Serpinc1 irna compositions and methods of use thereof

Abstract

To provide methods of treating a bleeding event in a subject having hemophilia.SOLUTION: The invention relates to pharmaceutical compositions comprising an iRNA agent, e.g., double stranded ribonucleic acid (dsRNA) agent and methods of using such compositions to treat a bleeding event in a subject having hemophilia (e.g., with or without inhibitors).SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 793,020, filed January 16, 2019, the entire contents of which are incorporated herein by reference.

[0002] This application is related to International Application No. PCT / US2018 / 041400, filed July 10, 2018, U.S. Provisional Patent Application No. 62 / 530,518, filed July 10, 2017, U.S. Provisional Patent Application No. 62 / 599,223, filed December 15, 2017, U.S. Provisional Patent Application No. 62 / 614,111, filed January 5, 2018, and U.S. Provisional Patent Application No. 62 / 673,424, filed May 18, 2018. The entire contents of each of the above-mentioned patent applications are incorporated herein by reference.

[0003] This application is also related to U.S. Patent Application No. 15 / 371,300, filed December 7, 2016, International Application No. PCT / US2016 / 065245, filed December 7, 2016, U.S. Provisional Patent Application No. 62 / 264,013, filed December 7, 2015, U.S. Provisional Patent Application No. 62 / 315,228, filed March 30, 2016, U.S. Provisional Patent Application No. 62 / 366,304, filed July 25, 2016, and U.S. Provisional Patent Application No. 62 / 429,241, filed December 2, 2016. The entire contents of each of the above-mentioned patent applications are incorporated herein by reference.

[0004] Additionally, this application is related to U.S. Provisional Patent Application No. 61 / 992,057, filed May 12, 2014, U.S. Provisional Patent Application No. 62 / 089,018, filed December 8, 2014, U.S. Provisional Patent Application No. 62 / 102,281, filed January 12, 2015, and International Application No. PCT / US2015 / 030337, filed May 12, 2015. The entire contents of each of the above-mentioned patent applications are incorporated herein by reference.

[0005] This application is also related to U.S. Provisional Patent Application No. 61 / 638,952, filed April 26, 2012, U.S. Provisional Patent Application No. 61 / 669,249, filed July 9, 2012, U.S. Provisional Patent Application No. 61 / 734,573, filed December 7, 2012, U.S. Provisional Patent Application No. 13 / 837,129, filed March 15, 2013, now U.S. Patent No. 9,127,274, U.S. Provisional Patent Application No. 13 / 837,129, filed July 22, 2015, now U.S. Patent No. 9,127,274, No. 14 / 806,084, now U.S. Patent No. 9,376,680, U.S. Patent Application No. 15 / 070,358, filed March 15, 2016, U.S. Patent Application No. 15 / 955,873, filed April 18, 2018, U.S. Patent Application No. 16 / 220,157, filed December 14, 2018, and International Application No. PCT / US2013 / 038218, filed April 25, 2013. This application is also related to International Application No. PCT / US2012 / 065601, filed November 16, 2012. The entire contents of each of the above-mentioned patent applications are incorporated herein by reference.

[0006] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. Said ASCII copy, created on January 7, 2020, is named 117811_03020_SL.TXT and is 20,997 bytes in size. [Background technology]

[0007] Serpinc1 is a member of the serine proteinase inhibitor (serpin) superfamily. Serpinc1 mediates the regulation of thrombin as well as other activating serine proteins in the hemocoagulation system. It is a plasma protease inhibitor that regulates the blood coagulation cascade by inhibiting phosphoproteases such as factors X, IX, XI, XII and VII. The anticoagulant activity of Serpinc1 is enhanced by the presence of heparin and other related glycosaminoglycans that catalyze the formation of the thrombin:antithrombin (TAT) complex.

[0008] Bleeding disorders are conditions in which there is insufficient blood clotting, whether inherited or acquired. Hemophilia, for example, is a group of inherited genetic bleeding disorders that impair the body's ability to control blood clotting or coagulation. Hemophilia A is a recessive X-linked genetic disorder with a deficiency of functional clotting factor VIII and represents 80% of hemophilia cases. Hemophilia B is a recessive X-linked genetic disorder with a deficiency of functional clotting factor IX. It comprises approximately 20% of hemophilia cases. Hemophilia C is an autosomal genetic disorder with a deficiency of functional clotting factor XI. Hemophilia C is not fully recessive, and heterozygous individuals also exhibit increased bleeding.

[0009] There is currently no cure for hemophilia, but it can be controlled by regular infusions of the missing clotting factor, e.g., factor VIII in hemophilia A. However, some hemophiliacs become refractory to the replacement clotting factor by producing antibodies (inhibitors) against the administered replacement factor. Thus, bleeding in such subjects cannot be adequately controlled.

[0010] The investigational once-monthly subcutaneously administered RNAi therapeutic targeting antithrombin (AT) Fitusiran has recently been developed for the treatment of hemophilia A and B with and without inhibitors, and stable pharmaceutical compositions comprising such therapeutics are needed in the art as alternative treatments for subjects with bleeding disorders, e.g., hemophilia. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention is based at least in part on the discovery of a stable pharmaceutical composition comprising double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of Serpinc1 gene, which has improved stability, efficacy, durability and ease of administration compared with other compositions that comprise double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of Serpinc1 gene.Such pharmaceutical composition is useful for treating subjects with bleeding disorder, for example hemophilia. [Means for solving the problem]

[0012] Accordingly, in one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, wherein the pH of the pharmaceutical composition is suitable for subcutaneous administration to a subject, and the dsRNA agent comprises a serine / stearate molecule consisting of the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, where a ligand is conjugated to the 3' end of the sense strand via a linker, and the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in the free acid form.

[0013] In another aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, wherein the pH of the pharmaceutical composition is suitable for subcutaneous administration to a subject, and the dsRNA agent is a serine / stearate complex consisting of the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in a salt form.

[0014] In one aspect, the invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, wherein the pH and osmolality of the pharmaceutical composition are suitable for subcutaneous administration to a subject, and the dsRNA agent is selected from the group consisting of the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in the free acid form.

[0015] In another aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, wherein the pH and osmolality of the pharmaceutical composition are suitable for subcutaneous administration to a subject, and the dsRNA agent has the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the structure: [ka] Here, the dsRNA agent is in a salt form.

[0016] The salt form of the dsRNA may be a sodium salt form.

[0017] In one embodiment, substantially all of the phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion. In another embodiment, all of the phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0018] The concentration of PBS in the pharmaceutical composition may be between about 2 mM and about 7 mM; between about 3 to about 6 mM; or about 5 mM.

[0019] The pH of the pharmaceutical composition may be between about 5.0 and about 8.0; between about 6.0 and about 8.0; between about 6.5 and about 7.5; or between about 6.8 and about 7.2.

[0020] The osmolality of the pharmaceutical composition is between about 50 and about 400 mOsm / kg; between about 100 and about 400 mOsm / kg; between about 240 and about 390 mOsm / kg; or between about 240 and about 390 mOsm / kg. It may be between 90 and about 320 mOsm / kg.

[0021] The concentration of the dsRNA agent in the pharmaceutical composition can be between about 50 mg / mL and about 150 mg / mL; between about 80 mg / mL and about 110 mg / mL; or about 100 mg / mL.

[0022] In one embodiment, the composition is stable for about 6 months to about 36 months when stored at about 2° C. to about 8° C. In another embodiment, the composition is stable for about 6 months to about 36 months when stored at about 25° C. and 60% relative humidity (RH). In yet another embodiment, the composition is stable for about 6 months when stored at about 40° C. and 75% relative humidity (RH).

[0023] In other embodiments, the composition is stable for up to about 36 months when stored at about 2° C. to about 8° C. In other embodiments, the composition is stable for up to about 36 months when stored at about 25° C. and 60% relative humidity (RH). In yet other embodiments, the composition is stable for up to about 6 months when stored at about 40° C. and 75% relative humidity (RH).

[0024] In one embodiment, the composition contains at least (NLT) about 95.0 area % duplex and at most (NMT) about 5 area % total duplex impurities as determined by purity non-denaturing IPRP-HPLC.

[0025] In one embodiment, the composition contains at least (NLT) about 85.0 area % total single strands as determined by purity denaturing AX-HPLC.

[0026] In one embodiment, the composition contains at least (NLT) about 80.0 area % total single strands as determined by purity denaturing IPRP-HPLC.

[0027] The invention also provides vials and syringes containing the pharmaceutical compositions of the invention.

[0028] The vial may contain about 0.5 mL to about 2.0 ml of the pharmaceutical composition; or about 0.8 ml of the pharmaceutical composition.

[0029] The syringe of the present invention may be a 1 ml syringe; or a 3 ml syringe. In one embodiment, the syringe is a 1 ml disposable syringe.

[0030] The syringe of the present invention may include a 29G needle; or a 30G needle. In one embodiment, the needle is a 29G needle.

[0031] In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 100 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and the dsRNA agent comprises a sense strand and a 5'-u strand consisting of the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of sUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and the ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in the free acid form.

[0032] In another embodiment, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and the dsRNA agent comprises a sense strand and a 5'-u strand consisting of a nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of sUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and the ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in a salt form.

[0033] In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 100 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent has the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in the free acid form.

[0034] In another aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpincl gene, comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent has the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the structure: [ka] Here, the dsRNA agent is in a salt form.

[0035] In one embodiment, the salt form is a sodium salt form.

[0036] In one embodiment, substantially all of the phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion. In another embodiment, all of the phosphodiester and phosphorothioate groups in the agent include a sodium counterion.

[0037] In one embodiment, the composition is stable for about 6 months to about 36 months when stored at about 2° C. to about 8° C. In another embodiment, the composition is stable for about 6 months to about 36 months when stored at about 25° C. and 60% relative humidity (RH). In yet another embodiment, the composition is stable for about 6 months when stored at about 40° C. and 75% relative humidity (RH).

[0038] In one embodiment, the composition is stable for up to about 36 months when stored at about 2° C. to about 8° C. In another embodiment, the composition is stable for up to about 36 months when stored at about 25° C. and 60% relative humidity (RH). In yet another embodiment, the composition is stable for up to about 6 months when stored at about 40° C. and 75% relative humidity (RH).

[0039] In one embodiment, the composition comprises at least (NLT) about 95.0 area % duplex and at most (NMT) about 5 area % total duplex impurities as determined by purity non-denaturing IPRP-HPLC.

[0040] In one embodiment, the composition comprises at least (NLT) about 85.0 area % total single strands as determined by purity denaturing AX-HPLC.

[0041] In one embodiment, the composition comprises at least (NLT) about 80.0 area % total single strands as determined by purity denaturing IPRP-HPLC.

[0042] The present invention also provides a vial containing the pharmaceutical composition described above. The vial may contain about 0.5 mL to about 2.0 ml of the pharmaceutical composition; or about 0.8 ml of the pharmaceutical composition.

[0043] The present invention further provides a syringe comprising the above-described pharmaceutical composition.

[0044] The syringe of the present invention may be a 1 ml syringe; or a 3 ml syringe. In one embodiment, the syringe is a 1 ml disposable syringe.

[0045] The syringe of the present invention may include a 29G needle; or a 30G needle. In one embodiment, the needle is a 29G needle.

[0046] In one embodiment, the syringe is a pre-filled syringe.

