached to the RNAi agent via a monovalent, divalent, 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.

[0167] 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.

[0168] In certain embodiments, the ligand is: [ka]

[0169] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0170] In one embodiment, X is O.

[0171] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0172] 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 a 5'-UU and an antisense strand comprising the nucleotide sequence GAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0173] 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 ribose A, C, G, or U; 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.

[0174] 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 ribose A, C, G, or U; 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.

[0175] 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.

[0176] 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).

[0177] In another aspect, the present invention provides kits for carrying out the methods of the present invention, which may include an RNAi agent of the present invention, instructions for use, and, optionally, a means for administering the RNAi agent to a subject.

[0178] In one aspect, the present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpincl expression. The method includes administering to the subject a fixed dose of about 50 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and the double-stranded RNAi agent has a 5'-G The antibody comprises a sense strand comprising the nucleotide sequence GUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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 the 3' end, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0179] In another aspect, the present invention provides a method for treating a subject with a disorder that would benefit from reduced Serpinc1 expression. The method includes administering a fixed dose of about 50 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0180] In one embodiment, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression. The method includes administering a fixed dose of about 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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 in a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0181] In another aspect, the present invention provides a method for treating a subject with a disorder that would benefit from reduced Serpinc1 expression. The method includes administering a fixed dose of about 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0182] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject reduces Serpincl activity in the subject by about 70% to about 95%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more than 95%.

[0183] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0184] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0185] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%, 45%, 50%, 55%, or about 60%.

[0186] In yet another embodiment, administration of the dose of the double-stranded RNAi agent to the subject reduces the subject's on-demand annualized bleeding rate (ABR) by about 80% to about 95%, about 80% to about 85%, about 85% to about 90%, or about 90% to about 95%, when compared to the median historical ABR of subjects who have not been administered the double-stranded RNAi agent and who have a disorder that would benefit from reduced Serpinc1 expression.

[0187] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0188] In one embodiment, the subject is a human.

[0189] The disorder may be an acquired or inherited bleeding disorder, for example, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0190] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0191] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0192] In one embodiment, the double-stranded RNAi agent is administered chronically to the subject.

[0193] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0194] 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.

[0195] In one embodiment, each strand is no more than 30 nucleotides in length.

[0196] 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, e.g., 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, e.g., 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 at least one nucleotide. Including overhang.

[0197] 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.

[0198] 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.

[0199] In certain embodiments, the ligand is: [ka]

[0200] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0201] In one embodiment, X is O.

[0202] In one embodiment, the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuU The antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; 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.

[0203] 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 ribose A, C, G, or U; 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.

[0204] 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).

[0205] In another aspect, the present invention provides kits for carrying out the methods of the present invention, which may include an RNAi agent of the present invention, instructions for use, and, optionally, a means for administering the RNAi agent to a subject.

[0206] In one aspect, the invention provides a method for preventing at least one symptom of a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject about 50 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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, thereby preventing at least one symptom of a subject with a disorder that would benefit from reduced Serpincl expression. At least one symptom in the subject is prevented.

[0207] In another aspect, the invention provides a method of treating a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject about 50 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0208] In one aspect, the invention provides a method of preventing at least one symptom in a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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 wherein the sense strand is conjugated to a ligand attached at its 3' end, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0209] In another aspect, the invention provides a method of treating a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject about 80 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and an antisense strand comprising 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, thereby treating the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0210] In one aspect, the invention provides a method of preventing at least one symptom in a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject a fixed dose of about 50 mg of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and an antisense strand comprising the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), wherein A, C, G, and U are ribose A, C, G, or U; a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; and Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U. or U; s is a phosphorothioate linkage, and the sense strand is conjugated to a ligand that binds at the 3' end, thereby preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0211] In another aspect, the invention provides a method of treating a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject about 50 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and a nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14). The antisense strand comprises a nucleotide sequence, wherein A, C, G, and U are ribose A, C, G, or U; 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, and the sense strand is conjugated to a ligand attached at the 3' end, thereby treating a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0212] In one aspect, the invention provides a method of preventing at least one symptom in a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject about 80 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and wherein the double-stranded RNAi agent has a sense strand comprising the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and a nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14). wherein A, C, G, and U are ribose A, C, G, or U; 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 attached at the 3' end, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0213] In another aspect, the invention provides a method of treating a subject with hemophilia, e.g., hemophilia A (with or without inhibitors), hemophilia B (with or without inhibitors), or hemophilia C (with or without inhibitors). The method includes administering to the subject about 80 mg of a fixed dose of a double-stranded ribonucleic acid (RNAi) agent, wherein the fixed dose of the double-stranded RNAi agent is administered to the subject approximately once a month, and wherein the double-stranded RNAi agent comprises a sense strand comprising the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and a nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14). The antisense strand comprises a nucleotide sequence, wherein A, C, G, and U are ribose A, C, G, or U; 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, and the sense strand is conjugated to a ligand attached at the 3' end, thereby treating a subject with a disorder that would benefit from reduced Serpinc1 expression.

[0214] In one embodiment, administering the dose of the double-stranded RNAi agent to the subject reduces Se in the subject. rpinc1 activity is reduced by about 70% to about 95%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or greater than 95%.

[0215] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0216] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0217] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%, 45%, 50%, 55%, or about 60%.

[0218] In yet another embodiment, administration of the dose of the double-stranded RNAi agent to the subject reduces the subject's on-demand annualized bleeding rate (ABR) by about 80% to about 95%, about 80% to about 85%, about 85% to about 90%, or about 90% to about 95%, when compared to the median historical ABR of subjects who have not been administered the double-stranded RNAi agent and who have a disorder that would benefit from reduced Serpinc1 expression.

[0219] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0220] In one embodiment, the subject is a human.

[0221] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0222] In one embodiment, the double-stranded RNAi agent is administered chronically to the subject.

[0223] In certain embodiments, the ligand is: [ka]

[0224] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0225] In one embodiment, X is O.

[0226] 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 ribose A, C, G, or U; 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.

[0227] 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).

[0228] In another aspect, the present invention provides kits for carrying out the methods of the present invention, which may include an RNAi agent of the present invention, instructions for use, and, optionally, a means for administering the RNAi agent to a subject.

[0229] In one aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpincl expression. The method includes administering a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent to the subject, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that binds at the 3' end of the sense strand.

[0230] In another aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced Serpincl expression. 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, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that binds at the 3' end of the sense strand.

[0231] The double-stranded RNAi agent can be administered to a subject in two or more doses.

[0232] 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.

[0233] 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.

[0234] A double-stranded RNAi agent can be administered to a subject as a fixed dose of, for example, 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 40 mg and about 80 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 40 mg and about 85 mg, between about 40 mg and about 75 mg, between about 40 mg and about 65 mg, or between about 40 mg and about 55 mg.

