RU2801263C2 - Methods and compositions for treatment of bleeding phenomenon in hemophilia patients - Google Patents

Abstract

FIELD: medicine; hematology; hereditary diseases.

SUBSTANCE: group of inventions is intended for preventive treatment of patients with hemophilia. The following is described: a method of treating a bleeding episode in a patient with hemophilia A or B with inhibitors undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; where the sense strand contains the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the antisense strand contains the nucleotide sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; and where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram:

the method comprises the step of administering to a patient a therapeutically effective amount of an activated prothrombin complex concentrate (aPCC), wherein the therapeutically effective amount of aPCC is not more than about 50 U/kg per dose. Besides, alternative methods of the prophylactic treatment of a patient with hemophilia A or hemophilia B with inhibitors are described.

EFFECT: group of inventions provides an increase in the efficiency of prophylactic treatment of patients with hemophilia.

36 cl, 29 dwg, 5 tbl, 6 ex

Description

Related Applications

The present application claims priority under U.S. Provisional Application No: 62/530518 filed July 10, 2017, U.S. Provisional Application No: 62/599223 filed December 15, 2017, Provisional Application for U.S. Patent No.: 62/614111, filed Jan. 5, 2018, and U.S. Provisional Application No.: 62/673,424, filed May 18, 2018, the entire contents of each of the above patent applications are incorporated herein by links.

This application is also related to International Application No. PCT/US2016/065245 filed December 7, 2016, US Provisional Application No: 62/264013 filed December 7, 2015, US Provisional Application No: 62/ 315228, filed March 30, 2016, with U.S. Provisional Application No.: 62/366304, filed July 25, 2016, and with U.S. Provisional Application No.: 62/429241, filed December 2, 2016, in full the contents of each of the above patent applications are hereby incorporated herein by reference.

Further, this application is related to U.S. Provisional Application No: 61/992057 filed May 12, 2014, U.S. Provisional Application No: 62/089018 filed December 8, 2014, Provisional Patent Application U.S. No: 62/102281, filed January 12, 2015, and International Application No. PCT/US2015/030337, filed May 12, 2015, the entire contents of each of the above patent applications are hereby incorporated by reference herein.

This application is also related to U.S. Provisional Application No: 61/638952, filed Apr. 26, 2012, U.S. Provisional Application No.: 61/669249, filed July 9, 2012, U.S. Provisional Application No. : 61/734573, filed December 7, 2012, U.S. Patent Application No.: 13/837129, filed March 15, 2013, now U.S. Patent Application No.: 9127274, U.S. Patent Application No.: 14/806084, filed July 22, 2015, now US Patent No: 9376680, US Patent Application No: 15/070358 filed March 15, 2016, and International Application No. PCT/US2013/038218 filed April 25, 2013 is also related to International Application No. PCT/US2012/065601, filed November 16, 2012, the entire contents of each of the above patent applications are incorporated herein by reference.

List of Sequences

This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on July 2, 2018, is named 117811-02720_SL.TXT and has a size of 21147 bytes.

State of the Art Inventions

Serpinc1 is a member of the superfamily of serine proteinase inhibitors (serpins). Serpinc1 is a plasma protease inhibitor that inhibits thrombin as well as other activated serine proteases of the coagulation system such as factors X, IX, XI, XII and VII and thus regulates the coagulation cascade. The anticoagulant activity of Serpinc1 is enhanced in the presence of heparin and other related glycosaminoglycans, which catalyze the formation of thrombin:antithrombin (TAT) complexes.

Coagulation disorders, either hereditary or acquired, are conditions in which insufficient blood clotting occurs. For example, hemophilia is a group of inherited genetic clotting disorders that interfere with the body's ability to regulate blood clotting or coagulation. Hemophilia A is an X-linked recessive genetic disorder associated with the absence of functional coagulation factor VIII and accounts for 80% of cases of hemophilia. Hemophilia B is an X-linked recessive genetic disorder associated with the absence of a functional coagulation factor IX. It covers approximately 20% of hemophilia cases. Hemophilia C is an autosomal genetic disorder associated with the absence of functional clotting factor XI. Hemophilia C is not completely recessive, as heterozygous individuals also show increased bleeding.

Although there is currently no cure for hemophilia, it can be managed with regular infusions of the missing clotting factor, such as factor VIII in hemophilia A. However, some people with hemophilia develop antibodies (inhibitors) against the replacement factors they are given and thus become unresponsive to replacement clotting factor. Accordingly, bleeding cannot be properly controlled in such patients.

The production of high titer inhibitors, such as factor VIII and other coagulation factors, is the most severe complication associated with the treatment of hemophilia and makes the management of bleeding very difficult. Currently, the only strategies to stop bleeding in these patients are the use of "bypass agents" such as shunt activity in factor VIII inhibitors (FEIBA) and activated recombinant factor VII (rFVIIa), plasmapheresis, permanent factor replacement, and induction of immune tolerance, none of which which is not completely effective. Accordingly, there is a need in the art for alternative treatments for patients with a bleeding disorder such as hemophilia.

The essence of the invention

The present invention is based at least in part on the surprising discovery that in patients with hemophilia without inhibitors who are administered a therapeutically effective amount of an mRNA composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of Serpinc1 gene RNA transcripts, bleeding can be treated with a therapeutically effective an amount of a replacement factor, such as Factor VIII or Factor XI, less than the recommended effective amount of a replacement factor, such as that recommended by the World Hemophilia Federation (see, for example, 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 FDA; and that in hemophilia patients with inhibitors and who are administered a therapeutically effective amount of an mRNA composition that affects to RNA-induced silencing complex (RISC)-mediated cleavage of Serpinc1 RNA transcripts, bleeding can be treated with a therapeutically effective amount of a bypass agent, such as activated prothrombin complex concentrate (aPCC) or recombinant factor VIIa (rFVIIa), less than the recommended effective amount of bypass agent , such as recommended by the World Federation of Hemophilia (see, for example, 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 FDA.

Accordingly, in one aspect, the present invention provides a method of treating bleeding in a patient having a bleeding disorder such as hemophilia without inhibitors. The method includes administering to the patient a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced from the recommended effective amount of the replacement factor, thereby treating bleeding in a hemophilia patient without inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient having a bleeding disorder such as hemophilia with inhibitors. The methods include administering to the patient a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced compared to the recommended effective amount of the bypass agent, thereby treating bleeding in the hemophilia patient with inhibitors.

In one aspect, the present invention provides a method of treating bleeding in a patient having a bleeding disorder such as hemophilia without inhibitors. The method includes administering to the patient a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced from the recommended effective amount of the replacement factor, thereby treating bleeding in a hemophilia patient without inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient having a bleeding disorder such as hemophilia with inhibitors. The methods include administering to the patient a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits the expression of Serpinc1, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced compared to the recommended effective amount of the bypass agent, thereby treating bleeding in the hemophilia patient with inhibitors.

The double-stranded RNAi agent may be administered to a patient in two or more doses.

In some embodiments, the double-stranded RNAi agent is administered to the patient once a month, once every five weeks, once every six weeks, once every seven weeks, once every 2 months, once a quarter, or as needed.

In one embodiment, the double-stranded RNAi agent is administered to the patient once a month. In another embodiment, the double-stranded RNAi agent is administered to the patient once every six weeks. In one embodiment, the double-stranded RNAi agent is administered to the patient once every 2 months. In yet another embodiment, the double-stranded RNAi agent is administered to the patient once every quarter.

The double-stranded RNAi agent may be administered to the patient, for example, as a fixed dose of about 25 mg to about 100 mg, for example, about 25 mg to about 95 mg, about 25 mg to about 90 mg, about 25 mg to about 85 mg. , about 25 mg to about 80 mg, about 25 mg to about 75 mg, about 25 mg to about 70 mg, about 25 mg to about 65 mg, about 25 mg to about 60 mg, about 25 mg up to about 50 mg, from about 50 mg to about 100 mg, from about 50 mg to about 95 mg, from about 50 mg to about 90 mg, from about 50 mg to about 85 mg, from about 50 mg to about 80 mg, from about 30 mg to about 100 mg, from about 30 mg to about 90 mg, from about 30 mg to about 80 mg, from about 40 mg to about 100 mg, from about 40 mg to about 90 mg, from about 40 mg to about 80 mg, from about 60 mg to about 100 mg, from about 60 mg to about 90 mg, from about 25 mg to about 55 mg, from about 25 mg to about 65 mg, from about 30 mg to about 95 mg, from about 30 mg to about 85 mg, about 30 mg to about 75 mg, about 30 mg to about 65 mg, about 30 mg to about 55 mg, about 40 mg to about 95 mg, about 40 mg to about 85 mg, about 40 mg to about 75 mg, about 40 mg to about 65 mg, about 40 mg to about 55 mg, or about 45 mg to about 95 mg.

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

In some embodiments, the double-stranded RNAi agent is administered to the patient at a fixed dose of approximately 25 mg; or in a fixed dose of approximately 50 mg; or in a fixed dose of approximately 80 mg; or in a fixed dose of approximately 100 mg.

In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the patient.

In one embodiment, the patient is a human.

Hemophilia can be hemophilia A, hemophilia B, or hemophilia C.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the modified nucleotides are independently selected from the group consisting of 2'-deoxy-2'-fluoro-modified nucleotide, 2'-deoxy-modified nucleotide, blocked nucleotide, denuclearized nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholinonucleotide, phosphoramidate and nucleotide containing non-natural base.

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

In one embodiment, the region of complementarity is 19 to 21 nucleotides in length. In another embodiment, the complementarity region is 21 to 23 nucleotides in length.

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

At least one strand of the double-stranded RNAi agent may comprise a 3' overhang of at least 1 nucleotide or a 3' overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10 , 11, 12, 13, 14 or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent contains a 5' overhang of at least 1 nucleotide. In some embodiments, at least one strand contains a 5' overhang of at least 2 nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides . In other embodiments, both the 3' and 5' ends of one strand of the RNAi agent contain an overhang of at least 1 nucleotide.

In some embodiments, the ligand is N-acetylgalactosamine (GalNAc). The ligand may be one or more GalNAcs attached to the RNAi agent via a monovalent, bivalent or trivalent branched linker. The ligand can 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.