[0047] In another embodiment, the invention provides a 2 ml vial containing about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpincl gene, wherein the pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and wherein the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and wherein the dsRNA agent has the amino acid sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0048] In another aspect, the present invention provides a 1 ml pre-filled disposable syringe with a 29G needle, wherein the syringe contains about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpincl gene, the pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, the pH of the pharmaceutical composition is about 6.8 to about 7.2, the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf- and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0049] In another embodiment, the present invention provides a 2 ml vial containing about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpinc1 gene, wherein the pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent has the structure: [ka] where Am, Gm, Cm and Um are 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf are 2'-fluoro A, G, C, U; s is a phosphorothioate bond; and L96 is a ligand and linker having the structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0050] In another aspect, the present invention provides a 1 ml pre-filled disposable syringe with a 29G needle, wherein the syringe contains about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpincl gene, the pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, the pH of the pharmaceutical composition is about 6.8 to about 7.2, the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent has the structure [ka] having where Am, Gm, Cm and Um are 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf are 2'-fluoro A, G, C, U; s is a phosphorothioate bond; and L96 is a ligand and linker having the structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0051] In another embodiment, the present invention provides a 2 ml vial containing about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpincl gene, wherein the pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and Agent A has a sense strand consisting of the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:941) and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0052] In another embodiment, the present invention provides a 1 ml pre-filled disposable syringe with a 29G needle, wherein the syringe contains about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpincl gene, the pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and wherein the dsRNA agent has the structure: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941). and an antisense strand consisting of the nucleotide sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and a ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0053] In another embodiment, the present invention provides a 2 mL vial containing about 0.8 mL of a pharmaceutical composition for inhibiting expression of the Serpincl gene, wherein the pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, and Agent A has the structure [ka] having where Am, Gm, Cm, and Um are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; s is a phosphorothioate bond; and L96 is a ligand and linker having the structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion.

[0054] In another embodiment, the present invention provides a 1 ml pre-filled disposable syringe with a 29G needle, wherein the syringe contains about 0.8 ml of a pharmaceutical composition for inhibiting expression of the Serpincl gene, the pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, the pH of the pharmaceutical composition being about 6.8 to about 7.2, and the dsRNA agent having the structure [ka] having where Am, Gm, Cm, and Um are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; s is a phosphorothioate bond; and L96 is a ligand and linker having the structure: [ka] Here, the dsRNA agent is in sodium salt form, and all phosphodiester and / or phosphorothioate groups in the agent include a sodium counterion. [Brief description of the drawings]

[0055] [Figure 1]Representative non-denaturing ion-pair reversed-phase high performance liquid chromatography (IP RP-HPLC) chromatograms of the Fitusiran formulation are shown. Incidental slight cleavage of the Fitusiran duplex peak was observed due to partial decomposition of different stereoisomers of phosphorothioates in the siRNA duplex. Mass spectrometry analysis of the Fitusiran peak confirmed the presence of the expected masses of both single strands in equal proportions throughout the duplex peak. [Diagram 2] Representative denaturing anion exchange high performance liquid chromatography (AX-HPLC) chromatograms of single strands in double strands in a Fitusiran formulation are shown. [Diagram 3] Representative denaturing ion-pair reversed-phase high performance liquid chromatography (IP RP-HPLC) chromatogram profiles of single strands in duplexes in Fitusiran formulations are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The present invention provides pharmaceutical compositions comprising iRNA agents that induce RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of Serpinc1 gene.The present invention is based, at least in part, on the discovery of stable pharmaceutical compositions comprising dsRNA agents that inhibit the expression of Serpinc1 gene, which have improved stability, efficacy, durability and ease of administration compared to other compositions comprising such agents.Such pharmaceutical compositions are useful for treating subjects with disorders that would benefit from the inhibition of the expression of Serpinc1 gene and / or the inhibition or reduction of the expression of Serpinc1 gene, such as bleeding disorders, for example, hemophilia.

[0057] The following detailed description discloses methods for making and using compositions comprising iRNAs that inhibit expression of the Serpinc1 gene, as well as compositions, uses and methods for treating subjects with diseases and disorders that would benefit from inhibition and / or reduction of expression of this gene.

[0058] 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 given, any intermediate values ​​or ranges of the given values ​​are also intended to be part of the present invention.

[0059] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0060] The term "about" is used herein within the scope of typical acceptance in the art. For example, "about" is understood to mean about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is present before a series of numbers or ranges, it is understood that "about" can modify each of the series of numbers or ranges.

[0061] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0062] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless clearly indicated otherwise.

[0063] As used herein, the term "pharmaceutical composition" refers to a composition useful for treating a disease or disorder in a subject, such as a human subject.

[0064] The term "pharmaceutical administration" refers to the delivery of the composition comprising dsRNA agent to a subject for treating disease or disorder.Therefore, "suitable for pharmaceutical administration", for example "suitable for subcutaneous administration", describes the composition comprising dsRNA agent that can be used to treat disease or disorder in a subject by subcutaneous administration of the pharmaceutical composition.The pharmaceutical composition is suitable for pharmaceutical administration, for example suitable for subcutaneous administration.

[0065] The term "osmolarity" refers to the number of osmoles of solute per kg of solvent. It is expressed in units of osmol / kg or Osm / kg. "Osmole" is a unit of measurement that describes the number of moles of a compound that contribute to the osmotic pressure of a chemical solution.

[0066] As used herein, "Serpinc1" refers to a specific polypeptide expressed in cells. Serpinc1 is also known as serpin peptidase inhibitor, clade C (antithrombin; AT), member 1; antithrombin III; AT3; antithrombin; and heparin cofactor 1. The sequence of human Serpinc1 mRNA transcript can be found, for example, in GenBank Accession No. GI:254588059 (NM_000488; SEQ ID NO:1). The sequence of rhesus monkey Serpinc1 mRNA can be found, for example, in GenBank Accession No. GI:157167169 (NM_001104583; SEQ ID NO:2). The sequence of mouse Serpinc1 mRNA can be found, for example, in GenBank Accession No. 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).

[0067] The term "Serpinc1" as used herein also refers to a specific polypeptide that is expressed in cells due to the naturally occurring DNA sequence variation of Serpinc1 gene, for example, single nucleotide polymorphism in Serpinc1 gene.A number of SNPs have been identified in Serpinc1 gene, and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).Non-limiting examples of SNPs in Serpinc1 gene can be found in NCBI dbSNP accession numbers rs677;rs5877;rs5878;rs5879;rs941988;rs941989;rs1799876;rs19637711;rs2008946;and rs2227586.

[0068] As used herein, a "subject" is an animal, e.g., a mammal, such as a primate (e.g., a human, a non-human primate, e.g., a monkey and a chimpanzee), a non-primate (e.g., a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In certain embodiments, the subject is a human, a human being treated or evaluated for a disease, disorder or condition that would benefit from reduced Serpinc1 expression; a human at risk for a disease, disorder or condition that would benefit from reduced Serpinc1 expression; a human having a disease, disorder or condition that would benefit from reduced Serpinc1 expression; and / or a human being treated for a disease, disorder or condition described herein that would benefit from reduced Serpinc1 expression.

[0069] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0070] As used herein, the phrase "inhibiting expression of Serpinc1" includes inhibition of expression of any Serpinc1 gene (e.g., mouse Serpinc1 gene, rat Serpinc1 gene, monkey Serpinc1 gene or human Serpinc1) as well as variants or mutants of the Serpinc1 gene that encode the Serpinc1 protein.

[0071] "Inhibiting expression of the Serpinc1 gene" includes any level of inhibition of the Serpinc1 gene, for example at least partial suppression of expression of the Serpinc1 gene, for example 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.

[0072] The expression of Serpinc1 gene is evaluated based on the level of any variable related to Serpinc1 gene expression, such as Serpinc1 mRNA level, Serpinc1 protein level, or, for example, thrombin:antithrombin complex level, bleeding time, prothrombin time (PT), platelet count and / or activated partial thromboplastin time (aPTT) as indicators of thrombin generation potential. Inhibition is evaluated by the reduction of the absolute or relative level of one or more of these variables compared to a control level. The control level may be any kind of control level used in the art, such as the baseline level before administration or the level determined from a similar subject, cell or sample that is not treated or is treated with a control (such as a buffer only control or a non-activating agent control).

[0073] In one embodiment, at least partial suppression of expression of the Serpinc1 gene is assessed by a reduction in the amount of Serpinc1 mRNA isolated or detected in a first cell or group of cells in which the Serpinc1 gene is transcribed and which has been treated to inhibit expression of the Serpinc1 gene, compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated. The degree of inhibition can be measured by:

number

[0074] As used herein, the phrase "contacting a cell with an RNAi agent" such as dsRNA includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo.Contacting can be performed directly or indirectly.Thus, for example, the RNAi agent can be physically contacted with a cell by the individual who performs the method, or can be placed in a situation that allows or causes the RNAi agent to contact with a cell thereafter.

[0075] The contacting of cells in vitro is performed, for example, by incubating the cells with an RNAi agent. The contacting of cells in vivo is performed, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into other areas, such as the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue where the contacted cells are located. For example, the RNAi agent may contain and / or be bound to a ligand, such as GalNAc3, that directs the RNAi agent to a site of interest, such as the liver. A combination of in vitro and in vivo contact methods is also possible. For example, the cells may be contacted with the RNAi agent in vitro and then transplanted into a subject.

[0076] II. Pharmaceutical Compositions of the Present Invention The present invention provides a stable pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of Serpinc1 gene.The pharmaceutical composition of the present invention comprises the dsRNA agent described herein and phosphate buffered saline, and is suitable for subcutaneous administration to a subject.The pharmaceutical composition comprising the dsRNA agent is useful for treating diseases or disorders related to the expression or activity of Serpinc1 gene, such as Serpinc1-related diseases, such as hemophilia.The pharmaceutical composition of the present invention is administered at a dose sufficient to inhibit the expression of Serpinc1 gene.

[0077] In one embodiment, the pharmaceutical composition of the present invention comprises the dsRNA agent of the present invention in free acid form. In another embodiment, the pharmaceutical composition of the present invention comprises the dsRNA agent of the present invention in sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the agent as counterions (to maintain electrical neutrality) for substantially all of the phosphodiester and / or phosphorothioate groups present in the agent. Agents in which substantially all phosphodiester and / or phosphorothioate linkages have sodium counterions include phosphodiester and / or phosphorothioate linkages that do not have at most 5, 4, 3, 2 or 1 sodium counterion. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester and / or phosphorothioate linkages present in the agent.

[0078] The pharmaceutical compositions of the invention may comprise a dsRNA agent at a concentration of about 50 mg / mL to about 200 mg / mL, about 50 mg / mL to about 150 mg / mL; about 90 mg / mL to about 110 mg / mL, about 90 mg / mL to about 100 mg / mL, or about 80 mg / mL to about 110 mg / mL, e.g., about 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, In one embodiment, the pharmaceutical composition of the present invention may comprise at a concentration of about 200 mg / mL, 105 mg / mL, 106 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL, 160 mg / mL, 165 mg / mL, 170 mg / mL, 175 mg / mL, 180 mg / mL, 185 mg / mL, 190 mg / mL, 195 mg / mL or about 200 mg / mL. The dsRNA agent is contained at a concentration of about 100mg / mL. Values ​​intermediate to the above ranges and values ​​are also intended to be part of the present invention.Furthermore, ranges of values ​​using any combination of the above values ​​as upper and / or lower limits are also intended to be included.

[0079] The pharmaceutical composition of the present invention may comprise PBS. In one embodiment, the PBS comprises sodium chloride and sodium phosphate, but does not comprise potassium chloride and / or potassium phosphate. In another embodiment, the PBS comprises sodium chloride, sodium phosphate and potassium chloride. In yet another embodiment, the PBS comprises sodium chloride, sodium phosphate and potassium phosphate. In one embodiment, the PBS comprises sodium chloride, sodium phosphate, potassium chloride and potassium phosphate. In a particular embodiment, for example, when the PBS comprises sodium chloride and sodium phosphate, the PBS may be at a concentration of about 1 mM to about 10 mM; or about 3 mM to about 6 mM, for example about 1 mM, 1.5 mM, 2 mM, 2.5 mM. It may be at a concentration of 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 6.5 mM, 7 mM, 7.5. mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM or about 10 mM PBS. In one embodiment of the present invention, the pharmaceutical composition of the present invention comprises at least about 5 mM (e.g., about 0.64 mM NaH 2 PO 4 , about 4.36mM Na 2 HPO 4 , about 85mM NaCl) in PBS. Values ​​intermediate to the above ranges and values ​​are also intended to be part of the invention. Additionally, ranges of values ​​using a combination of any of the above values ​​as upper and / or lower limits are also intended to be included.