[0235] In some embodiments, the double-stranded RNAi agent can be administered as a fixed dose of 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, or about 90 mg.

[0236] In some embodiments, the double-stranded RNAi agent is administered to the subject as a fixed dose of about 40 mg; or as a fixed dose of about 50 mg; or as a fixed dose of about 80 mg; or as a fixed dose of about 90 mg.

[0237] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0238] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by about 70% to about 95%.

[0239] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0240] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0241] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%.

[0242] In yet another embodiment, administration of the dose of the double-stranded RNAi agent to the subject reduces the subject's on-demand annualized bleeding rate (ABR) by about 80 to about 95% when compared to the median historical ABR of subjects not administered the double-stranded RNAi agent who have a disorder that would benefit from reduced Serpinc1 expression.

[0243] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0244] In one embodiment, the subject is a human.

[0245] The disorder may be an acquired or inherited bleeding disorder, for example, a bleeding disorder such as hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C.

[0246] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0247] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0248] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0249] 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.

[0250] The region of complementarity may be at least 17 or 19 nucleotides in length.

[0251] 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.

[0252] In one embodiment, each strand is no more than 30 nucleotides in length.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] In certain embodiments, the ligand is: [ka]

[0257] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0258] In one embodiment, X is O.

[0259] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0260] 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).

[0261] 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 ribose A, C, G, or U; 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.

[0262] 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 ribose A, C, G, or U; 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.

[0263] 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.

[0264] 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).

[0265] In another aspect, the present invention provides kits for carrying out the methods of the present invention, which may include an RNAi agent of the present invention, instructions for use, and, optionally, a means for administering the RNAi agent to a subject.

[0266] In one aspect, the present invention provides a method for inhibiting expression of Serpincl in a subject, 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, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that is attached at the 3' end of the sense strand.

[0267] In another aspect, the present invention provides a method for inhibiting expression of Serpincl in a subject, 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, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 double-stranded RNAi agent comprises a ligand, e.g., the sense strand of the double-stranded RNAi agent is conjugated to a ligand that is attached at the 3' end of the sense strand.

[0268] The double-stranded RNAi agent can be administered to a subject in two or more doses.

[0269] In some embodiments, the double-stranded RNAi agent is administered to the subject once a month, once every five weeks, once every six weeks, or once a month. It is administered once weekly, once every seven weeks, once every two months, once quarterly, or as needed.

[0270] 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.

[0271] A double-stranded RNAi agent can be administered to a subject as a fixed dose of, for example, 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 40 mg and about 80 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 40 mg and about 85 mg, between about 40 mg and about 75 mg, between about 40 mg and about 65 mg, or between about 40 mg and about 55 mg.

[0272] In some embodiments, the double-stranded RNAi agent can be administered as a fixed dose of 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, or about 90 mg.

[0273] In some embodiments, the double-stranded RNAi agent is administered to the subject as a fixed dose of about 40 mg; or as a fixed dose of about 50 mg; or as a fixed dose of about 80 mg; or as a fixed dose of about 90 mg.

[0274] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0275] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject reduces Serpinc1 activity in the subject by about 70% to about 95%.

[0276] In one embodiment, the subject is a human.

[0277] In one embodiment, the subject has a disorder that would benefit from reduced Serpincl expression. The disorder may be an acquired or inherited bleeding disorder, such as a hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C.

[0278] In one embodiment, the subject has hemophilia A and is an inhibitor subject. In another embodiment, the subject has hemophilia B and is an inhibitor subject. In yet another embodiment, the subject has hemophilia C and is an inhibitor subject.

[0279] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without a disorder that would benefit from reduced Serpinc1 expression.

[0280] In one embodiment, administration of the dose of the double-stranded RNAi agent to a subject is sufficient to achieve peak thrombin formation levels in the subject at approximately the same level as that achieved by administration of Factor VIII to the subject.

[0281] In another embodiment, administration of the dose of the double-stranded RNAi agent to the subject is sufficient to achieve a peak thrombin formation level in the subject of greater than about 40%.

[0282] In yet another embodiment, administration of the dose of double-stranded RNAi agent to a subject comprises administering to the subject a dose of Serp The subject's on-demand annualized bleeding rate (ABR) is reduced by about 80 to about 95% when compared to the median historical ABR of subjects not receiving the double-stranded RNAi agent who have a disorder that would benefit from reduced inc1 expression.

[0283] In one embodiment, the double-stranded RNAi agent is administered with a buffer solution, such as a buffer solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0284] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0285] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0286] 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.

[0287] The region of complementarity may be at least 17 or 19 nucleotides in length.

[0288] 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.

[0289] In one embodiment, each strand is no more than 30 nucleotides in length.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] In certain embodiments, the ligand is: [ka]

[0294] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka] X is O or S.

[0295] In one embodiment, X is O.

[0296] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0297] 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).

[0298] 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 ribose A, C, G, or U; 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.

[0299] 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 ribose A, C, G, or U; 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.

[0300] 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.

[0301] 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).

[0302] In another aspect, the present invention provides kits for carrying out the methods of the present invention, which may include an RNAi agent of the present invention, instructions for use, and, optionally, a means for administering the RNAi agent to a subject. [Brief explanation of the drawings]