In some embodiments, the double-stranded RNAi agents of the invention comprise a plurality of, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently attached to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

In some embodiments, the ligand is

.

In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following scheme

and where X represents O or S.

In one embodiment, X is O.

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

In one embodiment, the double-stranded RNAi agent contains a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and the antisense strand contains the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15 ).

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

In one embodiment, the sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:13) and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14), where a, c, g, and u represent 2'-O-methyl (2'-OMe) A, C, G or U; Af, Cf, Gf or Uf represent 2'-fluoro A, C, G or U; and s represents a phosphorothioate bond; and while the sense strand is conjugated to the ligand, as shown in the following diagram

,

where X represents O or S.

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

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

In one embodiment, administering a dsRNA agent to a patient reduces Serpinc1 activity by about 75% or more.

In one embodiment, the replacement factor is factor VIII. A therapeutically effective amount of factor VIII administered to a patient 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, 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 the patient is from about one and a half times to about five times less than the recommended effective amount of factor VIII, for example, a dose of from about 5 IU/kg to about 20 IU/kg, or from about 10 IU/kg to about 20 IU/kg, such as about 5, 10, 15 or 20 IU/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

In another embodiment, the replacement factor is factor IX. A therapeutically effective amount of factor IX 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, 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 approximately 10 IU/kg. In one embodiment, the therapeutically effective amount of factor IX administered to the patient is from about two times to about six times less than the recommended effective amount of factor IX, for example, a dose of from about 10 IU/kg to about 30 IU/kg, or from about 20 to about 30 IU/kg, for example about 10, 15, 20, 25 or about 30 IU/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

In one embodiment, the bypass agent is an activated prothrombin complex concentrate (aPCC). A therapeutically effective amount of 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 the patient is from about two times to about three times less than the recommended effective amount of aPCC, eg, a dose of about 30 to about 50 U/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

In another embodiment, the bypass agent is recombinant factor VIIa (rFVIIa). A therapeutically effective amount of bypass agent may be less than about 120 µg/kg, or less than about 110 µg/kg, or less than about 100 µg/kg, or less than about 90 µg/kg, or less than about 80 µg/kg, or less than about 70 µg/kg, or less than about 60 µg/kg, or less than about 50 µg/kg, or less than about 40 µg/kg, or less than about 30 µg/kg, or less than about 20 µg/kg. In one embodiment, the therapeutically effective amount of rFVIIa administered to the patient is approximately half the recommended effective amount of rFVIIa, eg, a dose of approximately 45 μg/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

In one aspect, the present invention provides a method of treating bleeding in a patient with hemophilia, eg, hemophilia A, hemophilia B, or hemophilia C, without inhibitors. The method includes administering, for example, subcutaneously administering to a patient a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' ( SEQ ID NO:13) and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14) with a, c, g and u representing 2'-O-methyl (2'-OMe) A, C , G or U; Af, Cf, Gf or Uf represent 2'-fluoro A, C, G or U; and s represents a phosphorothioate bond; and while the 3'-end of the sense strand is conjugated to the ligand, as shown in the following scheme

,

where X represents O or S; and administering to the patient a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced from the recommended effective amount of the replacement factor, thereby treating bleeding in a hemophilia patient without inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient with hemophilia, eg hemophilia A, hemophilia B or hemophilia C with inhibitors. The method includes administering, for example, subcutaneously administering to a patient a fixed dose of approximately 80 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' ( SEQ ID NO:13) and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14) with a, c, g and u representing 2'-O-methyl (2'-OMe) A, C , G or U; Af, Cf, Gf or Uf represent 2'-fluoro A, C, G or U; and s represents a phosphorothioate bond; and while the 3'-end of the sense strand is conjugated to the ligand, as shown in the following scheme

,

where X represents O or S; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced compared to the recommended effective amount of the bypass agent, thereby treating bleeding in the hemophilia patient with inhibitors.

In one embodiment, a fixed dose of the RNAi agent is administered subcutaneously to the patient.

In one embodiment, a fixed dose of the RNAi agent is administered to the patient once a month.

Hemophilia can be hemophilia A, hemophilia B, or hemophilia C.

Brief Description of Drawings

In FIG. 1A is a graph showing the effect of a single subcutaneous dose of 0.03 mg/kg AT3SC-001 on plasma thrombin formation levels in one healthy human subject.

In FIG. 1B is a graph showing the effect of a single subcutaneous dose of 0.03 mg/kg AT3SC-001 on plasma thrombin formation levels in one healthy human subject.

In FIG. 1C is a graph showing the effect of a single subcutaneous dose of 0.03 mg/kg AT3SC-001 on plasma thrombin formation levels in one healthy human subject.

In FIG. 1D is a graph showing the effect of a single subcutaneous dose of 0.03 mg/kg AT3SC-001 on plasma thrombin formation levels in one healthy human subject.

In FIG. 2A is a graph showing the effect of a single subcutaneous dose of 0.03 mg/kg AT3SC-001 on plasma levels of AT protein (Serpinc1) in one healthy human subject.

In FIG. 2B is a graph showing the effect of a single subcutaneous dose of 0.03 mg/kg AT3SC-001 on plasma levels of AT protein (Serpinc1) in one healthy human subject.

In FIG. 3 is a graph showing the relationship between the percentage of AT (Serpinc1) knockdown and the percentage increase in peak thrombin production in healthy patients treated with a single subcutaneous dose of 0.03 mg/kg AT3SC-001.

In FIG. 4 is a graph showing the effect of multiple doses of 0.015 mg/kg, 0.045 mg/kg, or 0.075 mg/kg of AT3SC-001 on plasma levels of AT protein (Serpinc1) in human patients with hemophilia A or B.

In FIG. 5A is a graph showing the effect of multiple doses of 0.225 mg/kg, 0.450 mg/kg, 0.900 mg/kg, 1.800 mg/kg, or 80 mg of AT3SC-001 on plasma levels of the AT protein (Serpinc1) in human patients with hemophilia A or B .

In FIG. 5B is a graph showing the dose-dependent effect of AT3SC-001 on plasma levels of AT protein (Serpinc1) in human patients.

In FIG. 6A is a graph showing the effect of multiple doses of 0.015 mg/kg or 0.045 mg/kg of AT3SC-001 on peak thrombin levels in humans with hemophilia A or B.

In FIG. 6B is a graph showing the effect of multiple doses of 0.015 mg/kg or 0.045 mg/kg of AT3SC-001 on thrombin formation as percentage change from baseline in human patients with hemophilia A or B.

In FIG. 7 is a graph showing the effect of multiple doses of 0.045 mg/kg AT3SC-001 on clot formation time and clotting time in one patient with hemophilia A (patient 101-009).

In FIG. 8 is a graph showing the mean maximum AT reduction at a monthly equivalent dose.

In FIG. 9 is a graph showing the effect of multiple doses of AT3SC-001 on thrombin generation by AT reduction quartiles. Squares indicate the median and interquartile range. % change in peak TG: p<0.001, Mann-Whitney test when compared with the ATZ group is lower than <25%.

In FIG. 10A is a graph showing the relative activity of AT in relation to the percent peak thrombin formation achieved by factor VIII as determined in a patient administered 225 μg/kg qM AT3SC-001.

In FIG. 10B is a graph showing the relative activity of AT in relation to the percent peak thrombin formation achieved by factor VIII as determined in a patient administered 1800 μg/kg qM AT3SC-001.

In FIG. 10C is a graph showing the relative activity of AT in relation to the percent peak thrombin formation achieved by factor VIII as determined in a patient administered 80 mg qM AT3SC-001.

In FIG. 11 is a graph showing the effect of multiple doses of AT3SC-001 on bleeding by quartile of AT reduction. ABR, mean annual bleeding rate; SEM, standard error of the mean. Number of patients with time spent in the quartile; For each patient, the ABR in each quartile is calculated by 365.24*(number of bleeding events/number of days in the quartile).

In FIG. 12 is a table showing bleeding data for patients enrolled in the Phase CI portion of the AT3SC-001 clinical trial. PPx - prevention; OD - on demand; ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate; Post hoc analysis of treated bleeding events during onset (day 0-28) and follow-up (day 29 - to last study visit or last dose + 56 days, whichever comes first; pre-study ABR derived from medical records; ** - excluded patients C5-4 who dropped out of the analysis.

In FIG. 13A is a graph showing the mean annual bleeding rate (ABR) before study entry, study baseline, and study follow-up for all dosing cohorts in the CI phase portion of the AT3SC-001 clinical trial. PPx - prevention; OD - on demand; ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate; Post hoc analysis of treated bleeding events during onset (day 0-28) and follow-up (day 29 - to last study visit or last dose + 56 days, whichever comes first; pre-study ABR derived from medical records; ** - excluded patients C5-4 who dropped out of the analysis.

In FIG. 13B is a graph showing the mean annual bleeding rate (ABR) before study entry, study baseline, and study follow-up for the 80 mg (80 mg qM x3) monthly cohort in the CI phase portion of the AT3SC-001 clinical trial. PPx - prevention; OD - on demand; ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate; Post hoc analysis of treated bleeding events during onset (day 0-28) and follow-up (day 29 - to last study visit or last dose + 56 days, whichever comes first; pre-study ABR derived from medical records; ** - excluded patients C5-4 who dropped out of the analysis.

In FIG. 14A is a graph showing the relative activity of AT in relation to the percent peak thrombin generation achieved by factor VIII as measured in an inhibitor patient receiving a fixed monthly dose of 50 mg of AT3SC-001.

In FIG. 14B is a graph showing the relative activity of AT in relation to the percent peak thrombin generation achieved by factor VIII as determined in an inhibitor patient receiving a fixed monthly dose of 50 mg of AT3SC-001.

In FIG. 14C is a graph showing the relative activity of AT in relation to the percent peak thrombin generation achieved by factor VIII as measured in an inhibitor patient receiving a fixed monthly dose of 50 mg of AT3SC-001.

In FIG. 14D is a graph showing the relative activity of AT in relation to the percent peak thrombin formation achieved by factor VIII as measured in an inhibitor patient receiving a fixed monthly dose of 50 mg of AT3SC-001.