[0080] In one embodiment, the pharmaceutical composition of the invention is preservative-free. In another embodiment of the invention, the pharmaceutical composition of the invention comprises a preservative.

[0081] The pH of the pharmaceutical compositions of the present invention is suitable for subcutaneous administration and may be about 5.0 to about 8.0, about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, about 5.0 to about 7.5, about 5.5 to about 7.5, about 6.0 to about 7.5, about 6.5 to about 7.5, about 5.0 to about 7.2, about 5.25 to about 7.2, about 5.5 to about 7.2, about 5.75 to about 7.2, about 6.0 to about 7.2, about 6.5 to about 7.2, or about 6.8 to about 7.2. Ranges and values ​​intermediate to the above ranges and values ​​are also contemplated as part of the present invention.

[0082] The osmolality of the pharmaceutical composition of the invention may be suitable for subcutaneous administration, e.g. about 400 mOsm / kg or less, e.g. between 50 and 400 mOsm / kg, between 75 and 400 mOsm / kg, between 100 and 400 mOsm / kg, between 125 and 400 mOsm / kg, between 150 and 400 mOsm / kg, between 175 and 400 mOsm / kg, between 200 and 400 mOsm / kg, between 250 and 400 mOsm / kg, between 300 and 400 mOsm / kg, between 50 and 375 mOsm / kg, between 75 and 375 mOsm / kg, Between 100 and 375mOsm / kg, Between 125 and 375mOsm / kg, Between 150 and 375mOsm / kg, Between 175 and 375mOsm / kg, Between 200 and 375mOsm / kg, Between 250 and 375mOsm / kg, Between 300 and 375mOsm / kg, Between 50 and 350mOsm / kg, Between 75 and 350mOsm / kg, Between 100 and 350mOsm / kg, between 125 and 350mOsm / kg, between 150 and 350mOsm / kg, between 175 and 350mOsm / kg, between 200 and 350mOsm / kg, between 250 and 350mOsm / kg, between 50 and 325mOsm / kg, between 75 and 325mOsm / kg, between 100 and 325mOsm / kg, between 125 and 325mOsm / kg, between 150 and between 175 and 325mOsm / kg, between 200 and 325mOsm / kg, between 250 and 325mOsm / kg, between 300 and 325mOsm / kg, between 300 and 350mOsm / kg, between 50 and 300mOsm / kg, between 75 and 300mOsm / kg, between 100 and 300mOsm / kg, between 125 and 300mOsm / kg between 150 and 300 mOsm / kg, between 175 and 300 mOsm / kg, between 200 and 300 mOsm / kg, between 250 and 300 mOsm / kg, between 50 and 250 mOsm / kg, between 75 and 250 mOsm / kg, between 100 and 250 mOsm / kg, between 125 and 250 mOsm / kg, between 150 and 250 mOsm / kg, between 175 and 350 mOsm / kg, between 200 and 250 mOsm / kg, for example about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 , 110, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 295, 300, 305, 310, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or about 400 mOsm / kg. Ranges and values ​​intermediate to the above-recited ranges and values ​​are also intended to be part of the invention. Moreover, ranges using a combination of any of the above-recited values ​​as upper and / or lower limits are intended to be included.

[0083] The pharmaceutical composition of the present invention is physically and chemically stable.

[0084] The term "stable" as used herein refers to a pharmaceutical composition and / or a dsRNA agent within such pharmaceutical composition that essentially maintains its physical stability and / or chemical stability and / or biological stability.Various analytical techniques for measuring the stability of a composition and the dsRNA agent therein are available in the art and are described herein.

[0085] A pharmaceutical composition (or a dsRNA agent in such a composition) "retains its physical stability" if it shows, e.g., substantially no signs of increased impurities, e.g., upon visual inspection of color and / or clarity and UV inspection, or as measured, e.g., by HPLC analysis, e.g., denaturing IP RP-HPLC, non-denaturing IP RP-HPLC and / or denaturing AX-HPLC analysis.

[0086] A dsRNA agent "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time is such that the dsRNA is still considered to retain its biological stability. Chemical stability is evaluated, for example, by detecting and / or quantifying the chemically altered form of the dsRNA duplex and / or the chemically altered form of the sense strand and / or the antisense strand. Chemical alteration may involve size modification and / or sodium content change, evaluated, for example, by duplex retention time and / or by determining the molecular weight of the single strand that forms the duplex, for example, using non-denaturing IP RP-HPLC, by melting temperature, for example, using thermal UV spectrophotometry, and / or by determining the sodium content (on anhydrous basis), for example, using flame atomic absorption spectrometry (flame AAS) / inductively coupled plasma optical emission spectrometry (ICP-OES).

[0087] A dsRNA agent "retains its biological activity" in a pharmaceutical composition if the dsRNA agent in the composition is biologically active for its intended purpose. For example, biological activity is retained if the biological activity of the dsRNA agent in the composition is within about 30%, about 20%, or about 10% (within the error of the assay) of the biological activity exhibited when the composition was prepared (e.g., as determined by an in vitro RT-PCR assay).

[0088] For example, in some embodiments, the compositions of the present invention are stable for about 6 months to about 36 months when stored at about 2° C. to about 8° C. In other embodiments, the compositions of the present invention are stable for about 6 months to about 36 months when stored at about 25° C. and 60% relative humidity (RH). In yet another embodiment, the compositions of the present invention are stable for about 6 months when stored at about 40° C. and 75% relative humidity (RH).

[0089] In some embodiments, the compositions of the present invention are stable for up to about 36 months when stored at about 2° C. to about 8° C. In other embodiments, the compositions of the present invention are stable for up to about 36 months when stored at about 25° C. and 60% relative humidity (RH). In yet other embodiments, the compositions of the present invention are stable for up to 6 months when stored at about 40° C. and 75% relative humidity (RH).

[0090] In one embodiment, the compositions of the invention contain at least (NLT) about 95.0 area% duplex and at most (NMT) about 5 area% total duplex impurities as determined by purity non-denaturing IPRP-HPLC. In another embodiment, the pharmaceutical compositions of the invention contain at least (NLT) about 85.0 area% total single strands as determined by purity denaturing AX-HPLC. In yet another embodiment, the pharmaceutical compositions of the invention contain at least (NLT) about 80.0 area% total single strands as determined by purity denaturing IPRP-HPLC.

[0091] In one embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of the Serpinc1 gene. The pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, where the pH and osmolality of the pharmaceutical composition are suitable for subcutaneous administration to a subject, the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprises a region complementary to an mRNA encoding Serpinc1, the region comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15) by no more than 3 nucleotides, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached to the 3'-terminus, and the dsRNA agent is in free acid form.

[0092] In another embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of the Serpinc1 gene. The pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, where the pH and osmolality of the pharmaceutical composition are suitable for subcutaneous administration to a subject, the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprises a region of complementarity to an mRNA encoding Serpinc1 that comprises 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), substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated to a ligand attached to the 3'-terminus, and the dsRNA agent is in a salt form.

[0093] In one embodiment, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0094] 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.

[0095] The region of complementarity may be at least 17 nucleotides in length or 19 nucleotides in length.

[0096] 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.

[0097] In one embodiment, each strand is 30 nucleotides or less in length.

[0098] At least one strand of the double-stranded RNAi agent may comprise a 3' overhang of at least one nucleotide or 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 other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0099] In certain embodiments, the ligand is N-acetylgalactosamine (GalNAc).The ligand may be one or more GalNAcs 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.

[0100] In some embodiments, the double-stranded RNAi agent comprises multiple, eg, 2, 3, 4, 5, or 6, GalNAc, each independently linked to multiple nucleotides of the double-stranded RNAi via multiple monovalent linkers.

[0101] In certain embodiments, the ligand is [ka] It is.

[0102] In one embodiment, the RNAi agent is conjugated to the ligand via a linker, and the ligand and linker are represented by the following scheme: [ka] Conjugated to an RNAi agent as shown in In the formula, X is O or S.

[0103] In one embodiment, X is O.

[0104] In one embodiment, the region of complementarity consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15).

[0105] 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).

[0106] In one embodiment, the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf or Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

[0107] In one embodiment, the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, or Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and the sense strand is conjugated to a ligand via a linker, and the ligand and linker are represented by the following scheme: [ka] Conjugated to an RNAi agent as shown in In the formula, X is O or S.

[0108] In one embodiment, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpincl gene. The pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, wherein the pH and osmolality of the pharmaceutical composition are suitable for subcutaneous administration to a subject, and the dsRNA agent comprises a sense strand consisting of the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941) and a nucleotide sequence 5'-usUfsgAfaG and an antisense strand consisting of the nucleotide sequence of fuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and the ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in the free acid form.

[0109] In another aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of the Serpinc1 gene. The pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 50 mg / mL to about 200 mg / mL and phosphate buffered saline (PBS) at a concentration of about 1 mM to about 10 mM, where the pH and osmolality of the pharmaceutical composition are suitable for subcutaneous administration to a subject, and the dsRNA agent has a sense strand consisting of the nucleotide sequence of 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941) and an antisense strand consisting of the nucleotide sequence of 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is 0.01 to 0.15. The linkage is phosphorothioate, and the ligand is conjugated to the 3' end of the sense strand via a linker, the ligand and linker having the following structure: [ka] Here, the dsRNA agent is in a salt form.

[0110] In one embodiment, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpincl gene. The composition comprises a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 100 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent comprises a sense strand consisting of the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941) and a nucleotide sequence 5'-usUfsgAfaGf and an antisense strand consisting of the nucleotide sequence of uAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and the ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in the free acid form.

[0111] In another embodiment, the present invention provides a pharmaceutical composition for inhibiting expression of the Serpincl gene, the pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent at a concentration of about 106 mg / mL and phosphate buffered saline (PBS) at a concentration of about 5 mM, wherein the pH of the pharmaceutical composition is about 6.8 to about 7.2, the osmolality of the pharmaceutical composition is about 300 mOsm / kg, and the dsRNA agent comprises a sense strand consisting of the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941) and 5'-usUfsgAfaG and an antisense strand consisting of the nucleotide sequence of fuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2'-fluoro A, G, C, U; and s is a phosphorothioate linkage, and the ligand is conjugated to the 3' end of the sense strand via a linker, wherein the ligand and linker have the following structure: [ka] Here, the dsRNA agent is in a salt form.

[0112] The compositions of the present invention may further comprise other additive components conventionally found in pharmaceutical compositions at their art-established use levels.Thus, for example, the compositions may comprise additional compatible pharma- ceutically active substances, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may comprise additional substances useful for physically compounding the various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers.However, when added, such substances should not unduly interfere with the biological activity of the components of the compositions of the present invention.The formulations may be stabilized and, if desired, mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, colorants, flavorings and / or aromatic substances, etc., that do not adversely interact with the nucleic acid of the formulation.

[0113] In some embodiments, the pharmaceutical compositions featured in the present invention include (a) one or more iRNA compounds and (b) one or more agents that function by non-RNAi mechanisms and are useful for treating hemolytic disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-steatotic agents, anti-viral and / or anti-fibrotic agents. In addition, other substances commonly used to protect the liver, such as silymarin, may be used in combination with the iRNAs described herein. Other agents useful for treating liver disease include telbivudine, entecavir and protease inhibitors, such as telaprevir, and others disclosed, for example, in Tung et al., U.S. Patent Application Publication Nos. 2005 / 0148548, 2004 / 0167116 and 2003 / 0144217; and Hale et al., U.S. Patent Application Publication No. 2004 / 0127488.

[0114] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as procedures for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0115] III. iRNAs for Use in Pharmaceutical Compositions of the Invention The compositions of the invention include RNAi agents that target the Serpinc1 gene and inhibit expression of the Serpinc1 gene in a cell, e.g., in a subject, e.g., a mammal, e.g., a human having a Serpinc1-associated disorder, e.g., a bleeding disorder, e.g., hemophilia.