[0303] [Figure 1A] FIG. 1A is a graph showing the effect of a single subcutaneous dose of 0.03 mg / kg AD-57213 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 AD-57213 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 AD-57213 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 AD-57213 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 AD-57213 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 AD-57213 on plasma AT (Serpincl) protein levels in one healthy human subject. [Figure 3] FIG. 3 is a graph showing the relationship 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 AD-57213. [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 AD-57213 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 AD-57213 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 AD-57213 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 AD-57213 on peak thrombin levels in human subjects with hemophilia A or B. [Figure 6B] FIG. 6B is a graph showing the effect of multiple doses of 0.015 mg / kg or 0.045 mg / kg AD-57213 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 AD-57213 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 AD-57213 on thrombin generation by quartile decline in AT. [Figure 10A] FIG. 10A is a graph showing the relative AT activity in relation to the percentage of peak thrombin formation achieved by factor VIII as measured in subjects administered 225 mcg / kg qM AD-57213. [Figure 10B] FIG. 10B 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 1800 mcg / kg qM AD-57213. [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 AD-57213. [Figure 11] FIG. 11 is a graph showing the effect of multiple doses of AD-57213 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 a Phase I clinical trial of AD-57213. [Figure 13A] Figure 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 AD-57213 before the start of the study, during the initiation portion of the study, and during the observation 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 AD-57213. [Figure 14A] FIG. 14A 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 AD-57213 at 50 mg 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 AD-57213 at 50 mg 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 AD-57213 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 AD-57213 at 50 mg once monthly. [Figure 14E] FIG. 14E is a graph showing the relative AT activity in relation to the percentage of peak thrombin formation achieved by factor VIII, measured in inhibitor subjects receiving a fixed dose of AD-57213 at 50 mg 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 AD-57213 once monthly. [Figure 15] FIG. 15 is a graph showing the effect of multiple doses of 50 mg or 80 mg of AD-57213 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 AD-57213 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 AD-57213. [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 AD-57213 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 AD-57213 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 AD-57213. [Figure 19A] Figure 19A is a graph showing the effect of multiple doses of 50 mg or 80 mg of AD-57213 on mean AT (Serpinc1) activity relative to baseline in human subjects with hemophilia A or B without inhibitors in a Phase II open-label extension (OLE) study of AD-57213. [Figure 19B] 19B is a graph showing the effect of multiple doses of 50 mg or 80 mg of AD-57213 on peak thrombin formation in human subjects with hemophilia A or B without inhibitors in a Phase II open-label extension (OLE) study of AD-57213. The shaded area of ​​the graph represents the range of peak thrombin levels observed in healthy human volunteers (HV) administered AD-57213, indicating less than 25% AT knockdown in the Phase I study of AD-57213 described in Example 1. The dotted line within the HV range represents the median peak thrombin levels observed in healthy human volunteers (HV), indicating less than 25% AT knockdown with administration of AD-57213 in the Phase I study of AD-57213 described in Example 1. [Figure 20A] FIG. 20A is a table showing bleeding event data for subjects enrolled in a Phase II OLE clinical trial of AD-57213. [Figure 20B]FIG. 20B is a graph showing the median annualized bleeding rate (ABR) for all subjects in a Phase II OLE clinical trial of AD-57213 before the start of the study, during the initiation portion of the study, and during the observational portion of the study. DETAILED DESCRIPTION OF THE INVENTION

[0304] The present invention is based, at least in part, on the surprising discovery that very low doses (e.g., doses at least about 30-fold lower than those taught in the art) of GalNAc-linked double-stranded RNAi agents containing specific chemical modifications exhibit exceptional potency in inhibiting Serpincl expression, as well as exceptional duration of inhibition of Serpincl expression. Specifically, low doses of RNAi agents containing a GalNAc ligand and in which substantially all of the nucleotides are modified nucleotides, for example, RNAi agents containing one or more motifs of three identical modifications at three consecutive nucleotides, including one motif at or near the cleavage site of the agent, six phosphorothioate linkages, and a GalNAc ligand, are shown herein to be exceptionally effective and long-lasting in silencing the activity of the Serpincl gene.

[0305] Thus, the present invention provides methods for preventing at least one symptom, e.g., bleeding, in a subject with a disorder that would benefit from inhibiting or reducing expression of the Serpinc1 gene, e.g., a Serpinc1-associated disease, such as hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C), using iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the Serpinc1 gene. The present invention further provides methods for treating a subject with a disorder that would benefit from inhibiting or reducing expression of the Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C), using iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the Serpinc1 gene.

[0306] 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, The RNA strand (antisense strand) comprises an RNA strand having a region of 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the Serpinc1 gene.

[0307] 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.

[0308] 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.

[0309] The following detailed description describes how to make and use compositions containing iRNA to inhibit expression of the Serpincl gene, as well as compositions, uses, etc. for treating subjects with diseases and disorders that would benefit from the inhibition and / or reduction of expression of this gene. Also disclosed are methods.

[0310] 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.

[0311] 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.

[0312] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0313] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0314] 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 number GI:254588059 (NM_000488; SEQ ID NO:1). The sequence of rhesus monkey Serpinc1 mRNA 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 No. GI:58865629 (NM_001012027; SEQ ID NO: 4).

[0315] 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.

[0316] 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 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 disease, disorder that would benefit from reduced Serpincl expression. Or a human, such as a human being treated for a condition.

[0317] 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), and improvement or reduction in 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.

[0318] 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.

[0319] As used herein, " prevention " or " preventing ", when used in relation to a disease, disorder or condition that would benefit from the reduction of Serpincl gene expression, refers to the reduction in the likelihood that a subject will develop symptoms associated with the disease, disorder or condition, such as bleeding. For example, when an individual with one or more risk factors for bleeding does not develop bleeding, or develops bleeding with less severity, compared with a group of individuals with the same risk factors but who have not undergone treatment as described herein, the likelihood of developing bleeding is reduced. Effective prevention is considered when the onset of symptoms associated with such disease, disorder or condition is reduced (for example, by at least about 10% on the clinically recognized scale for the disease or disorder), or when symptoms are delayed (for example, by days, weeks, months, or years).

[0320] 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.

[0321] A "therapeutically effective amount," as used herein, refers to an RNAi agent that, when administered to a subject with a bleeding disorder and bleeding, is sufficient to result in 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpincl gene, and is a sequence of the primary transcription product. This includes mRNA, which is the product of RNA processing. 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 an mRNA molecule formed during transcription of the Serpincl gene.

[0327] 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 up to 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 contemplated as part of the invention.

[0328] 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.

[0329] "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.

[0330] 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.

[0331] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, e.g., a 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 a cell is degraded into siRNAs by a type III endonuclease known as Dicer. (Sharp et al., (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein et al., (2001) Nature 409:363). The siRNAs are 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). Therefore, in one aspect, the present invention relates to the single-stranded RNA (siRNA) that is formed in cells, which promotes the formation of RISC complex, and causes the silencing of target gene, i.e., Serpincl gene.Therefore, the term " siRNA " is used herein also to refer to the RNAi as described above.

[0332] In another embodiment, the RNAi agent may be a single-stranded siRNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al. (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any antisense nucleotide sequence described herein can be used as the single-stranded siRNA described herein or as a single-stranded siRNA chemically modified by the method described in Lima et al. (2012) Cell 150;:883-894.

[0333] 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.

[0334] 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.

[0335] As used herein, the term "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleoside linkage, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of a functional group or atom to the internucleoside linkage, sugar moiety, or nucleobase. Modifications suitable for use in the agents of the present invention include any type of modification disclosed herein or known in the art. For purposes of this specification and claims, any such modification used in siRNA-type molecules is referred to as a "RNP." It is included in the category of "AI drugs."

[0336] 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 It may range in length from 0 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0337] 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.

[0338] 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.

[0339] 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) Cell 107:309). )Genes Dev.15:188).

[0340] 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.

[0341] 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.

[0342] 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.

[0343] " 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.

[0344] 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., Serpincl 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 Serpincl nucleotide sequence as defined herein. A region of complementarity that is not completely complementary to a target sequence. In this case, mismatches may occur 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] As used herein, a "complementary" sequence is a complementary sequence with respect to its ability to hybridize. As long as the above requirements are met, the base pair may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, including, but not limited to, G:U wobble or Hoogsteen base pairing.