In FIG. 14E is a graph showing the relative activity of AT in relation to the percent peak thrombin generation achieved by factor VIII as measured in an inhibitor patient receiving a fixed monthly dose of 50 mg of AT3SC-001.

In FIG. 14F is a graph showing the relative activity of AT in relation to the percent peak thrombin generation achieved by factor VIII as measured in an inhibitor patient receiving a fixed monthly dose of 50 mg of AT3SC-001.

In FIG. 15 is a graph showing the effect of multiple doses of 50 mg or 80 mg of AT3SC-001 on mean AT (Serpinc1) activity relative to baseline in human hemophilia A or B patients with inhibitors.

In FIG. 16 is a graph showing that the AT-lowering effect of multiple doses of 50 mg AT3SC-001 correlates with increased thrombin production in a patient with hemophilia A.

In FIG. 17A is a table showing bleeding data for patients enrolled in the Phase DI part of the AT3SC-001 clinical trial. OLE - open extended study; ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate; Post hoc analysis of treated bleeding events during onset (day 0-28) and follow-up (day 29 - to last study visit or last dose + 56 days, whichever comes first; pre-study ABR derived from medical records; ^Patients progressing to phase 2 OLE since data collection was stopped.

In FIG. 17B is a graph showing the mean annual bleeding rate (ABR) before study entry, study baseline, and study follow-up for all patients in Phase I Part D of the AT3SC-001 clinical study. Median ABR, pre-study period: 31; Median ABR, follow-up period: 0; Patients reporting no bleeding: 9/16 (56%); Patients reporting no spontaneous bleeding (AsBR=0) 11/16 (69%). OLE - open extended study; ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate.

In FIG. 18 is a graph showing the effect of multiple doses of 80 mg of AT3SC-001 on mean AT (Serpinc1) activity relative to baseline in an inhibitor-free human hemophiliac in the phase II open-label extended (OLE) study of AT3SC-001.

In FIG. 19A is a graph showing the effect of multiple doses of 50 mg or 80 mg of AT3SC-001 on mean AT (Serpinc1) activity relative to baseline in human hemophilia A or B patients with or without inhibitors, in the Phase II Open-label Expanded (OLE) study of AT3SC -001.

In FIG. 19B is a graph showing the effect of multiple doses of 50 mg or 80 mg of AT3SC-001 on peak thrombin production in human patients with hemophilia A or B, with or without inhibitors, in the Phase II Open-label Expanded (OLE) study of AT3SC-001. The shaded portion of the graph depicts the range of peak thrombin levels observed in healthy human volunteers (HV) administered AT3SC-001 with less than 25% AT knockdown in the Phase I AT3SC-001 study described in Example 1. The dotted line through the HV region represents the median the peak thrombin level observed in healthy human volunteers (HV) with AT knockdown less than 25% who were administered AT3SC-001 in the Phase I AT3SC-001 study described in Example 1.

Fig. 20A: Summary of median ABR values in patients without inhibitors. In FIG. 20A is a graph showing the pre-study mean annual bleeding rate (ABR), at baseline, and during the follow-up phase of the study in hemophilia A or B patients without inhibitors from patients in the phase II OLE clinical trial AT3SC-001. Duration of the median value during the observation period: 13 months (range 2-19); The average activity of AT during the observation period (relative to the baseline): 22%. The follow-up period, defined as day 29 of treatment before the earliest of the data transfer, or 56 days after the last dose. ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate; PPx - prevention; OD - on demand.

Fig. 20B: Summary of median ABR values in patients with inhibitors. In FIG. 20B is a graph showing the pre-study mean annual bleeding rate (ABR) and during the follow-up phase of the study in hemophilia A or B patients with inhibitors from patients in the phase II OLE of clinical trial AT3SC-001. Duration of the median value during the observation period: 6 months (range 1-11); The average activity of AT during the observation period (relative to the baseline): 18%. The follow-up period, defined as day 29 of treatment before the earliest of the data transfer, or 56 days after the last dose. ABR, mean annual bleeding rate; AsBR, mean annual spontaneous bleeding rate; PPx - prevention; OD - on demand.

In FIG. 21 is a table showing the characteristics of bleeding occurring in patients with hemophilia A or B with or without inhibitors in the phase II OLE clinical trial AT3SC-001. Patients were treated for abdominal pain, as it was not certain whether it was bleeding.

In FIG. 22 is a table showing the management of bleeding occurring in patients with hemophilia A or B without inhibitors in the phase II OLE clinical trial AT3SC-001.FVIII factor VIII; FIX, factor IX. Replacement factor products used: Advate, Aimafix, Benefix, Eloctate, Haemate, Helixate, Immunate, Immunine, Octanate, Recombinate, Refacto.

In FIG. 23 is a table showing the characteristics of bleeding occurring in patients with hemophilia A or B with inhibitors in the phase II OLE clinical trial AT3SC-001. aPCC, activated prothrombin complex concentrate; rFVIIa, recombinant factor VIIa; BPA, shunt agent; Used bypass agents: FEIBA, NovoSeven, Prothromplex.

In FIG. 24A is a graph showing the effect of a monthly fixed dose of 50 mg or 80 mg of AT3SC-001 on the amount of factor VIII required to control bleeding in hemophilia A patients without inhibitors.

In FIG. 24B is a graph showing the effect of a monthly fixed dose of 50 mg or 80 mg of AT3SC-001 on the amount of factor IX required to control bleeding in hemophilia B patients without inhibitors.

In FIG. 24C is a graph showing the effect of a monthly fixed dose of 50 mg or 80 mg of AT3SC-001 on the amount of rFVIIa required to control bleeding in hemophilia A or B patients with inhibitors.

In FIG. 24D is a graph showing the effect of a monthly fixed dose of 50 mg or 80 mg of AT3SC-001 on the amount of aPCC required to control bleeding in hemophilia A or B patients with inhibitors.

In FIG. 25A is a graph showing the effect of aPCC addition on thrombin formation in plasma samples from a hemophilia A patient with inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 25B is a graph showing the effect of rFVIIa addition on thrombin generation in plasma samples from a hemophilia A patient with inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26A is a graph showing the effect of aPCC addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26B is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26C is a graph showing the effect of aPCC addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26D is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26E is a graph showing the effect of aPCC addition on thrombin generation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26F is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26G is a graph showing the effect of aPCC addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26H is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26I is a graph showing the effect of aPCC addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26J is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26K is a graph showing the effect of aPCC addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26L is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26M is a graph showing the effect of aPCC addition on thrombin generation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 26N is a graph showing the effect of rFVIIa addition on thrombin formation in plasma samples from a hemophilia A patient without inhibitors before (lower line) and after (upper line) administration of AT3SC-001 to a patient.

In FIG. 27A is a graph showing the effect of multiple doses of AT3SC-001 on thrombin generation by AT reduction quartiles in a phase I/II open-label extended (OLE) clinical trial.

In FIG. 27B is a graph showing the simulated effect of multiple doses of AT3SC-001 on thrombin generation by AT reduction quartiles.

In FIG. 27C is a scatterplot showing the strong correlation between simulated TG (in FIG. 27B) and measured TG (in FIG. 27A).

In FIG. 28A shows in silico simulated thrombin generation curves for different levels of AT and 0.1% Factor FVIII (Severe Hemophilia A simulation).

In FIG. 28B is a map of peak thrombin intensity at different doses of Factor VIII (single dose) and AT level for severe Hemophilia A.

In FIG. 28C is an image of a peak thrombin intensity map at different doses of FIX (single dose) and AT level for severe Hemophilia B.

In FIG. 29A is a graph showing simulated peak thrombin potential (nM) as a function of time for 5, 10, 20, and 50 IU/kg FVIII with AT at 100%.

In FIG. 29B is a graph showing simulated peak thrombin potential (nM) as a function of time for 5, 10, 20 IU/kg FVIII with AT at 20% baseline.

Detailed Description of the Invention

The present invention is based at least in part on the surprising discovery that in patients with hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C) without inhibitors, who are administered a therapeutically effective amount of an mRNA composition that affects RNA-induced silencing complex (RISC) mediated cleavage of Serpinc1 RNA transcripts, bleeding can be treated with a therapeutically effective amount of replacement factor, such as factor VIII or factor XI, less than the recommended effective amount of replacement factor, such as that recommended by the World Hemophilia Federation (see, for example, Srivastava, et al., "Guidelines for the Management of Hemophilia", Hemophilia Epub 6 July 2012; DOI:10,1111/j,1365-2516,2012,02909.x; all contents of which are incorporated herein by reference) and/or the Food and Drug Administration and drugs (see, for example, ADVATE (Antihemophilic Factor (Recombinant)) product insert; 11/2016; BeneFIX (Coagulation Factor IX (Recombinant) product insert; 11/2011; the entire contents of each of which are incorporated herein by reference). Accordingly, the present invention provides a method for treating bleeding in a patient with hemophilia without inhibitors. The method includes administering to the patient a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced from the recommended effective amount of the replacement factor, thereby treating bleeding in a hemophilia patient without inhibitors.

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

The present invention is also based at least in part on the discovery that in patients with hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C) with inhibitors administered a therapeutically effective amount of an mRNA composition that affects RNA-induced silencing complex (RISC) mediated cleavage of Serpinc1 RNA transcripts, bleeding can be treated with a therapeutically effective amount of a bypass agent, such as activated prothrombin complex concentrate (aPCC) or recombinant factor VIIa (rFVIIa), less than the recommended effective amount of the bypass agent, such as that recommended by the World Federation of Hemophilia (see , for example, 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 Food and Drug Administration agents (see e.g. NovoSeven RT, Coagulation FactorVIIA (Recombinant) product insert; 07/2014; FEIBA, Anti-Inhibitor Coagulation Complex product insert; 11/2013; the entire contents of each of which are incorporated herein by reference).

Accordingly, in another aspect, the present invention provides a method of treating bleeding in a patient with hemophilia with inhibitors. The methods include administering to the patient a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced from a recommended effective amount, e.g., approved by the Food and Drug Administration, of the bypass agent, thereby treating bleeding in the hemophilia patient with inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient with hemophilia with inhibitors. The methods include administering to the patient a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits the expression of Serpinc1, wherein the double-stranded RNAi agent contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides differing by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced from a recommended effective amount, e.g., approved by the Food and Drug Administration, of the bypass agent, thereby treating bleeding in the hemophilia patient with inhibitors.

mRNA tools for use in the methods of the invention typically comprise an RNA strand (antisense strand) having a region that is approximately 30 nucleotides or less in length, such as 15-30, 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19- 26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 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, and this region is essentially complementary to at least part of the mRNA transcript of the Serpinc1 gene.