[0116] As used herein, a "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpincl gene, e.g., an mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence is at least a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpincl gene. The portion is of sufficient length to act as a substrate for iRNA-dependent cleavage at or near the site.

[0117] The target sequence may be about 9-36 nucleotides in length, for example about 15-30 nucleotides in length. For example, the target sequence may be about 15-30 nucleotides, 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- The length may be 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 ranges and lengths are also considered part of the invention.

[0118] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide that comprises a strand of nucleotides described by a referenced sequence, using standard nucleotide nomenclature.

[0119] "G", "C", "A", "T" and "U" each generally refer to a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, or surrogate replacement moieties, as described in more detail below (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted for other moieties without substantially changing the base pairing properties of oligonucleotides that contain nucleotides carrying such replacement moieties. For example, but not limited to, a nucleotide that contains inosine as a base can base pair with a nucleotide that contains adenine, cytosine or uracil. Thus, a nucleotide that contains uracil, guanine or adenine is substituted, for example, by a nucleotide that contains inosine in the nucleotide sequence of dsRNA characterized in the present invention. In another example, adenine and cytosine in an oligonucleotide may be substituted at any position with guanine and uracil, respectively, to form a GU wobble base pair with a target mRNA. Sequences containing such substituted portions are suitable for the compositions and methods featured in the present invention.

[0120] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent" used interchangeably herein refer to agents that mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway, including RNA, as the term is 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 Serpinc1 in cells, e.g., cells in a subject, e.g., a mammalian subject.

[0121] 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, and causes 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 is a ribonuclease-III-like enzyme that processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic 2-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 helical fragments are inserted into the siRNA complex. A ricase unwinds 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 107:309). Dev.15:188). Thus, in one aspect, the present invention relates to a single-stranded RNA (siRNA) that is produced in cells, promotes the formation of RISC complex, and causes the silencing of target gene, i.e., Serpincl gene. Thus, the term "siRNA" is also used herein to refer to the above RNAi.

[0122] In other embodiments, the RNAi agent may be a single-stranded siRNA introduced into a cell or organism to inhibit target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease Argonaute2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the contents of each of which are incorporated herein by reference in their entirety. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs as described herein, or may be chemically modified and used by the methods described in Lima et al., (2012) Cell 150;:883-894.

[0123] In other embodiments, the "iRNA" for use in the compositions, uses and methods of the present invention is double-stranded RNA, and is referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure, comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations with respect to the target RNA, i.e., the Serpincl gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) causes the degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0124] Generally, the majority of the nucleotides in each strand of dsRNA molecule are ribonucleotides, but as described in detail herein, either or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, "RNAi agent" may contain ribonucleotides with chemical modifications; RNAi agent may contain substantial modifications in multiple nucleotides.

[0125] The term "modified nucleotide" as used herein refers to a nucleotide that has modified sugar moiety, modified internucleotidic bond and / or modified nucleobase independently.Thus, the term modified nucleotide includes, for example, the substitution, addition or removal of functional group or atom to internucleoside bond, sugar moiety or nucleobase.Suitable modifications for use in the agent of the present invention include all kinds of modifications disclosed herein or known in the art.All such modifications used in siRNA type molecules are included in "RNAi agent" for the purpose of this specification and claims.

[0126] The duplex region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can be from 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, 15-29, 25-30 ... 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, 19-2 The length may be in the range of 5, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the invention.

[0127] The two strands forming the duplex structure may be different parts of one larger RNA molecule or may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore joined by an uninterrupted stretch between the 3'-end of one strand and the 5'-end of the other, forming a duplex structure, the joined RNA strands are called "hairpin loops". The hairpin loop may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop may 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.

[0128] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules may be covalently linked, but not necessarily. The two strands are covalently linked by means other than an uninterrupted nucleotide chain between the 3'-end of one strand and the 5'-end of each other strand forming a duplex structure, and the linking structure is called "linker". The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest strand of dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may include one or more nucleotide overhangs.

[0129] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, e.g., a Serpincl target mRNA sequence, and directs 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 is a ribonuclease-III-like enzyme that processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic 2-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 RISC cleave the target, inducing silencing (Elbashir et al. (2001) Genes Dev. 15:188).

[0130] The term "nucleotide overhang" as used herein 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 may include an overhang of at least one nucleotide; alternatively, the overhang may be less than one nucleotide. The nucleotide overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogues, such as deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide of the overhang may be present on the 5'-end, the 3'-end or both ends of either the antisense strand or the sense strand of the dsRNA.

[0131] In one embodiment, the antisense strand of the dsRNA has a nucleotide overhang at the 3'-end and / or 5'-end of 1 to 10 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the sense strand of the dsRNA has a nucleotide overhang at the 3'-end and / or 5'-end of 1 to 10 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0132] In certain embodiments, the overhang on the sense strand or the antisense strand or both may comprise an extension of more than 10 nucleotides, for example, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the extended overhang are substituted with a nucleoside thiophosphate.

[0133] "Blunt" or "blunt end" means that there is no unpaired nucleotide at the end of double-stranded RNAi agent, i.e., there is no nucleotide overhang.A "blunt end" RNAi agent is a dsRNA that is double-stranded throughout its length, i.e., 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 (i.e., the agent that has one overhang and one blunt end) or the agent that has nucleotide overhang at both ends.

[0134] The term "antisense strand" or "guide strand" refers to the strand of iRNA, e.g., dsRNA, that comprises a region that is substantially complementary to a target sequence, e.g., Serpinc1 mRNA. The term "region of complementarity" as used herein refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a Serpinc1 nucleotide sequence as defined herein. When the region of complementarity is not completely complementary to the target sequence, mismatches may exist within the internal or terminal regions of the molecule. In general, the most tolerated mismatches are in the terminal regions, e.g., 5, 4, 3 or 2 nucleotides at the 5'- and / or 3'-end of iRNA.

[0135] The terms "sense strand" or "passenger strand," as used herein, refer 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.

[0136] The term "cleavage region" as used herein refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises 3 bases on either side of the cleavage site and immediately adjacent to it. In some embodiments, the cleavage region comprises 2 bases on either side of the cleavage site and immediately adjacent to it. In some embodiments, the cleavage site specifically occurs at the site bound to nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

[0137] Unless otherwise specified, the term "complementary" as used herein, when used to describe a first nucleotide sequence in relation 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 certain conditions, as understood by those skilled in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions that may be encountered in an organism. Those skilled in the art may determine the most appropriate set of conditions for testing the complementarity of two sequences, depending on the ultimate application of the hybridized nucleotides.

[0138] The complementary sequence in iRNA, for example, dsRNA described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences are 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 fully complementary or may form one or more, but generally at most 5, 4, 3 or 2 mismatched base pairs when hybridizing a duplex of up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to its final application, such as the inhibition of gene expression via the RISC pathway. However, when two oligonucleotides are designed to form one or more single-stranded overhangs when hybridizing, such overhangs should not be considered as mismatches for determining complementarity. For example, a dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length is referred to as "fully complementary" for purposes described herein, even if the longer oligonucleotide contains a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0139] As used herein, a "complementary" sequence may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements for hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairs.

[0140] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" are used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as understood from the context of use.

[0141] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding Serpinc1). For example, a polynucleotide is complementary to at least a portion of a Serpinc1 mRNA if the sequence is substantially complementary to an uninterrupted portion of an mRNA encoding Serpinc1.

[0142] Thus, in some embodiments, the antisense strand polynucleotide disclosed herein is fully complementary to the target Serpinc1 sequence.In other embodiments, the antisense strand polynucleotide disclosed herein is substantially complementary to the target Serpinc1 sequence, and comprises a contiguous nucleotide sequence that is at least about 80%, for example about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary to the corresponding region of the nucleotide sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 over its entire length.

[0143] In one embodiment, the RNAi agent of the present invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, wherein the antisense strand is complementary to a 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 throughout its length.

[0144] Suitable dsRNA agents capable of inhibiting the expression of target genes (i.e., Serpinc1 gene) in vivo include chemical modifications. In certain aspects of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all of the nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention in which "substantially all of the nucleotides are modified" may be mostly modified but not entirely modified, and may contain at most 5, 4, 3, 2 or 1 unmodified nucleotides.

[0145] iRNA agents for use in the methods of the invention will generally be about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-2 The present invention includes an RNA strand (antisense strand) having a region of 5, 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.

[0146] In other embodiments, one or both strands of the double-stranded RNAi agents of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, and have 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, the sense and antisense strands are 18-30 nucleotides in length. It forms a duplex of consecutive nucleotides.

[0147] In some embodiments, an iRNA agent for use in the methods of the invention includes an RNA strand (antisense strand) 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, and may be up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 in length. In some embodiments, such an iRNA agent with a longer antisense strand may 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.

[0148] An RNAi agent includes a sense strand and an antisense strand. Each strand of an RNAi agent can range from 12-30 nucleotides in length. For example, each strand can be 14-30 nucleotides in length, 17-30 nucleotides in length, 19-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0149] The sense and antisense strands typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of the RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region may be 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In other examples, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0150] In one embodiment, the RNAi agent may include 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 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long or 1-2 nucleotides long. 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 kinked. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence or other sequences. The first and second strands may also be joined, e.g., by additional bases to form a hairpin, or by other non-basic linkers.

[0151] In one embodiment, the nucleotides in the overhang region of the RNAi agent may each be independently modified or unmodified nucleotides, 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 may be the overhang sequence for either end of either strand. The overhang may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence or other sequences.

[0152] The 5'- or 3'-overhang of the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with phosphorothioate between the two nucleotides, where the two nucleotides may 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 at the antisense strand. In one embodiment, the 3'-overhang is present at the sense strand.

[0153] RNAi agents may only contain a single overhang, which may enhance the interference activity of RNAi without affecting the overall stability. For example, the single-stranded overhang may be located at the 3'-end of the sense strand or the 3'-end of the antisense strand. RNAi may also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or the opposite. In general, the antisense strand of RNAi has a nucleotide overhang at the 3'-end and the 5'-end is blunt. Without wishing to be bound by theory, the blunt end at the 5'-end of the asymmetric antisense strand and the 3'-end overhang of the antisense strand are favorable for the loading of the guide strand into the RISC process.

[0154] Any nucleic acid featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse ligation) or 3'-terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.); base modifications, such as stabilizing bases, destabilizing bases, or replacement with bases that base pair with an extended repertoire of partners, removal of bases (abasic nucleotides) or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; and / or backbone modifications, such as modification or replacement of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. The RNA with modified backbone includes, among others, those that do not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, modified RNA that does not have phosphorus atom in internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified iRNA has phosphorus atom in its internucleoside backbone.

[0155] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters and boranophosphates, their 2'-5' linked analogs and those with reversed 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.

[0156] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing linkages include, but are not limited to, U.S. Pat. No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5 ,264,423;No.5,276,019;No.5,278,302;No.5,286,717;No.5,321,131;No.5,399,676;No.5,40 No. 5,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,8 No. 21; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; 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;No.6,60 Nos. 8,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0157] Modified RNA backbones that do not contain phosphate atoms therein include backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and mixed N, O, S and CH 2Others include those that contain component parts.

[0158] Representative United States patents which teach the preparation of the above-mentioned oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; Nos. 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the contents of each of which are incorporated by reference in their entirety.

[0159] In other embodiments, suitable RNA mimics are considered for use in iRNA, in which the sugar and internucleoside linkages, i.e., backbone, of nucleotide units are replaced by novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide part 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 contents of each of which are incorporated herein by reference in their entirety. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0160] Some embodiments featured in the present invention include RNA with phosphorothioate backbones and heteroatom backbones, particularly the --CH of U.S. Pat. No. 5,489,677 referenced above. 2 --NH--CH 2 -, --CH 2 --N(CH 3 )--O--CH 2 -- [also known as the methylene (methylimino) or MMI backbone], --CH 2 --O--N(CH 3 )--CH 2 --, --CH 2 --N(CH 3 )--N(CH 3 )--CH 2 -- and --N(CH 3 )--CH 2 --CH 2 --[where the natural phosphodiester backbone is --O--P--O--CH 2 --] and oligonucleotides having the amide backbone of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structures of the above-referenced U.S. Patent No. 5,034,506.