[0350] 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.

[0351] 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, a polynucleotide is complementary to at least a portion of Serpinc1 mRNA if the sequence is substantially complementary to a contiguous portion of an mRNA encoding Serpinc1.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] The term "inhibiting," as used herein, is used synonymously with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes all levels of inhibition.

[0356] 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.

[0357] "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.

[0358] 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).

[0359] 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:

number

[0360] 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.

[0361] Contacting the cells in vitro can be done, for example, by incubating the cells with an RNAi agent. Contacting the cells in vivo can be done, for example, by incubating the cells with an RNAi agent. The RNAi agent can be injected into or near a certain tissue, or the RNAi agent can be injected into another area, for example, bloodstream or subcutaneous space, so that the RNAi agent can then reach the tissue of the cell that is contacted.For example, the RNAi agent can comprise and / or be combined with a ligand, for example, GalNAc3, that guides the RNAi agent to the target site, for example, liver.The method of contacting in vitro and in vivo can also be combined.For example, cell can be contacted with the RNAi agent in vitro, and then transplanted into subject.

[0362] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA to a cell" by promoting 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. Introducing the iRNA into a cell can be done in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved via beta-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Application Publication No. 2005 / 0281781, the entire contents of which 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.

[0363] II. The Methods of the Invention The invention provides therapeutic and prophylactic methods comprising administering an iRNA agent or a pharmaceutical composition comprising an iRNA agent of the invention to a subject having a Serpinc1-associated disease, e.g., a bleeding disorder, e.g., hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C). In some embodiments of the invention, the method further comprises administering an additional therapeutic agent to the subject.

[0364] In certain embodiments of the invention, for example, when a double-stranded RNAi agent contains one or more motifs of three identical modifications at three consecutive nucleotides, including one motif at or near the cleavage site of the agent, six phosphorothioate linkages, and a GalNAc ligand, such an agent may be administered at a dose of about 0.200 to 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 1.60 0 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 1.10 0 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 0.40 0 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 1.00 0 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, about 0.475 to about 1.500 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 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 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 to about 1.000 mg / kg, about 0.925 to about 1.825 The RNAi agent may be administered at a dose of 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. Values ​​and ranges intermediate to the above-mentioned recited values ​​are also contemplated as 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.

[0365] For example, the RNAi agent, e.g., the RNAi agent in the pharmaceutical composition, may be 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 g, 0.325mg / kg, 0.35mg / kg, 0.375mg / kg, 0.4mg / kg, 0.425mg / kg, 0.45mg / kg, 0.475mg / kg, Approximately 0.5mg / kg, 0.525mg / kg, 0.55mg / kg, 0.575mg / kg, approximately 0.6mg / kg, 0.625mg / kg, 0.65mg / kg, 0.6 75mg / kg, about 0.7mg / kg, 0.725mg / kg, 0.75mg / kg, 0.775mg / kg, about 0.8mg / kg, 0.925mg / kg, 0.95m g / kg, 0.975mg / kg, approximately 1.0mg / kg, 1.025mg / kg, 1.05mg / kg, 1.075mg / kg, approximately 1.1mg / kg, 1.125mg / 1.5 mg / kg, 1.15 mg / kg, 1.175 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 this invention.

[0366] Thus, in one aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced Serpinc1 expression, such as a bleeding disorder, e.g., hemophilia. The method includes administering to the subject a prophylactically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, of an iRNA agent, e.g., a dsRNA, of the present invention (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.

[0367] In another aspect, the invention provides a method of treating a subject having 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., a dose of about 0.200 mg / kg to about 1.800 mg / kg, of an iRNA agent that targets the Serpinc1 gene or a pharmaceutical composition comprising an iRNA agent that targets the Serpinc1 gene, thereby treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0368] In another aspect, the invention provides for the use of an iRNA, e.g., a dsRNA, of the invention at a prophylactically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, 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 Serpinc1 expression, such as hemophilia.

[0369] In another aspect, the invention provides use of an iRNA agent of the invention at a prophylactically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, 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 Serpinc1 expression, such as hemophilia.

[0370] In another aspect, the invention provides for the use of an iRNA agent of the invention at a therapeutically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, to treat a subject, e.g., a subject who would benefit from reduced and / or inhibited Serpincl expression.

[0371] In yet another aspect, the present invention provides a method for the treatment of a subject, e.g., a subject who would benefit from the reduction and / or inhibition of Serpincl expression, such as a subject with a bleeding disorder, e.g., hemophilia. The present invention provides the use of an iRNA agent of the invention that targets the Serpincl gene, e.g., a dsRNA, or a pharmaceutical composition comprising an iRNA agent that targets the Serpincl gene at a therapeutically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, in the manufacture of a medicament for treating a disease.

[0372] In some embodiments of the invention, for example, when a double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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' end, such an agent is administered at a dose of about 0.200 to 1.825 mg / kg, e.g., as a dose of about 0.200 mg / kg to about 0.250 mg / kg; or as a dose of about 0.425 mg / kg to about 0.475 mg / kg; or as a dose of about 0.875 mg / kg to about 0.925 mg / kg; or as a dose of about 1.775 mg / kg to about 1.825 mg / kg.

[0373] Thus, in one aspect, the present invention 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 dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, of a double-stranded ribonucleic acid (RNAi) agent, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) by no more than 3 nucleotides, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand that binds at its 3' end, thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced Serpinc1 expression.

[0374] In another aspect, the invention is a method of treating a subject having a disorder that would benefit from reduced Serpinc1 expression, e.g., a bleeding disorder, e.g., hemophilia, the method comprising administering to a subject, e.g., a human, a therapeutically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, of a double-stranded ribonucleic acid (RNAi) agent or iRNA agent that targets the Serpinc1 gene, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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' end, thereby providing a method of treating a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0375] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent 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 Serpinc1 expression, such as hemophilia, at a prophylactically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 of the sense strand The present invention provides for the use of double-stranded ribonucleic acid (RNAi) agents in which 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 the 3' end.

[0376] In a further aspect, the invention provides use of a double-stranded ribonucleic acid (RNAi) agent 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 Serpinc1 expression, such as hemophilia, at a prophylactically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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' end.

[0377] In another aspect, the invention provides for the use of a double-stranded ribonucleic acid (RNAi) agent at a therapeutically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, for treating a subject, e.g., a subject that would benefit from reduced and / or inhibited Serpincl expression, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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' end.