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, with a region of at least 19 adjacent nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the Serpinc1 gene. In some embodiments, the sense and antisense strands form a duplex of 18-30 adjacent nucleotides.

In some embodiments, mRNA agents for use in the methods of the invention comprise an RNA strand (antisense strand) that can be up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43 , 27-53 nucleotides in length, with a region of at least 19 adjacent nucleotides that is substantially complementary to at least a portion of the Serpinc1 mRNA transcript. In some embodiments, such mRNA tools having longer antisense strands may comprise a second strand of RNA (sense strand) 20-60 nucleotides in length, with the sense and antisense strands forming a duplex of 18-30 adjacent nucleotides.

The following detailed description discloses the preparation and use of compositions containing mRNA to inhibit Serpinc1 gene expression, as well as compositions, uses, and methods of treating patients with diseases and disorders that would benefit from inhibition and/or reduction of expression of this gene.

I. Definitions

In order for the present invention to be more easily understood, some terms must first be defined. In addition, it should be noted that whenever a value or range of values of a parameter is specified, it is intended that values and ranges intermediate to the specified values are also intended to form part of the present invention.

The singular forms within the scope of the present invention refer to one or more than one (ie at least one) grammatical form object. By way of example, "element" means one element or more than one element, such as a plurality of elements.

The term "including" in the context of the present invention means and is used interchangeably with the expression "including, but not limited to".

The term "or" in the context of the present invention means and is used interchangeably with the term "and/or", unless the context clearly indicates otherwise.

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

The term "Serpinc1" in the context of the present invention also refers to a specific polypeptide expressed in a cell by naturally occurring variants of the DNA sequence of the Serpinc1 gene, such as a single nucleotide polymorphism of the Serpinc1 gene. many SNPs have been identified within the Serpinc1 gene, which can be found, for example, in the NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the Serpinc1 gene can be found in NCBI dbSNP accession number rs677; rs5877; rs5878; rs5879; rs941988; rs941989; rs1799876; rs19637711; rs2008946; and rs2227586.

In the context of the present invention, a “sick” is an animal, e.g., a mammal, including a primate (e.g., a human, a non-human primate, e.g., macaque and chimpanzee), a non-primate (e.g., a cow, a pig, a camel, a llama, a horse, a goat). , rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale) or a bird (such as a duck or goose). In one embodiment, the patient is a human, eg, a human being treated for or being evaluated for a disease, disorder, or condition that would benefit from reduced Serpinc1 expression; a person at risk for a disease, disorder, or condition that would benefit from a reduction in Serpinc1 expression; a human having a disease, disorder or condition that would benefit from a reduction in Serpinc1 expression; and/or a person receiving treatment for a disease, disorder or condition that would benefit from a reduction in Serpinc1 expression as described herein.

As used herein, the terms “treatment” or “treatment” refer to a beneficial or desired result, including, but not limited to, alleviation or elimination of one or more symptoms, reduction in bleeding, stabilized (i.e., no worsening) bleeding condition, reduction or reduction of bleeding, whether detectable or non-detectable, or elimination of bleeding. "Treatment" can also mean prolonging life compared to life expectancy without treatment. In the methods of the invention, treatment includes on-demand treatment and management of bleeding episodes, perioperative management of bleeding, and routine prophylaxis to reduce the frequency of bleeding episodes.

The term "lower" in the context of a patient's Serpinc1 level or a disease marker or symptom refers to a statistically significant decrease in that level. The reduction may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 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 preferably up to a level accepted as the normal range for a person without such a disorder.

As used herein, "prevention" or "prevention" when used with reference to a disease, disorder, or condition thereof that would benefit from a reduction in expression of the Sertpinc1 gene, refers to reducing the likelihood that a patient will develop a symptom associated with such disease, disorder, or condition. or a condition, such as a symptom such as bleeding. The likelihood of bleeding is reduced, for example, when a person who has one or more risk factors for bleeding either does not develop bleeding or develops less severe bleeding relative to a population with the same risk factors and not receiving treatment, as described herein. Absence of development of a disease, disorder, or condition, or reduction in the development of a symptom associated with such disease, disorder, or condition (e.g., at least about 10% on a clinically accepted scale for that disease or disorder), or onset of delayed symptoms (e.g., for days, weeks, months or years) is considered effective prevention.

In the context of the present invention, the term "clotting disorder" is a disease or disorder that results in poor blood clotting and/or excessive bleeding. The bleeding disorder may be an inherited disorder, such as hemophilia or von Willebrand disease, or an acquired disorder associated with, for example, disseminated intravascular coagulation, pregnancy-associated eclampsia, vitamin K deficiency, an autoimmune disorder, inflammatory bowel disease, ulcerative colitis, a dermatological disorder (eg. , psoriasis, pemphigus), a respiratory disease (eg, asthma, chronic obstructive pulmonary disease), an allergic reaction to a drug, such as the result of drugs such as aspirin, heparin, and warfatin, diabetes, acute hepatitis B infection, acute hepatitis B infection C, malignant or solid tumor (eg, prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, melanoma, kidney and/or hematologic malignancy). In one embodiment, the inherited bleeding disorder is hemophilia, such as hemophilia A, B, or C. In one embodiment, the patient having the inherited bleeding disorder, such as hemophilia, is developing inhibitors, such as allogeneic antibody inhibitors, to coagulation replacement therapy, and in this document, he is referred to as "patient with inhibitor". In one embodiment, the patient with the inhibitor has hemophilia A. In another embodiment, the patient with the inhibitor has hemophilia B. In another embodiment, the patient with the inhibitor has hemophilia C.

In one embodiment, the bleeding disorder is a rare bleeding disorder (RBD). RBD can be acquired RBD or inherited RBD. Hereditary RBDs include disorders associated with deficiencies of fibrinogen, FII, FV, combined FV and FVIII, FVII, FX, FXI, FXIII, and congenital deficiency of vitamin K dependent factors (VKCFD). They are usually transmitted as autosomal recessive conditions, although in some cases, such as FXI and dysfibrinogenemia, they can be autosomal dominant. RBDs have been reported in most populations with a homozygous or double heterozygote frequency ranging from 1 in 500,000 for FVII deficiency to 1 in 2-3 million for prothrombin and FXIII. The relative frequency varies among populations, being higher where consanguineous or endogamous marriages are common, with an increased frequency of specific mutant genes.

Illustrative 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 deficiency of antithrombin III (thromboplastin; Factor III; tissue factor); parahemophilia (proaccelerin; Factor V; labile factor); Ovren's disease (proaccelerin; Factor V; labile factor); resistance to activated protein C (proaccelerin; Factor V; labile factor); Alexander's disease (stable factor proconvertin; Factor VII); congenital proconvertin/Factor VII deficiency (stable factor proconvertin; Factor VII); Stuart-Prower factor deficiency (Stuart-Prower factor; Factor X); congenital deficiency of Factor XIIIa/b (is a fibrin-stabilizing factor; Factor XIII); hereditary deficiency of Factor XIII (fibrin-stabilizing factor; Factor XIII); and fibrin-stabilizing factor deficiency (fibrin-stabilizing factor; Factor XIII).

The term "therapeutically effective amount" within the meaning of the present invention is intended to include the amount of an RNAi agent which, when administered to a patient having a bleeding disorder and bleeding disorder, is sufficient to effect treatment of a disease (e.g., by reducing, lessening, or stabilizing an existing disease, or one or more symptoms of the disease). The "therapeutically effective amount" may vary depending on the RNAi agent, the route of administration of the agent, the disease and its severity and history, age, weight, family history, gene set, types of prior or concomitant treatments, if any, and other individual characteristics of the patient, to be treated.

The term "prophylactically effective amount" within the meaning of the present invention is intended to include an amount of mRNA which, when administered to a patient having a bleeding disorder but not bleeding, e.g. a patient having a bleeding disorder scheduled for surgery (e.g., perioperative treatment), is sufficient to preventing or reducing the severity of the disease or one or more symptoms of the disease. amelioration of a disease includes slowing the progression of a disease or reducing the severity of a late-onset disease. The "prophylactically effective amount" may vary depending on the mRNA, route of administration, disease risk and history, age, weight, family history, gene set, types of prior or concomitant treatments, if any, and other individual characteristics of the patient being treated. .

The term "therapeutically effective amount" or "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 mRNA used in the methods of the present invention can be administered in sufficient amounts to obtain a reasonable benefit/risk ratio applicable to such treatment.

"Recommended therapeutically effective amount of replacement factor" and "Recommended therapeutically effective amount of bypass agent" are doses of replacement factor or bypass agent, respectively, sufficient to form thrombin and eliminate bleeding and/or sufficient to achieve peak plasma levels of the factor in a bleeding patient, proposed by the World Federation of Hemophilia (see, for example, 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)) product insert; 11/2016; and BeneFIX (Coagulation Factor IX (Recombinant) product insert; 11/2011. The entire contents of each of the above documents are incorporated herein by reference.

For example, the recommended dose of a factor replacement or bypass agent for a patient who has little bleeding is a dose sufficient to achieve a peak plasma level of factor VIII of approximately 10-40 IU/dl; the recommended dose of factor replacement or bypass agent for a patient with moderate bleeding is the dose sufficient to achieve a peak plasma factor VIII level of approximately 30-60 IU/dl; the recommended dose of a factor replacement or bypass agent for a patient who is bleeding heavily is a dose sufficient to achieve a peak plasma factor VIII level of approximately 60-100 IU/dl; The recommended dose of a factor replacement or bypass agent for a patient during surgery is a dose sufficient to achieve a peak plasma level of Factor VIII of approximately 30-60 IU/dL (see, for example, Tables 1 and 2 ADVATE (Antihemophilic Factor (Recombinant)) product insert; 11/2016).