[0161] Modified RNAs can also include 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 C 1 From C 10 Alkyl or C 2 From C 10 Alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are from 1 to about 10. In other embodiments, the dsRNA includes at the 2' position the following: 1 From C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents with similar properties. In some embodiments, the modification is 2'-methoxyethoxy (2'-O--CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Other exemplary modifications include O(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as shown below in the Examples herein. 2 ) 2 ON(CH 3 )2 groups and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH 2 --O--CH 2 --N(CH 2 ) 2 It is.

[0162] Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the 3' terminal nucleotide or in 2'-5' linked dsRNAs, and at the 5' position of the 5' terminal nucleotide. An iRNA can also have a sugar mimetic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Representative United States patents which 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; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670 and 5,700,920, certain of which are commonly owned with this application, the entire contents of each of which are incorporated herein by reference.

[0163] iRNAs may also contain nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 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, These include cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine and 3-deazaguanine and adenine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those described in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, edited by Herdewijn, P., Wiley-VCH, 2008; those described in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, edited by Kroschwitz, JL, John Wiley & Sons, 1990, those described in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those described in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, edited by Crooke, ST and Lebleu, B., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds characterized in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, YS, Crooke, ST, and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are particularly exemplary base substitutions when combined with 2'-O-methoxyethyl sugar modifications.

[0164] Representative United States patents which teach the preparation of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,50 No. 2,177; No. 5,525,711; No. 5,552,540; 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. 6,015,886; No. 6,147,200; No. 6,166,197; No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,52 Nos. 8,640; 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.

[0165] The RNA of the iRNA may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring that is modified by a bridge between two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside that has a sugar moiety that includes 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 the 2'-carbon of the sugar ring. Thus, in some embodiments, the agent of the invention may include an RNA of the iRNA that may be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety in which the ribose moiety includes an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-terminal structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0166] Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, but are not limited to, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention contain one or more bicyclic nucleosides that contain a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to 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 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 above are incorporated herein by reference.

[0167] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,84 Nos. 5, 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 Publication No. 2008 / 0039618, and U.S. Patent Publication No. 2009 / 0012281, the contents of each of which are incorporated by reference in their entirety.

[0168] Any of the bicyclic nucleosides described above may be prepared with one or more stereochemical sugar configurations including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0169] The RNA of an iRNA may be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S stereochemical configuration, referred to herein as "S-cEt."

[0170] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is long enough to place the oxygen in an optimal position for stability and affinity, making it less likely to result in puckering of the ribose ring.

[0171] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication No. WO 2013 / 036868, the contents of each of which are incorporated by reference herein in their entirety.

[0172] One or more nucleotides of the iRNA of the invention may also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as a "non-locked nucleic acid" ("UNA") modification.

[0173] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the contents of each of which are incorporated by reference in their entirety herein.

[0174] Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication WO 2011 / 005861.

[0175] In certain embodiments of the invention, the double-stranded RNAi agents of the invention include agents having chemical modifications disclosed, for example, in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or PCT / US2012 / 065691, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference.

[0176] The double-stranded RNA (dsRNA) agent of the present invention may be optionally conjugated to one or more ligands.Ligand may be bound to sense strand, antisense strand or both strands at 3'-end, 5'-end or both ends.For example, ligand may be conjugated to sense strand.In a preferred embodiment, ligand is conjugated to 3'-end of sense strand.

[0177] In one embodiment, the ligand is a carbohydrate conjugate, such as a monosaccharide. In one embodiment, the ligand is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In certain embodiments of the invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is linked to the iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the invention via a trivalent linker. Suitable ligands are disclosed, for example, in U.S. Patent Application No. 15 / 371,300 and U.S. Patent Publication No. 2009 / 0239814, the contents of each of which are incorporated herein by reference in their entirety with respect to suitable ligands.

[0178] In some embodiments, a ligand, e.g., a GalNAc ligand, is attached to the 3' end of the RNAi agent. [ka] As shown in Figure 1, the ligand is conjugated to a ligand, e.g., a GalNAc ligand, via a linker. wherein X is O or S. In one embodiment, X is O.

[0179] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,13 No. 8,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,6 No. 67,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5, No. 082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5 ,258,506;No.5,262,536;No.5,272,250;No.5,292,873;No.5,317,098;No.5,371,241,No.5,391,723;No.5,416,203,No.5, No. 451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928 and No. 5,688,941; No. 6,294,664; No. 6,320,017; No. Nos. 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the contents of each of which are incorporated herein by reference in their entirety.

[0180] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above-mentioned modifications may be incorporated in a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0181] In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each consisting of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA has been modified to confer to the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the target nucleic acid. Additional regions of the iRNA may act as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNaseH is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNaseH results in cleavage of the RNA target, thereby greatly enhancing the efficacy of iRNA inhibition of gene expression. As a result, shorter iRNAs often provide comparable results when using chimeric dsRNAs, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target is routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0182] In certain instances, the RNA of the iRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to enhance the activity, cellular distribution or cellular uptake of iRNA, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl -S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS, 1992, 10:111), Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. Exemplary conjugates include aryl, aryl, and aryl-co-aryl. The conjugation protocol involves the synthesis of RNA carrying an amino linker at one or more positions of the sequence. The amino group is then reacted with the conjugated molecule using an appropriate coupling or activation reagent. The conjugation reaction may be carried out with the RNA still bound to the solid support or after cleavage of the RNA in solution phase. Typically, purification of the RNA conjugate by HPLC results in a pure conjugate.

[0183] V. Uses of the Pharmaceutical Compositions of the Invention The pharmaceutical compositions of the invention are useful for therapeutic and prophylactic treatment of subjects with disorders that would benefit from reduced Serpinc1 expression, such as bleeding disorders, e.g., hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C).

[0184] The term "treat" or "treatment" as used herein refers to a beneficial or desired result, including, but not limited to, the alleviation or amelioration of one or more symptoms, the reduction in the severity of bleeding, the stabilization (i.e., not worsening) of the bleeding condition, the amelioration or alleviation of bleeding or the resolution of bleeding, whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival in the absence of treatment. In the methods of the present invention, treatment includes on-demand treatment and control of bleeding episodes, perioperative management of bleeding, and routine prophylaxis to reduce the frequency of bleeding episodes.

[0185] The term "lower" in the context of Serpincl levels or disease markers or symptoms in a subject refers to a statistically significant reduction in such levels. Reduction can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, and is preferably reduced to a level that is accepted as being within the normal range of individuals who do not have such disorder.

[0186] As used herein, "prevention" or "preventing", when used in relation to a disease, disorder or its symptoms 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 such disease, disorder or symptoms, such as bleeding.The likelihood of developing bleeding is reduced, for example, when an individual with one or more risk factors for bleeding either does not develop bleeding or develops less severe bleeding compared to a population with the same risk factors and does not undergo the treatment described herein.Not developing a disease, disorder or symptom, or showing a reduction in the development of symptoms associated with a disease, disorder or symptom (e.g., at least about 10% for a clinically approved scale for said disease or disorder) or a delay in symptoms (e.g., by days, weeks, months or years) is considered to be effective prevention.

[0187] Subjects who would benefit from the reduction and / or inhibition of Serpinc1 gene expression are subjects with a bleeding disorder, e.g., an inherited bleeding disorder or an acquired bleeding disorder as described herein. In one embodiment, the subject with an inherited bleeding disorder has hemophilia, e.g., hemophilia A, B, or C. In one embodiment, the subject with an inherited bleeding disorder, e.g., hemophilia, is an inhibitor subject (a subject who has become refractory 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. Treatment of subjects who would benefit from the reduction and / or inhibition of Serpinc1 gene expression can be therapeutic (e.g., where necessary, e.g., the subject is bleeding (spontaneous bleeding or bleeding as a result of trauma) and is unable to clot) treatment, as well as prophylactic (e.g., The subject is not bleeding and / or is undergoing surgery) procedures.

[0188] The term "bleeding disorder" as used herein refers to a disease or disorder that results in insufficient blood clotting and / or excessive bleeding.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, dermatological disorders (e.g., psoriasis, pemphigus), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease), allergic drug reactions, such as the result of medications such as aspirin, heparin and warfarin, diabetes, acute hepatitis B infection, acute hepatitis C infection, malignant or solid tumors (e.g., prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervical, breast, melanoma, kidney and / or hematological 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, develops 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.

[0189] In one embodiment, the bleeding disorder is a rare bleeding disorder (RBD). RBD may be acquired or inherited RBD. Inherited RBD includes disorders related to congenital deficiency of clotting factors fibrinogen, FII, FV, combined FV and FVIII, FVII, FX, FXI, FXIII and vitamin K-dependent factor (VKCFD). They are generally transmitted as autosomal recessive conditions, but in some cases, such as FXI and dysfibrinogenemia, may be autosomal dominant. RBD is reported in most populations with homozygous or double heterozygous incidence rates ranging from 1 in 500,000 for FVII deficiency to 1 in 2 to 3 million for prothrombin and FXIII deficiency. Relative frequencies vary between populations and are higher when consanguineous or intra-marriage is common, increasing the frequency of certain mutant genes.

[0190] Exemplary RBDs include afibrinogenemia (fibrinogen; factor I deficiency); hypofibrinogenemia (fibrinogen; factor I deficiency); dysfibrinogenemia (fibrinogen; factor I deficiency); hypodysfibrinogenemia (fibrinogen; factor I deficiency); hypoprothrombinemia (prothrombin; factor II deficiency); prothrombin deficiency (prothrombin; factor II deficiency); thrombophilia (prothrombin; factor II deficiency); congenital antithrombin III deficiency (thromboplastin; factor III; tissue factor); parahemophilia (proaccelerin; factor V; labile factor); orthrombocytopenia (ADHD) (ADHD). disease (proaccelerin; factor V; labile factor); activated protein C resistance (proaccelerin; factor V; labile factor); Alexander disease (stable factor proconvertin; factor VII); congenital proconvertin / factor VII deficiency (stable factor proconvertin; factor VII); Stuart-Prower deficiency (Stuart-Prower factor; factor X); congenital factor XIIIa / b deficiency (fibrin stabilizing factor; factor XIIIa); hereditary factor XIII deficiency (fibrin stabilizing factor; factor XIII); and fibrin stabilizing factor deficiency (fibrin stabilizing factor; factor XIII).

[0191] As used herein, "therapeutically effective amount" is intended to include the amount of RNAi agent sufficient to cause disease treatment (e.g., by reducing, ameliorating or maintaining existing disease or one or more symptoms of disease) when administered to a subject with bleeding disorder and bleeding. "Therapeutically effective amount" can vary depending on the RNAi agent, the method of administering the agent, the disease and its severity and the medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment if any, and other individual characteristics of the subject to be treated.

[0192] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA that, when administered to a subject having a bleeding disorder but who is not bleeding, e.g., a subject having a bleeding disorder and scheduled for surgery (e.g., peri-operative treatment), is sufficient to prevent or ameliorate a disease or one or more symptoms of a disease.

[0193] Amelioration of disease includes slowing the course of disease or reducing the severity of later onset disease. A "prophylactically effective amount" can vary depending on the iRNA, the manner in which the agent is administered, the degree of risk of disease and the medical history, age, weight, family history, genetic makeup of the subject being treated, types of prior or concurrent treatments, if any, and other individual characteristics of the subject being treated.