[0378] In yet another aspect, the invention provides use of a pharmaceutical composition comprising an iRNA agent, e.g., a dsRNA, or double-stranded ribonucleic acid (RNAi) agent of the invention targeting the Serpinc1 gene in a therapeutically effective dose, e.g., a dose of about 0.200 mg / kg to about 1.825 mg / kg, wherein the double-stranded ribonucleic acid (RNAi) agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 wherein the sense strand is conjugated to a ligand attached at its 3' end, 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.

[0379] In some embodiments, the iRNA agent of the present invention is administered to a subject as a fixed dose. A "fixed dose" (e.g., a dose in mg) means that a single dose of the 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 the iRNA agent of the present invention is based on a predetermined body weight or age.

[0380] 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 80 mg, between about 40 mg and about 100 mg, between about 40 mg and about 50 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.

[0381] In some embodiments, the RNAi agent is administered as 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.

[0382] Thus, in one aspect, the present invention 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 fixed dose of an iRNA agent, e.g., a dsRNA, of the present invention (e.g., a pharmaceutical composition comprising a dsRNA of the present invention) that is prophylactically effective, e.g., 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 fixed dose of an iRNA agent, e.g., a dsRNA, of the present invention (e.g., a pharmaceutical composition comprising a dsRNA of the present invention) that is prophylactically effective, e.g., about 50 mg, 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.

[0383] In another aspect, the present invention provides a method for 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., 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 of the invention, e.g., a dsRNA (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 comprises administering to the subject a therapeutically effective dose, e.g., a fixed dose of about 80 mg, of an iRNA agent of the invention, e.g., a dsRNA (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.

[0384] In another aspect, the present invention provides for 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 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 Serpinc1 expression, such as hemophilia. In one embodiment, the present invention provides for 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 Serpinc1 expression, such as hemophilia. In another embodiment, the present invention provides for 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 Serpinc1 expression, such as hemophilia. Provided is the use of a prophylactically effective dose, e.g., a fixed dose of about 80 mg, of an iRNA, e.g., a dsRNA, of the present invention to prevent at least one symptom in a subject suffering from a disorder that would benefit from reduced and / or inhibited pinc1 expression.

[0385] In a further aspect, the invention provides for the use of an iRNA agent of the invention in a prophylactically effective dose, e.g., a fixed dose of about 25 mg to about 100 mg, 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 Serpinc1 expression, such as hemophilia. In one embodiment, the invention provides for the use of an iRNA agent of the 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., a disorder that would benefit from reduced and / or inhibited Serpinc1 expression, such as hemophilia. In another embodiment, the invention provides for the use of an iRNA agent of the 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., a disorder that would benefit from reduced and / or inhibited Serpinc1 expression, such as hemophilia.

[0386] In another aspect, 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 25 mg to about 100 mg, to treat a subject, e.g., a subject who 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 who 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 who would benefit from reduced and / or inhibited Serpincl expression.

[0387] 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., a fixed dose of 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.

[0388] In some embodiments of the invention, for example, when a double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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' end, such an agent can be administered at a fixed dose of about 25 mg to about 100 mg, e.g., as a fixed dose of about 25 mg; or as a fixed dose of about 50 mg; or as a fixed dose of about 80 mg. or administered as a fixed dose of about 100 mg. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0389] Thus, in one aspect, the present invention 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 dose, e.g., a fixed dose of about 25 mg to about 100 mg, of a double-stranded ribonucleic acid (RNAi) agent, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 consecutive 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 that binds at its 3' end, thereby preventing at least one symptom in a subject with 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.

[0390] In another aspect, the present invention provides a method for 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., a fixed dose of about 25 mg to about 100 mg, of a double-stranded ribonucleic acid (RNAi) agent or an iRNA agent targeting the Serpinc1 gene, wherein the double-stranded ribonucleic acid (RNAi) agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 is conjugated to a ligand attached at its 3' end, thereby treating the subject with 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.

[0391] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for use in a prophylactically effective dose, e.g., a fixed dose of about 25 mg to about 100 mg, 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 Serpinc1 expression, such as hemophilia, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 consecutive 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' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0392] In a further aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent in the manufacture of a medicament for preventing at least one symptom of a subject suffering from a bleeding disorder, e.g., a disorder that would benefit from reduced and / or inhibited Serpinc1 expression, such as hemophilia, at a prophylactically effective dose, e.g., about 25 mg to about 100 mg fixed dose, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), and substantially Provided is the use of double-stranded ribonucleic acid (RNAi) agent, wherein all nucleotides and substantially all nucleotides of antisense strand are modified nucleotides, and sense strand is conjugated with the ligand that binds at 3' end.In one embodiment, the fixed dose is 50mg.In another embodiment, the fixed dose is 80mg.

[0393] In another aspect, the present invention provides the use of a double-stranded ribonucleic acid (RNAi) agent in a therapeutically effective dose, for example, a fixed dose of about 25 mg to about 100 mg, for treating a subject, for example, a subject who would benefit from reduced and / or inhibited Serpincl expression, the double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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.

[0394] In yet another aspect, the present invention provides use of an iRNA agent, e.g., a dsRNA, or a pharmaceutical composition comprising a therapeutically effective dose, e.g., about 25 mg to about 100 mg, of a double-stranded ribonucleic acid (RNAi) agent of the present 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. The double-stranded ribonucleic acid (RNAi) agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 consecutive 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' end. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

[0395] 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.

[0396] The reduction in gene expression can be evaluated by any method known in the art.For example, the reduction in Serpinc1 expression can be determined by using methods familiar to those skilled in the art, such as Northern blotting, qRT-PCR to determine the mRNA expression level of Serpinc1, by using methods familiar to those skilled in the art, such as Western blotting, immunological techniques, etc. to determine the protein level of Serpinc1, and / or by confirming the biological activity of Serpinc1, for example, by affecting one or more molecules related to the blood coagulation mechanism of cells (or blood coagulation itself in an in vivo environment).In one embodiment, for example, whole blood ROTEM® Thromboelast® can be used to evaluate Serpinc1 expression. Thrombin formation time, clot formation time and / or clotting time are determined using thrombometry analysis.

[0397] 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%.

[0398] 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.

[0399] 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.

[0400] 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.

[0401] 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.

[0402] 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.

[0403] In one embodiment, a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 wherein the sense strand is conjugated to a ligand attached at its 3' end, is administered to a subject as a monthly dose of about 0.200 mg / kg to about 0.250 mg / kg, e.g., about 0.225 mg / kg.

[0404] In another embodiment, a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 wherein the sense strand is conjugated to a ligand attached at its 3' end, is administered to a subject as a monthly dose of about 0.425 mg / kg to about 0.475 mg / kg, e.g., about 0.450 mg / kg.

[0405] In yet another embodiment, a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 wherein the sense strand is conjugated to a ligand attached at its 3' end, is administered to a subject as a monthly dose of about 0.875 mg / kg to about 0.925 mg / kg, e.g., about 0.900 mg / kg.