The recommended dose of a factor replacement or bypass agent for a patient who has little bleeding is a dose sufficient to achieve a factor IX peak plasma level of approximately 10-30 IU/dL; the recommended dose of a replacement factor or bypass agent for a patient with moderate bleeding is a dose sufficient to achieve a peak plasma factor IX level of approximately 25-50 IU/dl; The recommended dose of factor replacement or bypass agent for a patient who is bleeding heavily is the dose sufficient to achieve a peak plasma factor IX level of approximately 50-100 IU/dL.

The expression "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms that are within the medical point of view, are suitable for use in contact with the tissues of human patients and animal patients without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit/risk ratio.

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

In the context of the present invention, "target sequence" refers to an adjacent portion of the nucleotide sequence of an mRNA molecule generated during transcription of the Serpinc1 gene, containing an mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target sequence region will be at least long enough to serve as a substrate for mRNA-directed cleavage at or near that region of the nucleotide sequence of the mRNA molecule generated during transcription of the Serpinc1 gene.

The target sequence may be approximately 9-36 nucleotides in length, for example, approximately 15-30 nucleotides in length. For example, the target sequence may be approximately 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, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19 -20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29 , 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 nucleotides in length. It is also contemplated that ranges and lengths intermediate to the above ranges and lengths are part of the invention.

In the context of the present invention, the term "strand containing a sequence" refers to an oligonucleotide containing a chain of nucleotides, which is described by reference to the sequence using standard nucleotide nomenclature.

Each of "G", "C", "A", "T" and "U" usually denotes a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as set out in more detail below, or to a mimic substitution fragment (see, for example, Table 1). It is well known to those skilled in the art that guanine, cytosine, adenine, and uracil can be replaced by other moieties without significantly altering the base pairing properties of an oligonucleotide containing a nucleotide carrying such a substitution moiety. For example, without limitation, a nucleotide containing inosine as its base can be paired with nucleotides containing adenine, cytosine, or uracil. As a result, nucleotides containing uracil, guanine or adenine can be replaced in the nucleotide sequences of the dsRNA of the invention with a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U ambiguous base pairing with the target mRNA. Sequences containing such replacement fragments are suitable for the compositions and methods of the invention.

The terms "mRNA", "RNAi agent", "mRNA agent", "RNA interference agent", which are used interchangeably herein, refer to a preparation that contains RNA as defined in this document and which mediates targeted cleavage of RNA- transcript via the RNA-induced silencing complex (RISC) pathway. RNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The mRNA modulates, eg inhibits, the expression of Serpinc1 in a cell, eg a cell of a patient, eg a mammalian patient.

In one embodiment, the RNAi agent of the invention comprises a single stranded RNA that interacts with a target RNA sequence, such as a Serpinc1 mRNA target sequence, to direct cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is cleaved into siRNA by a type III endonuclease known as Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into small interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then inserted into an RNA-induced silencing complex (RISC), where one or more helicases untangle the siRNA duplex, allowing a complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within RISC cleave the target to cause silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the invention relates to a single-stranded RNA (siRNA) created within a cell that promotes the formation of a RISC complex that affects the silencing of a target gene, ie, the Serpinc1 gene. Accordingly, the term "siRNA" is also used herein to refer to RNAi as described above.

In another embodiment, the RNAi agent may be a single stranded siRNA that is introduced into a cell or organism to inhibit the target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are usually 15-30 nucleotides long and are chemically modified. The construction and testing of single-stranded siRNAs is described in US Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single stranded siRNA as described herein or chemically modified using the methods described in Lima et al., (2012) Cell 150;:883-894 .

In another embodiment, "mRNA" for use in the compositions, uses, and methods of the invention is double-stranded RNA, which is referred to herein as "double-stranded RNAi agent", "dsRNA molecule (dsRNA)", "dsRNA agent" or " dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a dual structure containing two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA, i.e. the Serpinc1 gene. In some embodiments, the double-stranded RNA (dsRNA) triggers the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

In general, most of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as detailed herein, each or both strands may also contain one or more non-ribonucleotides, such as a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this description, "RNAi means" may contain ribonucleotides with chemical modifications; the RNAi agent may contain significant modifications at many nucleotides.

As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide bond, and/or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions, or deletions of, for example, a functional group or atom, in internucleoside bonds, sugar moieties, or nucleobases. Modifications suitable for use in the compositions of the invention include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, the term "RNAi agent" encompasses any such modifications that are used in an siRNA-type molecule.

The doubled region can be any length that allows specific degradation of the desired target RNA via the RISC pathway and can range from about 9 to 36 bp in length, e.g., about 15-30 bp 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. approximately 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15- 20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19- 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs in length. It is also contemplated that ranges and lengths intermediate to the above ranges and lengths are part of the invention.

The two strands that form the double structure may be different sections of the same large RNA molecule, or they may be separate RNA molecules. When two strands are part of the same large molecule and are therefore linked by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand, forming a twinned structure, the linking strand of RNA is called a "hairpin loop". The hairpin loop may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop may comprise 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 at least 10, at least 20, at least 23 or more unpaired nucleotides.

When two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules need not, but may be, covalently linked. When two strands are covalently linked by a chain of nucleotides other than a continuous chain between the 3' end of one strand and the 5' end of the corresponding other strand, forming a twinned structure, the linking structure is called a "linker". Strands of RNA may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs that are present in the duplex. In addition to the double structure, RNAi may contain overhangs of one or more nucleotides.

In one embodiment, the RNAi agent of the invention is a 24-30 nt dsRNA that interacts with a target RNA sequence, such as the Serpinc1 mRNA target sequence, to direct cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is cleaved into siRNA by a type III endonuclease known as Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into small interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then inserted into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, allowing a complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within RISC cleave the target to cause silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In the context of the present invention, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the doubled structure of an mRNA, such as dsRNA. For example, when the 3' end of one dsRNA strand protrudes beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang. The dsRNA may contain an overhang of at least one nucleotide; alternatively, the 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 a nucleotide/nucleoside analog containing a deoxynucleotide/nucleoside. The ledge(s) may be on the sense strand, the antisense strand, or any combination thereof. In addition, overhang nucleotide(s) may be present at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

In one embodiment, the dsRNA antisense strand has 1-10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, a 3' overhang and/or a 5' end. In one embodiment, the sense strand of the dsRNA is 1-10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, with a 3' and/or 5' overhang. In another embodiment, one or more nucleotides in the overhang are replaced with a thiophosphate nucleoside.

In some embodiments, the overhang on the sense strand or the antisense strand, or both, may be longer than 10 nucleotides, such as 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In some embodiments, the implementation of the expanded ledge is on the sense thread of the duplex. In some embodiments, the flared ridge is at the 3' end of the sense strand of the duplex. In some embodiments, the flared ridge is at the 5' end of the sense strand of the duplex. In some embodiments, the expanded ridge is on the antisense strand of the duplex. In some embodiments, the flared horn is located at the 3' end of the antisense strand of the duplex. In some embodiments, the flared horn is located at the 5' end of the antisense strand of the duplex. In some embodiments, one or more nucleotides in the extended overhang is replaced by a thiophosphate nucleoside.

"Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded RNAi agent, i.e., no nucleotide overhang. The vehicle for "blunt-ended" RNAi is dsRNA, which is double-stranded throughout its length, i.e. there is no nucleotide overhang at either end of the molecule. The RNAi agents of the invention comprise RNAi agents with nucleotide overhangs at one end (i.e., with one overhang and one blunt end), or with nucleotide overhangs at both ends.

The term "antisense strand" or "guide strand" refers to a strand of mRNA, eg dsRNA, that contains a region that is substantially complementary to a target sequence, eg Serpinc1 mRNA. As used herein, the term "region of complementarity" refers to a region on an antisense strand that is substantially complementary to a sequence, eg, a target sequence, eg, a Serpinc1 nucleotide sequence, as defined herein. When the region of complementarity is not fully complementary to the target sequence, there may be mismatches in the internal or terminal regions of the molecule. In general, the most tolerated mismatches are in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' end of the mRNA.

The term "sense strand" or "accompanying strand" as used herein refers to an mRNA strand that contains a region that is substantially complementary to that of an antisense strand as defined herein.

In the context of the present invention, the term "cleavage region" refers to the region that is in close proximity to the cleavage site. A cleavage site is a site in the target at which cleavage occurs. In some embodiments, the cleavage region contains three bases at each end of and in close proximity to the cleavage site. In some embodiments, the cleavage region contains two bases at each end of and in close proximity to the cleavage site. In some embodiments, the cleavage site is specifically located at the site limited by nucleotides 10 and 11 of the antisense strand, and the cleavage region contains nucleotides 11, 12 and 13.

For the purposes of the present invention, and unless otherwise indicated, the term "complementary", when used to describe a first nucleotide sequence with respect to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize and, under certain conditions, form a tandem with the oligonucleotide or polynucleotide, containing a second nucleotide sequence, as will be understood by a specialist. Such conditions, for example, may be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by rinsing (See e.g. "Molecular Cloning: Laboratory Manual, Sambrook, et al., (1989) Cold Spring Harbor Laboratory Press"). Other conditions may apply, such as physiologically relevant conditions that may occur in the body. One skilled in the art will be able to determine the set of conditions most suitable for testing the complementarity of two sequences according to the final application of the hybridized nucleotides.

Complementary sequences within an mRNA, for example within a dsRNA, as described herein, comprise the base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence throughout the entire length of one or both of the nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when the first sequence is referred to herein as "substantially complementary" to the second sequence, the two sequences may be completely complementary, or they may form one or more, but usually no more than 5, 4, 3, or 2 mismatched base pairs when hybridization for a duplex of up to 30 base pairs while retaining the ability to hybridize under the conditions most relevant to their end use, such as inhibition of gene expression via the RISC pathway. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered as mismatches with respect to the determination of complementarity. For example, a dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, with the longer oligonucleotide containing a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide, for the purposes described herein, may also be referred to as "completely complementary".

"Complementary" sequences within the scope of the present invention may also contain or be entirely formed from non-Watson-Crick base pairs and/or base pairs formed from unnatural and modified nucleotides, provided that the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, ambiguous or Hoogsteen G:U base pairings.

The terms "complementary", "fully complementary", and "substantially complementary" may be used herein to refer to a base match between a sense strand and a dsRNA antisense strand, or between an mRNA agent antisense strand and a target sequence, as will be understood from the context of their use.