[0194] A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNAs used in the methods of the invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0195] A "recommended therapeutically effective amount of replacement factor" and a "recommended therapeutically effective amount of bypassing agent" are doses of replacement factor or bypassing agent, respectively, sufficient to generate thrombin and resolve bleeding and / or achieve peak plasma factor levels in a subject having a bleed as set forth by the World Federation of Hemophilia (see, e.g., Srivastava et al., "Guidelines for the Management of hemophilia," Hemophilia Epub 6 July 2012; DOI:10.1111 / j.1365-2516.2012.02909.x; ADVATE (Antihemophilic Factor (Recombinant)) Package Insert; 11 / 2016; and BeneFIX (Coagulation Factor IX (Recombinant) Package Insert; 11 / 2011, the contents of each of the foregoing being incorporated herein by reference in their entirety.

[0196] For example, the recommended dose of replacement factor or bypassing agent for a subject with mild bleeding is a dose sufficient to achieve a peak plasma factor VIII level of about 10-40 IU / dL; the recommended dose of replacement factor or bypassing agent for a subject with moderate bleeding is a dose sufficient to achieve a peak plasma factor VIII level of about 30-60 IU / dL; the recommended dose of replacement factor or bypassing agent for a subject with severe bleeding is a dose sufficient to achieve a peak plasma factor VIII level of about 60-100 IU / dL; and the recommended dose of replacement factor or bypassing agent for a perioperative subject is a dose sufficient to achieve a peak plasma factor VIII level of about 30-60 IU / dL (see, e.g., Tables 1 and 2 of the ADVATE (Antihemophilic Factor (Recombinant)) Package Insert; 11 / 2016).

[0197] A recommended dose of replacement factor or bypassing agent for a subject with mild bleeding is a dose sufficient to achieve a peak plasma factor IX level of about 10-30 IU / dL; a recommended dose of replacement factor or bypassing agent for a subject with moderate bleeding is a dose sufficient to achieve a peak plasma factor IX level of about 25-50 IU / dL; and a recommended dose of replacement factor or bypassing agent for a subject with severe bleeding is a dose sufficient to achieve a peak plasma factor IX level of about 50-100 IU / dL.

[0198] The methods and uses of the pharmaceutical compositions of the invention generally involve administering a pharmaceutical composition of the invention to a subject having a Serpinc1-associated disorder, e.g., a bleeding disorder, e.g., hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C). In some embodiments of the invention, the methods further involve administering an additional therapeutic agent to the subject.

[0199] Thus, in one aspect, the present invention provides a method for treating cancer that benefits from a reduction in Serpincl expression. The present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression, such as a bleeding disorder, e.g., hemophilia. The method includes administering to the subject, e.g., a human, a pharmaceutical composition of the present invention comprising an iRNA agent, e.g., a dsRNA, of the present invention in a prophylactically effective amount, e.g., a fixed dose of about 25 mg to 100 mg, e.g., a fixed dose of about 80 mg, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

[0200] In other embodiments, 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 amount, e.g., a fixed dose of about 25 to about 100 mg, e.g., a fixed dose of about 80 mg, of a pharmaceutical composition of the invention comprising an iRNA agent targeted to the Serpinc1 gene, or a pharmaceutical composition comprising an iRNA agent targeted to the Serpinc1 gene, thereby treating the subject having a disorder that would benefit from reduced Serpinc1 expression.

[0201] In certain embodiments, therapeutic and prophylactic methods of the invention include administering to a subject a pharmaceutical composition comprising an iRNA agent of the invention in an amount that reduces Serpinc1 activity in a subject by about 75% or more, and a therapeutically effective amount of a replacement factor or bypassing agent that is reduced, e.g., compared to a recommended therapeutically effective amount (e.g., an amount sufficient to generate thrombin and resolve bleeding (form a clot)) of the replacement factor or bypassing agent recommended, e.g., by the World Federation of Hemophilia (see, e.g., Srivastava et al., "Guidelines for the Management of Hemophilia," Hemophilia Epub 6 July 2012; DOI:10.1111 / j.1365-2516.2012.02909.x) and / or the Food and Drug Administration (see, e.g., ADVATE (Antihemophilic Factor (Recombinant)) Package Insert; 11 / 2016; BeneFIX (Coagulation Factor IX (Recombinant) Package Insert; 11 / 2011). The contents of each of the above are incorporated herein by reference in their entirety.

[0202] Suitable replacement factors include Factor VIII, such as Advate, Eloctate, Haemate, Helixate, Immunate, Octanate, Recombinate and Refacto, or Factor IX, such as Aimafix, Benefix, Immunine and Refacto. Suitable bypass agents for use in the methods of the invention include activated prothrombin concentrates (aPCC), including, for example, FEIBA and Prothromplex, and recombinant Factor VIIa (rFVIIa), such as NovoSeven.

[0203] The replacement factor can be factor VIII, and a therapeutically effective amount of the replacement factor administered to a subject in the methods of the invention is a dose sufficient to achieve a peak plasma factor VIII level of about 10 to 100 IU / dL, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 IU / dL.

[0204] For example, a therapeutically effective amount of replacement factor VIII administered to a subject is less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, or less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg. In one embodiment, the therapeutically effective amount of factor VIII administered to a subject is about 1.5 to about 5 times lower than the recommended effective amount of replacement factor, e.g., about 5 to about 20 IU / kg or about 10 to about 20 IU / kg, e.g., 5, 10, 15 or 20 IU / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a severe bleeding event.

[0205] The replacement factor can be factor IX, and a therapeutically effective amount of the replacement factor administered to a subject in the methods of the invention is a dose that achieves a peak plasma factor IX level of about 10 to 100 IU / dL, for example, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 IU / dL.

[0206] For example, a therapeutically effective amount of a factor IX replacement factor may be less than about 200 IU / kg, or less than about 190 IU / kg, or less than about 180 IU / kg, or less than about 170 IU / kg, or less than about 160 IU / kg, or less than about 150 IU / kg, or less than about 140 IU / kg, or less than about 130 IU / kg, or less than about 120 IU / kg, or less than about 110 IU / kg, or less than about 100 IU / kg, or less than about 90 IU / kg, or less than about 80 IU / kg, or less than about 70 IU / kg, less than about 60 IU / kg, or less than about 50 IU / kg, or less than about 40 IU / kg, or less than about 30 IU / kg, or less than about 20 IU / kg, or less than about 10 IU / kg. In one embodiment, the therapeutically effective amount of factor IX administered to the subject is about half to about one sixth of the recommended effective amount of replacement factor, for example, about 10 to about 30 IU / kg or about 20 to about 30 IU / kg, for example, about 10, 15, 20, 25 or 30 IU / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a severe bleeding event.

[0207] The bypassing agent may be an aPCC, and the therapeutically effective amount of the bypassing agent administered to a subject in the methods of the invention is a dose sufficient to generate thrombin and resolve bleeding.

[0208] For example, the therapeutically effective amount of bypassing agent aPCC may be less than about 100 U / kg, or less than about 90 U / kg, or less than about 80 U / kg, or less than about 70 U / kg, or less than about 60 U / kg, or less than about 50 U / kg, or less than about 40 U / kg, or less than about 30 U / kg, or less than about 20 U / kg, or less than about 10 U / kg. In one embodiment, the therapeutically effective amount of aPCC administered to a subject is about half to about one third of the recommended effective amount of replacement factor, for example, about 30 to about 50 U / kg, for example, 30, 35, 40, 45 or 50 U / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a severe bleeding event.

[0209] The bypassing agent may be rFVIIa, and the therapeutically effective amount of the bypassing agent administered to a subject in the methods of the invention is a dose sufficient to generate thrombin and resolve bleeding.

[0210] For example, the therapeutically effective amount of the bypassing agent rFVIIa is less than about 120 μg / kg, or less than about 110 μg / kg, or less than about 100 μg / kg, or less than about 90 μg / kg, or less than about 80 μg / kg, or less than about 70 μg / kg, or less than about 60 μg / kg, or less than about 50 μg / kg, or less than about 40 μg / kg, or less than about 30 μg / kg, or less than about 20 μg / kg. In one embodiment, the therapeutically effective amount of rFVIIa administered to the subject is about half the recommended dose of replacement factor, for example, a dose of about 45 μg / kg. In one embodiment, the bleeding event is a moderate bleeding event. In another embodiment, the bleeding event is a severe bleeding event.

[0211] In some embodiments, the pharmaceutical composition comprising dsRNA agent is administered to the subject in a fixed dose. "Fixed dose" (e.g., dose in mg) means that one dose of iRNA agent is used for all subjects regardless of any specific subject-related factors, such as body weight. In a particular embodiment, the fixed dose of the iRNA agent of the present invention is based on a predetermined body weight or age.

[0212] In some embodiments, a pharmaceutical composition including an iRNA agent comprises between about 25 mg and about 100 mg, e.g., between about 25 mg and about 95 mg, between about 25 mg and about 90 mg, between about 25 mg and about 85 mg, between about 25 mg and about 80 mg, between about 25 mg and about 75 mg, between about 25 mg and about 70 mg, between about 25 mg and about 65 mg, between about 25 mg and about 60 mg, between about 25 mg and about 50 mg, between about 50 mg and about 100 mg, between about 50 mg and about 95 mg, between about 50 mg and about 90 mg, between about 50 mg and about 85 mg, between about 50 mg and about 80 mg, between about 30 mg and about 100 mg, 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 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.

[0213] In some embodiments, a pharmaceutical composition including an iRNA agent is administered at a fixed dose of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg.

[0214] In one embodiment, the RNAi agent is administered to the subject at a fixed dose of about 100 mg.

[0215] In one embodiment, the RNAi agent is administered to a subject at a dose that reduces Serpinc1 activity by about 75% or more.

[0216] The pharmaceutical composition containing the iRNA agent is administered to the subject in one or more doses.

[0217] The pharmaceutical composition comprising the iRNA is administered to the subject about once a month, about once every 5 weeks, about once every 6 weeks, about once every 2 months, or once a quarter.

[0218] In some embodiments, the single dose of pharmaceutical composition can be long-lasting, so that subsequent doses are administered at intervals of at most 1, 2, 3, 4, 5, 6, 7 or 8 weeks.In some embodiments of the present invention, the single dose of pharmaceutical composition is administered once a month.In one embodiment, the fixed dose of RNAi agent is suitable for administration to subject once a month, for example, at a fixed dose of 80mg once a month.

[0219] The methods and uses of the present invention include administering the compositions described herein to a subject having a cell line comprising at least one of the following: , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80 days. In one embodiment, expression of the target Serpinc1 gene is reduced for an extended period of time, 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.

[0220] The reduction in gene expression can be evaluated by any method known in the art. For example, the reduction in expression of Serpinc1 can be determined by determining the mRNA expression level of Serpinc1 using methods that are routine to those skilled in the art, such as Northern blotting, qRT-PCR, determining the protein level of Serpinc1 using methods that are routine to those skilled in the art, such as Western blotting, immunological techniques, and / or determining the biological activity of Serpinc1, such as the effect on one or more molecules related to the blood clotting mechanism of cells (or in in vivo conditions, in blood clotting itself). In one embodiment, thrombin generation time, clot formation time and / or clotting time are determined, for example using ROTEM® Thromboelastometry analysis of whole blood to evaluate Serpinc1 expression.

[0221] Administration of dsRNA by the method and use of the present invention can result in the reduction of the severity, signs, symptoms and / or markers of Serpincl-related disease in patients with such disease or disorder.In this context, "reduction" refers to the statistically significant reduction of such level.Reduction can be, for example, 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%.

[0222] The efficacy of disease treatment or prevention is evaluated, for example, by measuring disease progression, disease remission, symptom severity, bleeding frequency, pain reduction, quality of life, the dosage of medication required to maintain treatment effectiveness, the level of disease markers, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the capabilities of a person skilled in the art to monitor the efficacy of treatment or prevention by measuring any one or any combination of such parameters. For example, the efficacy of treatment of bleeding disorders is evaluated, for example, by periodically monitoring thrombin:antithrombin levels. Comparing the later readings to the earlier readings provides the physician with an indication of whether the treatment is effective. It is well within the capabilities of a person skilled in the art to monitor the efficacy of treatment or prevention by measuring any one or any combination of such parameters. "Effective against" a bleeding disorder, in relation to administering an iRNA targeting Serpinc1 or a pharmaceutical composition thereof, indicates that administration in a clinically relevant manner results in a beneficial effect in at least a statistically significant percentage of patients, such as amelioration of symptoms, cure, reduction in disease, extension of life span, improvement in quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of bleeding disorders and related causes.