[0406] In one embodiment, a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity 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 wherein the sense strand is conjugated to a ligand attached at its 3' end, is administered to a subject as a monthly dose of about 1.775 mg / kg to about 1.825 mg / kg, e.g., about 1.800 mg / kg.

[0407] In one embodiment, the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a complementary 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 as 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.

[0408] Administration can be repeated on a regular basis, for example, once a month for one, two, three, four, or longer periods. After the initial treatment regimen, treatment can also be administered on a less frequent basis. For example, administration once a month for three months. Dosing can then be repeated once quarterly for a year or longer.

[0409] Thus, in some embodiments, the RNAi agent is administered in a dosing 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.

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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, the patient can be monitored for undesirable immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0414] 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.

[0415] 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.

[0416] Alternatively, the iRNA of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0417] Subjects who would benefit from reducing and / or inhibiting Serpinc1 gene expression are those with a bleeding disorder described herein, e.g., an inherited bleeding disorder or an acquired bleeding disorder. In one embodiment, the subject with an inherited bleeding disorder is a subject with hemophilia, e.g., hemophilia. In one embodiment, a subject with an inherited 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 reducing and / or inhibiting Serpincl gene expression include therapeutic treatments (e.g., on-demand, e.g., the subject is bleeding (spontaneously or bleeding as a result of trauma) and not clotting) and prophylactic treatments (e.g., the subject is not bleeding and / or undergoing surgery).

[0418] 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.

[0419] 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.

[0420] The iRNA and additional therapeutic agent(s) and / or treatment(s) can be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent(s) can be administered as part of separate compositions or at separate times and / or by other methods known in the art or described herein.

[0421] In one embodiment, the present invention provides a method for the treatment of rheumatoid arthritis with the compound AD-57213 (sense strand: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and antisense The present invention provides a method for treating a subject suffering from a bleeding disorder, e.g., hemophilia, by subcutaneously administering to the subject the following sequence: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; 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.

[0422] In another embodiment, the present invention provides a method for the treatment of atopic dermatitis with a rhodopsin-like disease, comprising administering to a subject a subject a rhodopsin-like disease, comprising administering to a subject a rhodopsin-like disease, the method ... A method is provided for treating a subject suffering from a bleeding disorder, such as hemophilia, by subcutaneously administering to the subject CfaAf-3' (SEQ ID NO: 13) and the antisense strand: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where A, C, G, and U are ribose A, C, G, or U; 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.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] In one embodiment, the RNAi agent may 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 may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs may be the result of one strand being longer than the other, or the result of two strands of the same length being offset. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be another sequence. The first and second strands may also contain additional bases, e.g., to form a hairpin, or other non-basic linker. are joined by

[0428] 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.

[0429] 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.

[0430] 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.

[0431] 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.

[0432] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates, as well as chiral phosphonates, phosphinates, 3'-amino phosphoramidates and amino alkyl phosphonates. These include phosphoramidates, including alkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, where 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] In other embodiments, suitable RNA mimics are considered for use in iRNA, where both sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit, are replaced with novel groups.The base unit is maintained for hybridization with suitable nucleic acid target compounds.Such RNA mimics have been shown to have excellent hybridization properties. One type of oligomeric compound, RNA mimics, 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. Additional 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.

[0437] 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.

[0438] 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 10It 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 10 The 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.

[0439] 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; 5,567 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.

[0440] 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.

[0441] 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; 5,130,300; 5,134,066; 5,175,273; 5,367,716; ,066;No.5,432,272;No.5,457,187;No.5,459,255;No.5,484,908;No.5,502,177;No.5,525,711;No.5,552, No. 540; No. 5,587,469; No. 5,594,121; No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,8 Nos. 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.

[0442] 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).

[0443] 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.

[0444] 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; and 7,034,133. ; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; 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.

[0445] 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).

[0446] 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."

[0447] 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.

[0448] 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.

[0449] 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.

[0450] 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.

[0451] 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.

[0452] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the present invention, the double-stranded RNAi agents of the present invention can be prepared, for example, using the methods described in U.S. Pat. No. 6,499,499, filed Nov. 18, 2011, the contents of each of which are incorporated herein by reference in their entirety. This includes agents having chemical modifications disclosed in Provisional Patent Application No. 61 / 561,710, or PCT / US Patent Application Publication No. 2012 / 065691, filed November 16, 2012.

[0453] 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.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] 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.

[0458] 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; and 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, 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 a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the terminal three nucleotides on 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 alternating motifs.Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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.

[0463] 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.

[0464] 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. The term "wing modification" herein refers to a modification of a molecule at or near the cleavage site on the same strand. Wing modifications refer to motifs present in another part of the strand separated from the chief. The wing modifications are 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, and when motifs are separated by one or more nucleotides, the chemical structures can be the same or different. Two or more wing modifications can be present. For example, when two wing modifications are present, each wing modification can be present at one end of the first motif at or near the cleavage site or on either side of the lead motif.

[0465] 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.

[0466] 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.

[0467] 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.

[0468] 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.

[0469] 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.

[0470] 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.

[0471] Because nucleic acids are polymers of subunits, many of the modifications, such as modifications of the base, or the phosphate moiety, or the non-linked O of the phosphate moiety, occur at repeated positions within the nucleic acid. In some cases, modifications occur at all of the intended positions in the nucleic acid, but often this is not the case. For example, modifications may occur only at the 3' or 5' terminal positions, or only in terminal regions, such as positions on the terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded region of the RNA, or in the RNA. For example, phosphorothioate modifications at non-linked O positions may be present only at one or both ends, or may be present only at the terminal regions, such as at the terminal nucleotide positions or the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may be present in both double-stranded and single-stranded regions, particularly at the ends. The 5' end or both ends may be phosphorylated.

[0472] 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.

[0473] 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.

[0474] 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.

[0475] In one embodiment, Na 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.

[0476] 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...".

[0477] In one embodiment, the RNAi agent of the present invention comprises a modification pattern of alternating motifs in the sense strand that is shifted relative to the modification pattern of alternating motifs in the antisense strand.This shift can be such that the modified group of the nucleotides in the sense strand corresponds to the differently modified group of the nucleotides in the antisense strand, or vice versa.For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start from "ABABAB" from the 5' to the 3' of the strand. For example, the alternating motif in the antisense strand may begin with "BABABA" from 5' to 3' of the strand within the duplex region. As another example, the alternating motif in the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand within the duplex region, such that there is a complete or partial shift in the modification pattern between the sense and antisense strands.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] 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.