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 an adjacent region of an mRNA of interest (eg, 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 a contiguous portion of the mRNA encoding Serpinc1.

Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the Serpinc1 target sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target sequence of Serpinc1 and contain an adjacent nucleotide sequence that is at least about 80% complementary throughout its length to an equivalent region of the nucleotide sequence of SEQ ID NO:1 or fragment SEQ ID NO: 1, for example, is about 85% complementary, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99%.

In one embodiment, the RNAi agent of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is in turn complementary to a Serpinc1 target sequence, and wherein the sense strand polynucleotide contains an adjacent nucleotide sequence that is at least complementary throughout its length. at least about 80% equivalent region of the nucleotide sequence of SEQ ID NO:5 or a fragment of any one of SEQ ID NO:5, for example, about 85% complementary, about 86%, about 87%, about 88%, about 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99%.

In one aspect of the invention, the agent for use in the methods and compositions of the invention is a single stranded antisense RNA molecule that inhibits a target mRNA through an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to the sequence within the target mRNA. Single stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing with mRNA and physically interfering with the translation mechanism, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. A single stranded antisense RNA molecule may be from about 15 to about 30 nucleotides in length and have a sequence that is complementary to the target sequence. For example, a single-stranded antisense RNA molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20 or more adjacent nucleotides from any one of the antisense sequences described herein.

The term "inhibition" is used interchangeably with "reduction", "silencing", "down regulation", "suppression" and other similar terms in the context of the present invention, and includes any level of inhibition.

The expression "inhibition of Serpinc1 expression" within the meaning of the present invention includes inhibition of the expression of any Serpinc1 gene (such as, for example, mouse Serpinc1 gene, rat Serpinc1 gene, macaque Serpinc1 gene or human Serpinc1 gene), as well as variants or mutants of the Serpinc1 gene, which encode the Serpinc1 protein.

"Inhibition of Serpinc1 gene expression" includes any level of inhibition of the Serpinc1 gene, such as at least partial suppression of Serpinc1 gene expression, such as inhibition of 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%.

Serpinc1 gene expression can be assessed based on the level of any variable associated with Serpinc1 gene expression, e.g. Serpinc1 mRNA level, Serpinc1 protein level, or e.g. thrombin:antithrombin complex levels as a measure of thrombin formation potential, bleeding time, prothrombin time (PT) , platelet count, and/or activated partial thromboplastin time (aPTT). Inhibition can be assessed as a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level that is used in the art, for example, a baseline level before dosing or a level determined in a similar patient, in a cell or sample, not receiving treatment or receiving control treatment (such as, for example, control only with buffer or control with inactive agent).

In one embodiment, at least partial suppression of Serpinc1 gene expression is measured by a decrease in the amount of Serpinc1 mRNA that can be isolated or detected in the first cell or group of cells in which the Serpinc1 gene is transcribed and which is or has been treated in such a way as to inhibit the expression of the Serpinc1 gene by compared with a second cell or group of cells essentially identical to the first cell or group of cells, but which or which have not been so treated (control cells). The degree of inhibition can be expressed in terms of

The term "contacting a cell with an RNAi agent" such as dsRNA in the sense of the present invention includes contacting the cell with any possible means. Contacting a cell with an RNAi agent includes contacting a cell with an mRNA in vitro or contacting a cell with an mRNA in vivo. The introduction into contact can be done directly or indirectly. Thus, for example, the RNAi agent can be placed in physical contact with the cell by individually performing the method, or alternatively, the RNAi agent can be placed in a situation that subsequently enables or allows it to come into contact with the cell.

Contacting a cell in vitro can be accomplished, for example, by incubating the cell with an RNAi agent. Contacting a cell in vivo can be accomplished, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, such as the bloodstream or subcutaneous space, such that the drug subsequently reaches tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be associated with a ligand, such as GalNAc3, that directs the RNAi agent to a site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, a cell can also be contacted in vitro with an RNAi agent and subsequently transplanted into a patient.

In one embodiment, contacting a cell with an mRNA includes "introducing" or "delivering the mRNA into the cell" by facilitating or influencing uptake or absorption into the cell. Absorption or uptake of mRNA can be carried out through self-diffusion or active cellular processes or with the help of assistive means or devices. The introduction of mRNA into the cell may be in vitro and/or in vivo. For example, for in vivo administration, the mRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved using a beta-glucan delivery system such as those described in US Pat. Introducing into a cell in vitro includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

II. Methods according to the invention

The present invention provides therapeutic methods for treating bleeding in a patient with hemophilia (e.g., hemophilia A, hemophilia B, or hemophilia C), which comprises administering to the patient an mRNA agent or a pharmaceutical composition containing an mRNA agent of the invention, for example, in an amount that reduces Serpinc1 activity in of the patient by about 75% or more, and a therapeutically effective amount of the replacement factor or bypass agent that is reduced from the recommended therapeutically effective amount of the replacement factor or bypass agent, such as that recommended by the World Federation of Hemophilia (see, for example, 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 Food and Drug Administration ((see e.g. , ADVATE (Antihemophilic Factor (Recombinant)) product insert; 11/2016; BeneFIX (Coagulation Factor IX (Recombinant) product insert; 11/2011) (for example, in an amount sufficient to form thrombin and eliminate bleeding (clot formation). The entire contents of each of the above documents are incorporated herein by reference.

As described in the examples below, it has been unexpectedly found that in patients with hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C, with or without inhibitors, administration of an RNAi agent that inhibits Serpinc1 expression in an amount that reduces Serpinc1 activity in the patient by approximately by 75% or more, reduces the mean annual bleeding rate and the spontaneous annual bleeding rate, and that bleeding can be controlled (thrombin is produced and bleeding is stopped) with a replacement factor or bypass agent in a therapeutically effective amount that is reduced from the recommended therapeutically effective amount replacement factor or bypass agent.

Suitable replacement factors for use in the methods of the invention include Factor VIII such as Advat, Elokat, Gaemat, Helixate, Immunate, Octanate, Recombinant and Refacto, or Factor IX such as Aimafix, Benefix, Immunin and Refacto. Suitable bypass agents for use in the methods of the invention include activated prothrombin complex concentrates (aPCC), eg FEIBA and Prothroplex, and Recombinant Factor VIIa (rFVIIa), eg NovoSeven.

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

For example, a therapeutically effective amount of Factor VIII replacement factor administered to a patient 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, 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 the patient is from about one and a half times to about five times less than the recommended effective amount of the replacement factor, for example, a dose of about 5 to about 20 IU/kg, or about 10 to about 20 IU/kg, such as 5, 10, 15 or 20 IU/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

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

For example, a therapeutically effective amount of 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. 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 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 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 kg, or less than about 10 IU/kg. In one embodiment, the therapeutically effective amount of factor IX administered to the patient is from about two times to about six times less than the recommended effective amount of the replacement factor, e.g., 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 is moderate bleeding. In another embodiment, the bleeding is major bleeding

The bypass agent may be aPCC, and the therapeutically effective amount of the bypass agent administered to the patient in the methods of the invention is a dose sufficient to generate thrombin and eliminate bleeding.

For example, a therapeutically effective amount of the aPCC bypass agent 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 the patient is from about two times to about three times less than the recommended effective amount of the replacement factor, e.g., a dose of about 30 to about 50 U/kg, e.g., about 30, 35, 40 , 45 or 50 U/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

The bypass agent may be rFVIIa, and the therapeutically effective amount of the bypass agent administered to the patient in the methods of the invention is a dose sufficient to generate thrombin and eliminate bleeding.

For example, a therapeutically effective amount of the rFVIIa bypass agent 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, 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 patient is approximately half the recommended effective amount of the replacement factor, eg, a dose of approximately 45 μg/kg. In one embodiment, the bleeding is moderate bleeding. In another embodiment, the bleeding is major bleeding.

In some embodiments, the RNAi agent is administered at a fixed dose of about 25 mg to about 100 mg, such as about 25 mg to about 95 mg, about 25 mg to about 90 mg, about 25 mg to about 85 mg, about 25 mg to about 80 mg, about 25 mg to about 75 mg, about 25 mg to about 70 mg, about 25 mg to about 65 mg, about 25 mg to about 60 mg, about 25 mg to about 50 mg mg, about 50 mg to about 100 mg, about 50 mg to about 95 mg, about 50 mg to about 90 mg, about 50 mg to about 85 mg, about 50 mg to about 80 mg, about 30 mg to about 100 mg, about 30 mg to about 90 mg, about 30 mg to about 80 mg, about 40 mg to about 100 mg, about 40 mg to about 90 mg, about 40 mg to about 80 mg , about 60 mg to about 100 mg, about 60 mg to about 90 mg, about 25 mg to about 55 mg, about 25 mg to about 65 mg, about 30 mg to about 95 mg, about 30 mg up to about 85 mg, from about 30 mg to about 75 mg, from about 30 mg to about 65 mg, from about 30 mg to about 55 mg, from about 40 mg to about 95 mg, from about 40 mg to about 85 mg, about 40 mg to about 75 mg, about 40 mg to about 65 mg, about 40 mg to about 55 mg, or about 45 mg to about 95 mg.

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

In one embodiment, the RNAi agent is administered to the patient at a dose that reduces Serpinc1 activity by approximately 75% or more.