[0223] Treatment or prevention effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening or progression of symptoms that would otherwise be expected. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more of the measurable parameters of the disease may indicate effective treatment. A given iRNA drug or a combination of such drugs may also be judged using an experimental animal model for a given disease, as known in the art. When using an experimental animal model, the efficacy of treatment is demonstrated when a statistically significant reduction in markers or symptoms is observed.

[0224] Alternatively, efficacy is measured by a reduction in disease severity, as determined by those skilled in the diagnostic field based on clinically accepted disease severity assessment scales. For example, any positive change resulting in a reduction in disease severity, as measured using an appropriate scale, is indicative of appropriate treatment with the iRNA or iRNA formulations described herein.

[0225] The present invention further provides methods and uses for the use of iRNAs or pharmaceutical compositions thereof in combination with other pharmaceutical agents and / or other therapeutic methods, such as known pharmaceutical agents and / or known therapeutic methods, such as those currently used to treat such disorders, to treat subjects who would benefit from reducing and / or inhibiting Serpinc1 expression, e.g., subjects with bleeding disorders.

[0226] For example, in certain embodiments, an iRNA targeting Serpinc1 is administered in combination with an agent useful for treating a bleeding disorder, eg, as described elsewhere herein. For example, further 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 high doses of FVIII or FIX along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasma exchange, with or without antifibrinolytic therapy, in combination with immunosuppression and FVIII or FIX infusion; immune tolerance induction (ITI) with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone and / or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytic agents, such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrates (PCC); antihemophilic agents; corticosteroids; immunosuppressants; and estrogens.

[0227] The iRNA and additional therapeutic agents and / or treatments may be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agents may be administered as part of separate compositions or at separate times and / or by other methods known in the art or described herein.

[0228] VI. Containers of the Present Invention The invention also provides a container, such as a vial, syringe, autoinjector pen or needleless administration device, which comprises a pharmaceutical composition of the invention.

[0229] In one embodiment, the compositions of the invention are used for self-administration, for example using a pre-filled syringe or an auto-injection device.

[0230] In one embodiment, the container containing the pharmaceutical composition of the present invention is a vial. The vial may contain about 0.5 mL to about 2.0 ml of the pharmaceutical composition. In one embodiment, the vial contains about 0.8 ml of the pharmaceutical composition. In one embodiment, the vial is a 2R vial (i.e., a 2 ml injection vial) containing a single dose of the pharmaceutical composition. In one embodiment, the 2R vial contains about 0.80 ml (e.g., about 0.96 to about 1.05 mL) of the pharmaceutical composition of the present invention, which contains a single dose of 80 mg of the composition.

[0231] In one embodiment, the container of the present invention comprises a syringe, e.g., a pre-filled syringe. In one embodiment, the pre-filled syringe comprises a needlestick safety feature (PFS-S). A suitable syringe may be a 1 ml syringe or a 3 ml syringe and may include a 29G or 30G needle. In one embodiment, the syringe is a disposable 3 ml glass syringe with a 29G or 30G needle. In one embodiment, the pre-filled syringe holds about 0.80 ml ( For example, about 0.84 ml or 0.8 to 0.84 ml).

[0232] An exemplary pre-filled syringe of the invention may include a syringe, e.g., a BD Neopak with a 29G x ½″ needle; a rigid needle shield (RNS); a plunger, e.g., a BD4023 plunger with FluroTec coating; a safety system, e.g., BD UltraSafelm Plus; a plunger rod, e.g., a BD UltraSafe passive plunger rod; and a finger flange, e.g., a BD UltraSaferm passive add-on finger.

[0233] VII. Kits of the Invention The present invention also provides a kit comprising the pharmaceutical composition. Such a kit comprises one or more vials or one or more pre-filled syringes comprising the pharmaceutical composition of the present invention and instructions for use, such as instructions for administering a prophylactically or therapeutically effective amount of an RNAi agent. The kit may optionally further comprise a means for administering the RNAi agent (e.g., an injection device) or a means for measuring inhibition of Serpinc1 (e.g., a means for measuring inhibition of Serpinc1 mRNA, Serpinc1 protein and / or Serpinc1 activity). Such a means for measuring inhibition of Serpinc1 may comprise a means for obtaining a sample, such as a plasma sample, from a subject. The kit of the present invention may optionally further comprise a means for determining a therapeutically or prophylactically effective amount.

[0234] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those skilled in the art usually understand in the technical field to which the present invention belongs.Methods and materials similar or equivalent to those described herein can be used in carrying out or testing the iRNA and methods characterized in the present invention, and suitable methods and materials are described below.All publications, patent applications and other references mentioned herein are incorporated herein by reference in their entirety.Furthermore, materials, methods and examples are illustrative only and are not intended to be limiting.

[0235] This invention is further illustrated in the following examples, which should not be construed as limiting.All references, patents and published patent applications, and figures cited throughout this application are incorporated by reference. EXAMPLES

[0236] [Table 1]

[0237] Example 1: Fitusiran formulation The Fitusiran formulation (Fitusiran) is a sterile solution containing 100 mg / mL Fitusiran (equivalent to 106 mg / mL Fitusiran sodium) in 5 mM phosphate buffered saline (PBS) for subcutaneous administration. The formulation is generally made of Teflon-coated butyl rubber stretch. It is commercially supplied as a 0.8 mL solution in a 2R Type I glass vial with a cap and central tear-over seal. The formulation contains no preservatives and is intended for single use.

[0238] The composition of the Fitusiran formulation is outlined in Table 2.

[0239] [Table 2]

[0240] The chemical structure of Fitusiran, shown below, is depicted with an expanded structural formula showing the phosphate backbone. The bases involved in base pairing are connected by dotted lines. The structure of L96, a GalNAc-containing ligand, and the linker that conjugates the ligand to the 3'-end of the sense strand are also shown below. The molecular formulas and masses of the duplex and single strands (AD-116858, sense strand; A-116861, antisense strand) of Fitusiran duplex (AD-57213) are also shown in the table below.

[0241] [Table 3]

[0242] Example 2: Physilan Formulation Development The Fitusiran formulation was designed for subcutaneous administration. Formulations designed for subcutaneous administration should not be too acidic or too basic to avoid the risk of increased irritation and chemical incompatibility. The formulation was designed to be as close to physiological as possible, with due consideration given to osmolality, pH and viscosity. The pH of aqueous solutions of the Fitusiran formulation at 100 mg / mL varies from 5.0 to 6.8. The presence of sodium counterions with the anionic phosphodiester contributes a certain amount of osmolality that depends on the concentration of the aqueous solution. In the target formulation of the drug substance at 100 mg / mL, the counterions result in a solution of approximately 118 mOsm / kg. To maintain the isotonicity and buffer capacity of the formulation, the drug substance was dissolved in 5 mM phosphate buffered saline (0.64 mM NaH2PO4, 4.36 mM Na2HPO4, 84 mM NaCl).

[0243] The above pharmaceutical formulation has the following physicochemical properties: pH of about 6.8 to about 7.2; osmolality of about 300 mOsm / kg; and density of about 1.038 g / mL.

[0244] The manufacture of the Fitusiran formulation consisted of dissolving the required amount of powdered (lyophilized) Fitusiran drug substance in 5 mM phosphate-buffered saline and adjusting the pH to approximately 7.0 with sodium hydroxide or phosphoric acid, followed by sterile filtration and filling.

[0245] Formulations used in early development, e.g., nonclinical and Phase 1 / 2 clinical trials, were supplied as a 100 mg / mL (fitusiran sodium) solution in a nominal 0.5 mL per vial. Formulations intended for commercial production, used in Phase 3 trials, were supplied as a 100 mg / mL (fitusiran free acid, equivalent to 106 mg / mL fitusiran sodium) solution in a nominal 0.8 mL per vial. The table below summarizes the differences in the fitusiran formulation formulations.

[0246] [Table 4]

[0247] Example 3: Analytical analysis of Fitusiran formulations Various analytical evaluations of the Fitusiran formulation were carried out to demonstrate that the formulation is physically and chemically stable.

[0248] exterior The Fitusiran formulations were visually inspected for color, homogeneity and particulate matter against black and white backgrounds under diffuse uniform illumination.

[0249] Appearance testing on Fitusiran formulation was conducted using the same visual test across different batches and the results met the specification of "a clear, colorless to pale yellow solution, essentially free of particles."

[0250] Identifying duplex retention time The Fitusiran formulations were analyzed by non-denaturing IP RP-HPLC along with the Fitusiran reference standard, and the duplex retention times of the samples were compared to the reference standard. All Fitusiran formulation batches manufactured to date have met the specification of "retention time consistent with reference standard," confirming their identity as annealed siRNA duplexes.

[0251] Assay of Physilan formulations by UV The UV absorption method was used to determine the assay (mg / mL) of Fitusiran in the Fitusiran formulation. The absorbance of the formulation appropriately diluted in 0.9% saline was measured at 260 nm using a UV spectrophotometer. C = (A × F × M) / (ε × b) where A is the measured absorbance, F is the dilution factor, b is the path length of the cell (1 cm), ε is the molar absorptivity of the duplex reference standard, M is the molecular weight, and C is the concentration (mg / mL). To account for duplex purity, the Phytusilan Assay for a given formulation is Results were corrected for purity factor (multiply by (non-denaturing IP-RP HPLC area %) / 100).

[0252] Assay of the fissiran formulation (mg / mL) was determined from a UV spectrophotometer and non-denaturing IP Corrected for duplex purity from the RP-HPLC method, results are reported based on the concentration of the H-form (free acid) of the duplex. All results were within the specification limits of 90 to 110 mg / mL (measured as the free acid form), with a mean assay value of 101.5 mg / mL and a standard deviation of 3.4%. Results showed good comparability between assay values ​​for all Phthalan lots tested.

[0253] pH of Fitusiran formulation The pH of the Fitsiran formulations was measured directly. Comparative pH results for the Fitsiran formulation lots were observed to be pH 7.1 with a standard deviation of 0.0. All results met the current specification of pH 6.0-8.0 for Fitsiran formulations. Analysis of the pH data for the Fitsiran formulation batches showed a high degree of comparability between Fitsiran lots.

[0254] Osmolality of Fitusiran formulations The osmolality of Fitusiran formulations is based on the principle of freezing point depression. Osmolality was reported as mOsm / kg values. The formulations had a fixed salt concentration from sodium phosphate buffer and Fitusiran duplex, and the observed osmolality values ​​showed only a narrow range. Osmolality results for Fitusiran formulation batches ranged from 297 to 310 (mOsm / kg), with a mean of 304 mOsm / kg and a standard deviation of 5.3%. All results were within the specification of 240 to 390 mOsm / kg for the osmolality of Fitusiran formulations.

[0255] Particulate matter in Fitusiran formulations Fitusiran formulations were analyzed for the number of subvisible particulate matter per container by light obscuration and results were reported as total number of particles (≥10 μm and ≥25 μm) per container. For particles ≥10 μm in Fitusiran formulations, the observed range was approximately 29 to 588 particles (≥10 μm), with a mean of approximately 188 particles and a standard deviation of 268.2%. All results were within the specification of NMT 6,000 per container for Fitusiran formulations.

[0256] For particles 25 μm and larger, the observed range was approximately 0 to 46 particles, with a mean of approximately 13 particles and a standard deviation of 22.4%. All results were within the specification of NMT600 per container for the Fitusiran formulation.

[0257] Container volume for Fitusiran formulation The volume of the solution containing the Fitusiran formulation was measured with the specification limit set at at least (NLT) 0.8 mL. The volume of the container observed in the different Fitusiran formulation lots showed a good degree of comparability between Fitusiran formulation batches with a standard deviation of 0.0.