[0482] In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. The internucleotide linkage modification may also be formed to connect the overhang nucleotide with the terminal paired nucleotide in the duplex region. For example, at least 2, 3, 4, or all of the overhang nucleotides are linked by a phosphorothioate or methylphosphonate internucleotide linkage, and optionally There can be additional phosphorothioate or methylphosphonate internucleotide linkages that connect overhanging nucleotides with the paired nucleotides adjacent to the overhanging nucleotides.For example, there can be at least two phosphorothioate internucleotide linkages between the three terminal nucleotides, and two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.These three terminal nucleotides can 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.

[0483] 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] 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 oligonucleotide sequences that independently contain 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.

[0489] In one embodiment, N a and / or N b contains alternating patterns of modifications.

[0490] 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).

[0491] 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).

[0492] 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.

[0493] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0494] 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 can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0495] Each of X, Y, and Z can be the same or different from each other.

[0496] 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

[0497] 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.

[0498] 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 oligonucleotide sequences 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

[0499] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0500] 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.

[0501] In one embodiment, the Y'Y'Y' motifs are all 2'-OMe modified nucleotides.

[0502] 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.

[0503] 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

[0504] When the antisense strand is represented by formula (IIb), N b' is an oligonucleotide containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N represents a nucleotide sequence. a ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0505] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0506] 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 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.

[0507] 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' (Ia) is expressed by

[0508] 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.

[0509] Each of X', Y', and Z' can be the same or different from each other.

[0510] 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.

[0511] 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.

[0512] 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.

[0513] 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.

[0514] 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.

[0515] 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.

[0516] 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-Nb -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:

[0517] 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.

[0518] 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.

[0519] 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.

[0520] When the RNAi agent is represented by formula (IIId), each N b , Nb ' 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 , 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.

[0521] 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 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.

[0522] 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.

[0523] 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.

[0524] 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.

[0525] 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.

[0526] When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides can be base-paired with the corresponding X' nucleotide; or all three of the X nucleotides can be base-paired with the corresponding X' nucleotide.

[0527] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y′ nucleotide and The modification on the Z' nucleotide is different from the modification on the Z' nucleotide and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0528] 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.

[0529] 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.

[0530] 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.

[0531] 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.

[0532] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe) are linked to each other at one or both of the 5'-end and 3'-end, and are optionally conjugated to a ligand.Each agent can target the same gene or two different genes; or each agent can target the same gene at two different target sites.

[0533] Various publications describe multimeric RNAi agents for use in the methods of the present invention. Such publications include WO 2007 / 091269, U.S. Pat. No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887 and WO 2011 / 031520, the contents of each of which are incorporated herein by reference in their entirety.

[0534] 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.

[0535] 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.

[0536] 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.

[0537] In certain embodiments, an RNAi agent for use in the methods of the invention is 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 ribose A, C, G, or U; 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).

[0538] These agents may further comprise a ligand.

[0539] 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.

[0540] In one embodiment, the ligand is a carbohydrate conjugate, such as a monosaccharide. In one embodiment, the ligand is N-acetylgalactosamine (GalNAc), 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.

[0541] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the invention is

[0542] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0543] In one embodiment, the GalNAc or GalNAc derivative is GalNAc3: [ka] is.

[0544] 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.

[0545] A variety of substances can be attached to the RNAi agents of the invention. A preferred moiety is a ligand, which is attached either directly or indirectly, preferably covalently, via an intervening tether.

[0546] 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.

[0547] 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.

[0548] 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.

[0549] 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.

[0550] 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.

[0551] 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.

[0552] 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.

[0553] 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.

[0554] 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.

[0555] 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.

[0556] 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.

[0557] 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.

[0558] 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.

[0559] 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.

[0560] 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).

[0561] 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.

[0562] 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 phosphorylligands 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.

[0563] 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).

[0564] 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.

[0565] 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.

[0566] 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.

[0567] 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.

[0568] 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 phosph...

Claims

1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in preventing at least one symptom in a human subject having hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the subject about once every two months as a dose of 40 mg to 90 mg of the dsRNA molecule; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand.

2. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in treating a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the subject about once every two months as a dose of 40 mg to 90 mg of the dsRNA molecule; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand.

3. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the frequency of bleeding episodes in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the subject. administered as a dose of 40 mg to 90 mg of the dsRNA molecule about once every two months; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand.

4. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the annualized bleeding rate (ABR) in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the subject about once every two months as a dose of 40 mg to 90 mg of the dsRNA molecule; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand.

5. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the annual spontaneous bleeding rate (AsBR) in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the subject about once every two months as a dose of 40 mg to 90 mg of the dsRNA molecule; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

7. The ligand is 【Chemistry 1】 The pharmaceutical composition according to any one of claims 1 to 5, wherein

8. The pharmaceutical composition of any one of claims 1 to 5, wherein the dsRNA is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 2】 where X is O or S.

9. 9. The pharmaceutical composition of claim 8, wherein X is O.

10. The pharmaceutical composition according to any one of claims 1 to 9, which is administered subcutaneously to a subject.

11. The pharmaceutical composition of any one of claims 1 to 10, which is administered to a subject as a dose of 50 mg of the dsRNA molecule.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein administration of the pharmaceutical composition reduces SERPINC1 activity in a subject by 70% to 95%.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein administration of the pharmaceutical composition to a subject increases the peak thrombin level in the subject to within the range of peak thrombin levels in subjects without hemophilia A or B.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein administration of the pharmaceutical composition to a subject is sufficient to achieve a peak thrombin formation level in the subject that is the same as that achieved by administration of Factor VIII to the subject.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein administration of the pharmaceutical composition to a subject is sufficient to result in a peak thrombin formation level that is greater than 40% equivalent to administration of Factor VIII to the subject.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein administration of the pharmaceutical composition to a subject reduces the subject's on-demand annualized bleeding rate (ABR) by 80 to 95% when compared to the median historical ABR of subjects with hemophilia A or B who are not receiving the pharmaceutical composition.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the subject is a hemophilia A patient with an inhibitor.

18. The pharmaceutical composition according to any one of claims 1 to 16, wherein the subject is a hemophilia A patient without inhibitors.

19. The pharmaceutical composition according to any one of claims 1 to 16, wherein the subject is a hemophilia B patient with an inhibitor.

20. The pharmaceutical composition according to any one of claims 1 to 16, wherein the subject is a hemophilia B patient without inhibitors.

21. The pharmaceutical composition of any one of claims 1 to 20, in a non-buffered solution.

22. 22. The pharmaceutical composition of claim 21, wherein the non-buffered solution is saline or water.

23. The pharmaceutical composition according to any one of claims 1 to 20, which is in a buffer solution.

24. 24. The pharmaceutical composition of claim 23, wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

25. 24. The pharmaceutical composition of claim 23, wherein the buffer is phosphate buffered saline (PBS).

26. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in preventing at least one hemophilic symptom in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the human subject as a 50 mg dose of the dsRNA molecule about once every two months; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Transformation 3】 Here, X is O.

27. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in treating a human subject having hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the human subject about once every two months as a 50 mg dose of the dsRNA molecule; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 4】 Here, X is O.

28. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the frequency of bleeding episodes in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the human subject as a 50 mg dose of the dsRNA molecule about once every two months; The dsRNA molecule comprises a sense strand and an antisense strand, The sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUf the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Transformation 5】 Here, X is O.

29. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the annualized bleeding rate (ABR) in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the human subject as a 50 mg dose of the dsRNA molecule about once every two months; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the 3' end of the sense strand is conjugated to a ligand as shown in the following schematic diagram: 【Transformation 6】 Here, X is O.

30. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the annual spontaneous bleeding rate (AsBR) in a human subject with hemophilia A or hemophilia B, wherein the pharmaceutical composition is administered to the human subject as a 50 mg dose of the dsRNA molecule about once every two months; The dsRNA molecule comprises a sense strand and an antisense strand, the sense strand comprises the sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the antisense strand comprises the sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Transformation 7】 Here, X is O.

31. The pharmaceutical composition according to any one of claims 26 to 30, wherein the subject is a hemophilia A patient with an inhibitor.

32. The method according to any one of claims 26 to 30, wherein the subject is a hemophilia B patient with an inhibitor. The pharmaceutical composition described in

33. The pharmaceutical composition according to any one of claims 26 to 30, wherein the subject is a hemophilia A patient without inhibitors.

34. The pharmaceutical composition according to any one of claims 26 to 30, wherein the subject is a hemophilia B patient without inhibitors.

35. 30. The pharmaceutical composition of claim 4 or 29, wherein administration of the pharmaceutical composition reduces the subject's ABR to 1 or less.

36. 31. The pharmaceutical composition of claim 5 or 30, wherein administration of the pharmaceutical composition reduces the subject's AsBR to zero.

37. The pharmaceutical composition of any one of claims 26 to 36, which is in a buffer solution.

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

39. 1. A kit for use in preventing, treating, reducing the frequency of bleeding episodes, reducing the annualized bleeding rate (ABR), or reducing the annualized spontaneous bleeding rate (AsBR) in a human subject with hemophilia A or hemophilia B, comprising: a) a pharmaceutical composition according to any one of claims 1 to 38; b) instructions for use; and c) optionally, a means for administering the pharmaceutical composition to a subject; The kit comprises:

40. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in preventing at least one hemophilic symptom in a human subject with hemophilia A or B, with or without an inhibitor, comprising: the pharmaceutical composition is administered to a human subject about once every two months as a dose of about 50 mg of the dsRNA molecule; The dsRNA molecule has a sense strand, an antisense strand, and a ligand; the nucleic acid sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleic acid sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 9】 Here, X is O.

41. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in treating hemophilia A or B in a human subject, with or without an inhibitor, wherein the pharmaceutical composition is administered to the human subject about once every two months as a dose of about 50 mg of the dsRNA molecule; The dsRNA molecule has a sense strand, an antisense strand, and a ligand; the nucleic acid sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleic acid sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 10】 Here, X is O.

42. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for routine prophylaxis for preventing, reducing the frequency of, bleeding episodes in a human subject with hemophilia A or B, with or without an inhibitor, wherein the pharmaceutical composition is administered to the human subject about once every two months as a dose of about 50 mg of the dsRNA molecule; The dsRNA molecule has a sense strand, an antisense strand, and a ligand; the nucleic acid sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleic acid sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 11】 Here, X is O.

43. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the annualized bleeding rate (ABR) in a human subject with hemophilia A or B, with or without an inhibitor, wherein the pharmaceutical composition is administered to the human subject about once every two months as a dose of about 50 mg of the dsRNA molecule; The dsRNA molecule has a sense strand, an antisense strand, and a ligand; the nucleic acid sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleic acid sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 12】 Here, X is O.

44. 1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in reducing the annual spontaneous bleeding rate (AsBR) in a human subject with hemophilia A or B, with or without an inhibitor, wherein the pharmaceutical composition is administered to the human subject about once every two months as a dose of about 50 mg of the dsRNA molecule; The dsRNA molecule has a sense strand, an antisense strand, and a ligand; the nucleic acid sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleic acid sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 13】 Here, X is O.

45. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) molecule for use in preventing at least one hemophilic symptom in a human subject with hemophilia A or B, with or without an inhibitor, wherein the pharmaceutical composition is administered subcutaneously to a human subject in need thereof at a dose of about 80 mg of the dsRNA molecule about once every two months; The dsRNA molecule has a sense strand, an antisense strand, and a ligand; the nucleic acid sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleic acid sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14); a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; The pharmaceutical composition, wherein the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: 【Chemistry 14】 Here, X is O.

46. The pharmaceutical composition according to any one of claims 40 to 45, wherein the subject is a hemophilia A patient with an inhibitor.

47. The pharmaceutical composition according to any one of claims 40 to 45, wherein the subject is a hemophilia B patient with an inhibitor.

48. The pharmaceutical composition according to any one of claims 40 to 45, wherein the subject is a hemophilia A patient without inhibitors.

49. The pharmaceutical composition according to any one of claims 40 to 45, wherein the subject is a hemophilia B patient without inhibitors.

50. 44. The pharmaceutical composition of claim 43, wherein administration of the pharmaceutical composition reduces the subject's ABR to 1 or less.

51. 45. The pharmaceutical composition of claim 44, wherein administration of the pharmaceutical composition reduces the subject's AsBR to zero.

52. 52. The pharmaceutical composition of any one of claims 40 to 51, in a buffer solution.

53. 53. The pharmaceutical composition of claim 52, wherein the buffer is phosphate buffered saline (PBS).

54. (i) prevention of at least one symptom of hemophilia in a human subject with hemophilia A or B, with or without inhibitors; (ii) treatment of human subjects with hemophilia A or B, with or without inhibitors; (iii) general prophylaxis for the prevention or reduction of frequency of bleeding episodes in human subjects with hemophilia A or B, with or without inhibitors; (iv) reducing the annualized bleeding rate (ABR) in human subjects with hemophilia A or B, with or without inhibitors; or (v) reducing the annual spontaneous bleeding rate (AsBR) in human subjects with hemophilia A or B, with or without inhibitors; 54. A kit for use in the method of claim 40, comprising the pharmaceutical composition of any one of claims 40 to 53.

55. 55. The kit of claim 54, including instructions for use.

56. 56. The kit of claim 54 or 55, comprising a means for administering the pharmaceutical composition to a subject.

57. 57. The kit of any one of claims 54 to 56, comprising more than one dose of the dsRNA molecule.

58. 58. The kit of claim 56 or 57, wherein the means for administering is an injection device for subcutaneous administration.

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