Accordingly, in one aspect, the present invention provides a method of treating bleeding in a patient with hemophilia without inhibitors. The method includes administering to the patient a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression (e.g., an amount that reduces Serpinc1 activity in the patient by about 75% or more), the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the 5'-UUGAAGUAAAUGGUGUUAACCAG-3' nucleotide sequence (SEQ ID NO: 15 ), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced from the recommended effective amount of the replacement factor (e.g., the amount sufficient to achieve a peak plasma level of Factor VIII is approximately 10-100 IU/dL (e.g., dose less than about 200 IU/kg factor VIII, e.g., a dose of about 5 to about 20 IU/kg factor VIII); or an amount sufficient to achieve peak plasma factor IX levels of about 10-100 IU/dl (e.g. , a dose less than about 200 IU/kg factor IX, eg, a dose of about 10 to about 30 IU/kg factor IX)), thereby treating bleeding in a hemophiliac patient without inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient with hemophilia with inhibitors. The methods include administering to the patient a fixed dose of about 30 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression (e.g., an amount that reduces the patient's Serpinc1 activity by about 75% or more), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15 ), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced from the recommended effective amount of the bypass agent (e.g., an amount sufficient to form thrombin and eliminate bleeding, e.g., a dose less than about 100 U/kg aPCC (e.g., , a dose of about 30 to 50 U/kg PCC); a dose less than about 120 µg/kg rFVIIa (eg, a dose of about 45 µg/kg rFVIIa)), thereby treating bleeding in a hemophilia patient with inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient with hemophilia without inhibitors. The method includes administering to the patient a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression (e.g., an amount that reduces Serpinc1 activity in the patient by about 75% or more), the double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15 ), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the replacement factor, wherein the effective amount of the replacement factor is reduced from the recommended effective amount of the replacement factor (e.g., the amount sufficient to achieve a peak plasma level of Factor VIII is approximately 10-100 IU/dL (e.g., dose less than about 200 IU/kg factor VIII, e.g., a dose of about 5 to about 20 IU/kg factor VIII); or an amount sufficient to achieve peak plasma factor IX levels is about 10-100 IU/dl (e.g. , a dose of less than about 200 IU/kg factor IX, eg, a dose of about 10 to about 30 IU/kg factor IX)), thereby treating bleeding in a hemophiliac patient without inhibitors.

In another aspect, the present invention provides a method of treating bleeding in a patient with hemophilia with inhibitors. The methods include administering to the patient a fixed dose of about 40 mg to about 90 mg of a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression (e.g., an amount that reduces the patient's Serpinc1 activity by about 75% or more), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15 ), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end; and administering to the patient a therapeutically effective amount of the bypass agent, wherein the effective amount of the bypass agent is reduced from the recommended effective amount of the bypass agent (e.g., an amount sufficient to form thrombin and eliminate bleeding, e.g., a dose less than about 100 U/kg aPCC (e.g., , a dose of about 30 to 50 U/kg PCC); a dose less than about 120 µg/kg rFVIIa (eg, a dose of about 45 µg/kg rFVIIa)), thereby treating bleeding in a hemophilia patient with inhibitors.

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

In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, suitable for administration to a patient at a fixed dose of about 30 mg to 90 mg (e.g., an amount that reduces Serpinc1 activity in the patient by about 75% or more); and a bypass agent suitable for administration to the patient at a dose reduced from the recommended effective amount of the bypass agent (e.g., an amount sufficient to form thrombin and eliminate bleeding, e.g., a dose less than about 100 U/kg aPCC (e.g., a dose of about 30 to 50 U/kg PCC); a dose less than about 120 µg/kg rFVIIa (e.g., a dose of about 45 µg/kg rFVIIa)) for use in a method of treating bleeding in a patient with hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C with inhibitors. The RNAi tool contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, with the sense strand conjugated to a ligand attached at the 3' end.

In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, suitable for administration to a patient at a fixed dose of about 40 mg to 90 mg (e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more); and a replacement factor suitable for administration to the patient at a dose reduced from the recommended effective amount of the replacement factor (e.g., the amount sufficient to achieve peak plasma factor VIII levels is approximately 10-100 IU/dL (e.g., a dose less than about 200 IU/kg factor VIII, e.g., a dose of about 5 to about 20 IU/kg factor VIII); or an amount sufficient to achieve peak plasma levels of factor IX is about 10-100 IU/dl (e.g., dose less than than about 200 IU/kg of factor IX, e.g., a dose of about 10 to about 30 IU/kg of factor IX)) for use in a method of treating bleeding in a patient with hemophilia, for example, hemophilia A, hemophilia B, or hemophilia C without inhibitors. The RNAi tool contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, with the sense strand conjugated to a ligand attached at the 3' end.

In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, suitable for administration to a patient at a fixed dose of about 40 mg to 90 mg (e.g., an amount that reduces Serpinc1 activity in the patient by about 75% or more); and a bypass agent suitable for administration to the patient at a dose reduced from the recommended effective amount of the bypass agent (e.g., an amount sufficient to form thrombin and eliminate bleeding, e.g., a dose less than about 100 U/kg aPCC (e.g., a dose of about 30 to 50 U/kg PCC); a dose less than about 120 μg/kg rFVIIa (e.g., a dose of approximately 45 μg/kg rFVIIa)) for use in a method of treating bleeding in a patient with hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C with inhibitors. The RNAi tool contains a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, with the sense strand conjugated to a ligand attached at the 3' end.

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

In one embodiment, the double-stranded RNAi agent contains a sense strand containing the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 16) and the antisense strand contains the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15 ).

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

In one embodiment, the sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:13) and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14), where a, c, g, and u represent 2'-O-methyl (2'-OMe) A, C, G or U; Af, Cf, Gf or Uf represent 2'-fluoro A, C, G or U; and s represents a phosphorothioate bond; and while the sense strand is conjugated to the ligand, as shown in the following diagram

,

where X represents O or S.

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

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

In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, suitable for administration to a patient at a fixed dose of about 80 mg (eg, an amount that reduces Serpinc1 activity in the patient by about 75% or more); and a replacement factor suitable for administration to the patient at a dose reduced from the recommended effective amount of the replacement factor (e.g., the amount sufficient to achieve peak plasma factor VIII levels is approximately 10-100 IU/dL (e.g., a dose less than about 200 IU/kg factor VIII, e.g., a dose of about 5 to about 20 IU/kg factor VIII); or an amount sufficient to achieve peak plasma levels of factor IX is about 10-100 IU/dl (e.g., dose less than than about 200 IU/kg of factor IX, e.g., a dose of about 10 to about 30 IU/kg of factor IX)) for use in a method of treating bleeding in a patient with hemophilia, for example, hemophilia A, hemophilia B, or hemophilia C without inhibitors. The RNAi agent contains a sense strand and an antisense strand, wherein the sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:13) and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14), wherein a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G or U; Af, Cf, Gf or Uf represent 2'-fluoro A, C, G or U; and s represents a phosphorothioate bond; and while the 3'-end of the sense strand is conjugated to the ligand, as shown in the following scheme

,

where X represents O or S.

In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent that inhibits Serpinc1 expression, suitable for administration to a patient at a fixed dose of about 80 mg (eg, an amount that reduces Serpinc1 activity in the patient by about 75% or more); and a bypass agent suitable for administration to the patient at a dose reduced from the recommended effective amount of the bypass agent (e.g., an amount sufficient to form thrombin and eliminate bleeding, e.g., a dose less than about 100 U/kg aPCC (e.g., a dose of about 30 to 50 U/kg PCC); a dose less than about 120 μg/kg rFVIIa (e.g., a dose of approximately 45 μg/kg rFVIIa)) for use in a method of treating bleeding in a patient with hemophilia, e.g., hemophilia A, hemophilia B, or hemophilia C with inhibitors. The RNAi agent contains a sense strand and an antisense strand, wherein the sense strand contains 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:13) and the antisense strand contains 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14), wherein a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G or U; Af, Cf, Gf or Uf represent 2'-fluoro A, C, G or U; and s represents a phosphorothioate bond; and while the 3'-end of the sense strand is conjugated to the ligand, as shown in the following scheme

,

where X represents O or S.

In one embodiment, a fixed dose of an RNAi agent is suitable for subcutaneous administration.

In one embodiment, a fixed dose of an RNAi agent is suitable for administration to a patient once a month.

The present invention also provides methods for preventing at least one symptom in a patient having a disorder that would benefit from reduced expression of Serpinc1, such as a bleeding disorder, such as hemophilia. The methods include administering to the patient a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in the patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of an mRNA agent, e.g. composition containing dsRNA according to the invention), thereby preventing at least one symptom in a patient with a disorder that would benefit from a decrease in Serpinc1 expression. In one embodiment, the methods include administering to the patient a prophylactically effective dose, e.g., a fixed dose of approximately 50 mg, of an mRNA agent, e.g., dsRNA, of the invention (e.g., a pharmaceutical composition comprising dsRNA of the invention), thereby preventing at least one symptom in a patient with a disorder that would benefit from a reduction in Serpinc1 expression. In another embodiment, the methods comprise administering to the patient a prophylactically effective dose, e.g., a fixed dose of approximately 80 mg, of an mRNA agent, e.g., dsRNA, of the invention (e.g., a pharmaceutical composition comprising dsRNA of the invention), thereby preventing at least one symptom in a patient with a disorder that would benefit from a reduction in Serpinc1 expression.

In another aspect, the present invention provides methods for treating a patient having a disorder that would benefit from a reduction in Serpinc1 expression, such as a bleeding disorder, such as hemophilia, which comprises administering to the patient, such as a human, a therapeutically effective dose, such as an amount that reduces Serpinc1 activity in a patient by about 75% or more, for example, a fixed dose of about 25 mg to about 100 mg of an mRNA agent targeting the Serpinc1 gene or a pharmaceutical composition containing an mRNA agent targeting the Serpinc1 gene, thereby treating a patient with a disorder who would benefit from a reduction in Serpinc1 expression. In one embodiment, the methods include administering to a patient a therapeutically effective dose, e.g., a fixed dose of approximately 50 mg, of an mRNA agent, e.g., dsRNA, of the invention (e.g., a pharmaceutical composition comprising dsRNA of the invention), thereby treating the patient having the disorder. , which would benefit from decreased Serpinc1 expression. In another embodiment, the methods include administering to a patient a therapeutically effective dose, e.g., a fixed dose of approximately 80 mg, of an mRNA agent, e.g., dsRNA, of the invention (e.g., a pharmaceutical composition comprising dsRNA of the invention), thereby treating the patient having the disorder. , which would benefit from decreased Serpinc1 expression.

In another aspect, the invention provides options for administering a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg, mRNA, e.g., dsRNA, according to the invention for the prevention of at least one symptom in a patient with a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder, eg hemophilia. In one embodiment, the invention provides options for administering a prophylactically effective dose, e.g., a fixed dose of approximately 50 mg, of an mRNA, e.g., dsRNA, of the invention for the prevention of at least one symptom in a patient with a disorder who would benefit from reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder such as hemophilia. In another embodiment, the invention provides options for administering a prophylactically effective dose, e.g., a fixed dose of approximately 80 mg, of an mRNA, e.g., dsRNA, of the invention for the prevention of at least one symptom in a patient with a disorder who would benefit from reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder such as hemophilia.