[0258] Bacterial endotoxins and sterility in batches of Fitusiran formulations All batches of Fitusiran formulation met the microbial safety testing criteria for bacterial endotoxins (at most (NMT) 100 endotoxin units (EU) / mL), demonstrating adequate control over the Fitusiran formulation process and its microbial safety profile.

[0259] Duplex analysis by non-denaturing ion-pair reversed-phase high-performance liquid chromatography (IP RP-HPLC) purity Non-denaturing IP RP-HPLC resolves the duplex from any remaining single strands. The area % purity of the duplex is determined by this method. The identity of the drug substance in the Fitusiran formulation was established by retention time agreement with the duplex reference standard.

[0260] A non-denaturing IPRP HPLC method was used to identify the constituent single strands, sense strands and antisense strands in the formulation in parallel with mass spectrometry (ESI-MS). The duplex peak was resolved from the remaining single strands and duplex purity was determined by this method. A representative IPRP chromatogram of a Fitusiran formulation is shown in Figure 1.

[0261] Stationary phase: Waters XBridge C8 2.1×50 column, 2.5 μm particle size. Mobile phase A: 95 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 16 mM triethylamine (TEA), 5 μM ethylenediaminetetraacetic acid (EDTA) in water. Mobile phase B: 100% methanol containing 5 μM EDTA. Flow rate: 0.25mL / min. Column temperature: 15℃ gradient: [Table 5]

[0262] Detection: UV at 260 nm and MS in negative ion mode from 700 to 2700 Da

[0263] Sample preparation: Samples were prepared in 1×PBS to a concentration of approximately 0.1 mg / mL for single-stranded intermediates and 0.2 mg / mL for double-stranded drug substance (Fitusiran).

[0264] The injection volume is 20 μL.

[0265] Detection limits: Chromatography software was used to integrate and report all peaks ≧0.05 area %.

[0266] Purity calculations: The area % of the major duplex peak was calculated by the chromatography software and reported as duplex purity. The area % of remaining single stranded and other impurities was reported similarly.

[0267] Identity: The identity of the constituent single strands, sense and antisense, present under the same duplex peak was established by molecular weight determined from deconvolution of the duplex peak spectrum by liquid chromatography mass spectrometry (LC-MS) using chromatography software.

[0268] The native profile of the Fitusiran formulation by native IP RP-HPLC confirmed the presence of the formulation in duplex form. Duplex purity indicated the percentage of annealed duplex siRNA in the Fitusiran formulation. Duplex purity values ​​for the formulation batches included in this study ranged from about 98.9 to 99.5 area %. Analysis of the data gave a mean (n=4) purity of about 99.2% with a standard deviation of about 0.3%, indicating that duplex purity was consistent and similar to each other for all Fitusiran formulation batches compared in this report.

[0269] Total Impurities by Non-Denaturing Ion-Pair Reversed Phase High Performance Liquid Chromatography (IP RP-HPLC) Non-duplexed (non-annealed) impurities by native IP RP-HPLC were reported as the sum of all (non-duplexed) peaks ≥ 0.050 area %. Results for total impurities by native IP RP-HPLC were observed to be within 2% and met the specification of NMT 10.0 area % for all batches of Fitusiran formulation included in this study. The mean value (n=4) for total impurities by native IP RP-HPLC was 0.85% with a standard deviation of 0.3%. Overall, the results showed that the Fitusiran formulation lots tested in this report had very similar profiles in terms of both specific (single-stranded) and non-specific impurities.

[0270] Purity by denaturing anion exchange high performance liquid chromatography (AX-HPLC) To determine the purity of the single strands in the preparations, denaturing AX-HPLC analysis was performed.

[0271] The presence of multiple peaks for the antisense strand was due to phosphorothioate diastereomers. A representative AX-HPLC chromatogram of a Fitusiran formulation is shown in Figure 2.

[0272] Stationary phase: Dionex DNA Pac PA200 column, 4x250mm Mobile phase A: 20 mM sodium phosphate, 10% ACN, pH 11 Mobile phase B: 20 ​​mM sodium phosphate, 1 M NaBr, 10% ACN, pH 11 gradient: [Table 6]

[0273] Detection limits: Chromatography software was used to integrate and report all peaks ≧0.05 area %.

[0274] Purity calculation: The area % of each of the major peaks was calculated by the chromatography software, and the sum of the area % of the sense and antisense strands was reported as the purity. The sum of the area % of impurities greater than 0.050 area % was reported as the total impurities.

[0275] Identity: Single-stranded identity was confirmed by comparing the retention times of test samples with corresponding reference standards.

[0276] AX-HPLC denatures the Physilan duplex to form the constituent sense and antisense single strands. The area % purity of the single strands was determined by this method.

[0277] The denaturing AX-HPLC method measures the purity of the individual single strands that comprise the Fitusiran duplex. The sum of the single strand area % represents the denaturing purity of the Fitusiran formulation. Analysis of the sum of the sense and antisense strand area % of Fitusiran lots in this study yielded a mean value (n=4) of 94.2 area % with a standard deviation of 0.8%. All results from a representative Fitusiran formulation batch met the NLT 85.0 area % specification, indicating overall comparable purity results for the Fitusiran formulations.

[0278] Total impurities by denaturing anion exchange high performance liquid chromatography (AX-HPLC) Analysis of the data gave a mean value (n=4) of 5.6% for the sum of all impurities NLT0.050 area% with a standard deviation of 0.7%. The results showed that the total impurity values ​​for the Fitusiran batches included in this report were consistent and comparable.

[0279] Purity by denaturing ion-pair reversed-phase high performance liquid chromatography (IP RP-HPLC) IP RP-HPLC denatures the Physilan duplex to form the constituent sense and antisense single strands. The area % purity of the single strands is determined by this method.

[0280] The modified IP RP-HPLC method is orthogonal to AX-HPLC and measures the purity of the individual single strands that comprise the Fitusiran duplex in the formulation. The sum of the single strand area % represents the modified IP RP-HPLC purity of the Fitusiran formulation.

[0281] Denaturing IP RP-HPLC analysis was also performed to determine the purity of the single strands in the preparation.

[0282] Stationary phase: Waters XBridge C18 (OST or XP) 2.1×50 column, 2.5 μm particle size. Mobile phase A: 550 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 13 mM trimethylamine (TEA) and 5 M ethylenediaminetetraacetic acid (EDTA) in 90:10 water:methanol. Mobile phase B: 100% methanol Flow rate: 0.40ml / min Column temperature: 80℃ gradient: [Table 7]

[0283] Detection: UV at 260 nm and MS in negative ion mode from 700 to 2700 Da

[0284] Sample preparation: Prepare samples in 1XPBS to a concentration of approximately 0.1 mg / mL for single-stranded intermediate and 0.2 mg / mL for double-stranded drug substance (Fitusiran). Ta.

[0285] The injection volume was 25 μL.

[0286] Detection limit: All peaks ≧0.05 area % were used to integrate and report using the chromatography software.

[0287] Purity calculations: The area % of the major duplex was calculated by the chromatography software and reported as duplex purity. The area % of the remaining single strands and other impurities were reported similarly.

[0288] A representative modified IP RP-HPLC chromatographic profile of the Fitusiran formulation is shown in Figure 3.

[0289] Analysis of the combined area % of sense and antisense strands for Fitusiran lots in this study yielded a mean value (n=4) of 88.7 area % with a standard deviation of 1.4%. All results for Fitusiran formulation lots met the NLT specification of 80.0 area %, indicating comparable purity results for Fitusiran formulation lots within analytical variability.

[0290] Total Impurities by Denaturing Ion-Pair Reversed Phase High Performance Liquid Chromatography (IP RP-HPLC) Analysis of the data gave a mean value (n=4) of 11.0% for the sum of all impurities NLT0.050 area % with a standard deviation of 1.5%. The results indicate that the total impurity values ​​for all Fitusiran batches included in this report are consistent and comparable.

[0291] Example 4: Container closure system and compatibility of Fitusiran formulations The container closure system for the Fitusiran formulation was selected to protect the sterile product from microbial contamination. The vials were sterilized and depyrogenated by dry heat at ≥300°C for ≥5 min. Butyl rubber seals were autoclaved at ≥121–125°C for ≥60 min.

[0292] Butyl rubber stoppers were sterilized by autoclaving on a validated cycle. All components were of standard quality for parenteral products. Fitusiran formulation stability studies were performed with the formulations stored in the same container closure system.

[0293] Fitusiran is formulated for subcutaneous injection. Based on the estimated calculated dose to be administered, 1 ml or 3 ml syringes are used. Two types of syringes, one made of polycarbonate and the other made of polypropylene, were tested for compatibility with Fitusiran. The formulation filled in vials at 100 mg / mL was drawn into the syringes. One set of filled syringes was incubated at 25°C for 8 hours and the other set of filled syringes was incubated with the control at 2-8°C for 48 hours. After incubation, the formulations were tested for assay and purity by AX-HPLC and compared to the vialed drug. As shown in Table 3 below, there was no difference between the control formulation and the formulations incubated in the two types of syringes in terms of label claim and purity, indicating that Fitusiran is compatible with the intended injection device.

[0294] [Table 8]

[0295] The use of larger gauge (narrower bore) needles was also evaluated with syringes of identical construction materials as shown in Table 3 for both extraction and dispensing of the undiluted formulation. Two types of syringes and two dispensing speeds were analyzed. The results are shown in Table 4 and indicate that the integrity of the Fitusiran formulation was maintained when using 29 or 30G needles.

[0296] [Table 9]

[0297] Example 5: Storage stability analysis of Fitusiran formulations Storage stability data for Fitusiran formulations were collected at recommended storage conditions of 2-8 °C and one or more accelerated conditions. Stability studies were designed according to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q1A (R2) and samples were stored in container closures identical to those used for storage of clinical materials (i.e., 2 mL USP Type I glass vials with Teflon-faced butyl rubber stoppers). Stability data were collected as shown in Table 5.

[0298] [Table 10]

[0299] The stability of Fitusiran formulations was evaluated for trends using the following analytical procedures: visual appearance, assayed by UV spectrometry, pH, osmolality, double-stranded purity by non-denaturing IPRP-HPLC and single-stranded purity as measured by two orthogonal methods: purity by denaturing AX HPLC and purity by denaturing IPRP-HPLC.

[0300] To further elucidate the thermal stability of the formulation, several stability studies included a long-term evaluation of the formulation at 25° C. / 60% RH as well as a 6-month accelerated aging study conducted at 40° C. / 75% RH. Additional data (i.e., more than 6 months at 25° C. / 60% RH and all data collected at 40° C. / 75% RH) were collected to evaluate the suitability of the drug for long-term storage in ICH Zone II climate conditions.

[0301] Storage stability data for three representative lots of Fitusiran formulations (P02314, P02715, and P07916) are presented in Tables 6-14 and summarized below.

[0302] Stability data up to 36 months have been collected for the Fitusiran formulation at the recommended storage conditions of 2-8°C. No significant changes were identified for any of the parameters during storage at 2-8°C, indicating that these conditions are suitable for long-term storage of the formulation. Furthermore, no significant changes were observed during storage at 25°C / 60% RH or even during storage at 40°C / 75% RH. Based on this and the assessment of the suitability of the formulation for long-term storage in ICH Zone II climatic conditions (i.e., 25°C / 60% RH), a shelf life of 36 months has been assigned to the Fitusiran formulation stored at 2-8°C, which has been further confirmed by additional data collected since the last update.

[0303] [Table 11] [Table 12]

[0304] [Table 13] [Table 14]

[0305]

Table 15

[0306]

Table 16

Table 17

[0307]

Table 18

Table 19

[0308]

Table 20

[0309]

Table 21

Table 22

[0310]

Table 23

Table 24

[0311]

Table 25

Claims

【Claim 1】 The invention described in the specification of this application.