In a further aspect, the present invention provides options for administering a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of an mRNA agent of the invention in the manufacture of a medicament. for the prevention of at least one symptom in a patient with a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder, eg hemophilia. In one embodiment, the present invention provides options for using a prophylactically effective dose, e.g., a fixed dose of approximately 50 mg, of an mRNA agent of the invention in the manufacture of a medicament for the prevention of at least one symptom in a patient with a disorder that would benefit from reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder such as hemophilia. In another embodiment, the present invention provides options for using a prophylactically effective dose, e.g., a fixed dose of approximately 80 mg, of an mRNA agent of the invention in the manufacture of a medicament for the prevention of at least one symptom in a patient with a disorder that would benefit from reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder such as hemophilia.

In another aspect, the present invention provides options for administering a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of an mRNA agent of the invention, to treat a patient, for example, a patient who would benefit from a reduction and/or inhibition of Serpinc1 expression. In one embodiment, the present invention provides options for using a therapeutically effective dose, e.g., a fixed dose of approximately 50 mg, of an mRNA agent of the invention to treat a patient, e.g., a patient who would benefit from a reduction and/or inhibition of Serpinc1 expression. In another embodiment, the present invention provides options for using a therapeutically effective dose, e.g., a fixed dose of approximately 80 mg, of an mRNA agent of the invention to treat a patient, e.g., a patient who would benefit from a reduction and/or inhibition of Serpinc1 expression.

In yet another aspect, the present invention provides the use of an mRNA agent, e.g., dsRNA, according to the invention, targeting the Serpinc1 gene, or a pharmaceutical composition containing a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, for example, a fixed dose of about 25 mg to about 100 mg of a Serpinc1 gene targeting mRNA agent in the manufacture of a medicament for the treatment of a patient, e.g., a patient who would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a patient with a clotting, such as hemophilia. In one embodiment, the present invention provides the use of an mRNA agent, e.g., dsRNA, of the invention targeting the Serpinc1 gene, or a pharmaceutical composition containing a therapeutically effective dose, e.g., a fixed dose of approximately 50 mg, of an mRNA agent targeting the Serpinc1 gene, when the manufacture of a medicament for the treatment of a patient, for example, a patient who would benefit from the reduction and/or inhibition of Serpinc1 expression, such as a patient with a bleeding disorder, for example, hemophilia. In another embodiment, the present invention provides the use of an mRNA agent, e.g., dsRNA, of the invention targeting the Serpinc1 gene, or a pharmaceutical composition containing a therapeutically effective dose, e.g., a fixed dose of approximately 80 mg, of an mRNA agent targeting the Serpinc1 gene, when the manufacture of a medicament for the treatment of a patient, for example, a patient who would benefit from the reduction and/or inhibition of Serpinc1 expression, such as a patient with a bleeding disorder, for example, hemophilia.

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

Accordingly, in one aspect, the invention provides methods for preventing at least one symptom in a patient having a disorder that would benefit from reduced expression of Serpinc1, eg, a bleeding disorder, eg, hemophilia. The methods include administering to the patient a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in the patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent containing a sense strand and an antisense strand, and the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), and substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, wherein the sense strand is conjugated to a ligand attached at the 3' end (e.g., a pharmaceutical composition containing an RNAi agent), thereby preventing at least one symptom in a patient with a disorder that would benefit from a reduction in Serpinc1 expression. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

In another aspect, the present invention provides methods for treating a patient having a disorder that would benefit from a reduction in Serpinc1 expression, such as a bleeding disorder, such as hemophilia, which comprises administering to the patient, such as a human, a therapeutically effective dose, such as an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent containing a sense strand and an antisense strand, the antisense strand containing a region of complementarity to an mRNA encoding Serpinc1 , which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), and essentially all nucleotides of the sense strand and essentially all nucleotides of the antisense strand represent are modified nucleotides, and the sense strand is conjugated to a ligand attached at the 3' end, or a pharmaceutical composition containing an mRNA agent targeting the Serpinc1 gene, thereby treating a patient with a disorder who would benefit from reduced expression Serpinc1. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

In another aspect, the invention provides options for administering a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent containing a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15 ), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end to prevent at least one symptom in a patient with a disorder that would benefit from the reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder such as hemophilia. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

In a further aspect, the present invention provides options for administering a prophylactically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent, containing a sense strand and an antisense strand, and the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein essentially all nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end, in the manufacture of a medicament for the prevention of at least one symptom in a patient with a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder such as hemophilia. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

In another aspect, the present invention provides options for administering a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, e.g., a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent, containing a sense strand and an antisense strand, and the antisense strand contains a region of complementarity to the mRNA encoding Serpinc1, which contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end, to treat a patient, e.g., a patient who would benefit from reducing and/or inhibiting Serpinc1 expression. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

In yet another aspect, the present invention provides the use of an mRNA agent, e.g., dsRNA, according to the invention, targeting the Serpinc1 gene, or a pharmaceutical composition containing a therapeutically effective dose, e.g., an amount that reduces Serpinc1 activity in a patient by about 75% or more, for example, a fixed dose of about 25 mg to about 100 mg of a double-stranded ribonucleic acid (RNAi) agent containing a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1 that contains at least 15 contiguous nucleotides that do not differ more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein essentially all nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand , attached at the 3'-end, in the manufacture of a drug for the treatment of a patient, for example, a patient who would benefit from the reduction and/or inhibition of Serpinc1 expression, such as a patient with a bleeding disorder, for example, hemophilia. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

Methods and uses of the invention include administering the composition described herein such that Serpinc1 target gene expression is reduced, for example, within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or approximately 80 days. In one embodiment, Serpinc1 target gene expression decreases over a long period of time, e.g., at least about seven days or more, e.g., about one week, two weeks, three weeks, about four weeks, about 5 weeks, about 6 weeks, approximately 2 months, approximately quarters or longer.

The reduction in gene expression can be assessed using any of the methods known in this field. For example, a decrease in Serpinc1 expression can be determined by determining the level of expression of Serpinc1 mRNA using methods routine for one skilled in the art, e.g. as Western blotting, immunological methods and/or by determining the biological activity of Serpinc1, for example, the influence of one or more molecules associated with the cellular mechanism of blood coagulation (or blood coagulation itself in vivo). In one embodiment, to assess Serpinc1 expression, thrombin formation time, clot formation time, and/or clotting time are determined using, for example, the ROTEM® whole blood thromboelastometric assay.

The administration of dsRNA according to the methods and uses of the invention may result in a patient with a Serpinc1 associated disease to reduce the severity, signs, symptoms and/or markers of such diseases or disorders. By "decrease" in this context is meant a statistically significant decrease in that level. The reduction may 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 approximately 100%.

The effectiveness of treating or preventing a disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, bleeding frequency, pain reduction, quality of life, dose of drug required to maintain treatment effect, disease marker level, or any other measurable parameter appropriate for a given disease that is being treated or prevented. The person skilled in the art is well placed to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of parameters. For example, the effectiveness of the treatment of bleeding disorders can be assessed, for example, by periodically monitoring the levels of thrombin:antithrombin. Comparison of later scores with initial scores gives the physician an indication of whether the treatment is effective. The person skilled in the art is well placed to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of parameters. In connection with the administration of an mRNA targeted to Serpinc1 or a pharmaceutical composition thereof, "effective against" a bleeding disorder indicates that administration in a clinically appropriate manner results in a beneficial effect in at least a statistically significant proportion of patients, such as improvement in symptoms, cure, reduction in disease , life extension, improved quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of bleeding disorders and related causes.

A therapeutic or prophylactic effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or when symptoms fail to worsen or develop when they would otherwise be expected. By way of example, a favorable change of at least 10% in a measurable disease parameter, and preferably at least 20%, 30%, 40%, 50%, or more, may indicate an effective treatment. The efficacy of a given mRNA drug or formulation of the drug can also be evaluated using an animal model for a given disease known in the art. In an animal model, treatment efficacy is confirmed when there is a statistically significant reduction in a marker or symptom.

Alternatively, efficacy can be measured by reduction in disease severity, which is determined by one of skill in the art of diagnosis based on a clinically accepted disease severity scale. Any positive change resulting in, for example, a reduction in disease severity as measured using an appropriate scale is indicative of adequate treatment using the mRNA or mRNA formulation as described herein.

_And RNA (or pharmaceutical compositions containing mRNA) can be administered to a patient about once a week, about twice a month, about once every six weeks, about once every 2 months, or once a quarter.

The double-stranded mRNA agent may be administered to the patient in one or more doses. For example, the double-stranded mRNA agent may be administered to a patient at a monthly dose of about 0.200 mg/kg to about 1.825 mg/kg. Alternatively, the double-stranded mRNA agent may be administered to the patient at a fixed dose of about 25 mg to about 100 mg.

In one embodiment, a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1 that contains at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG -3' (SEQ ID NO: 15), wherein essentially all nucleotides of the sense strand and essentially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached at the 3' end, is administered is administered to the patient in a fixed dose of about 25 to about 100 mg, such as about 25 mg, 50 mg, 80 mg, or 100 mg. In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

The administration may be repeated, eg on a regular basis, eg monthly, for one month, two months, three months, four months or longer. After the initial course of treatment, treatment may be less frequent. For example, after administration once a month for three months, administration can be repeated once a quarter for a year or longer.

Accordingly, in some embodiments, the RNAi agent is administered in a regimen that includes a "loading phase" of frequent administrations, which may be followed by a "maintenance phase" in which the RNAi agent is administered at longer intervals.

The loading application schedule and/or the maintenance application schedule may optionally be repeated over one or more iterations. The number of iterations may depend on achieving the desired effect, for example, suppressing the Serpinc1 gene and/or achieving a therapeutic or prophylactic effect, for example, increasing blood clotting, reducing clot formation time and/or reducing clotting time.

The introduction of mRNA can reduce levels of Serpinc1, for example, in a cell, tissue, blood, urine or other patient compartment by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% , 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46 %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or at least about 99% or more.

mRNA can be administered by intravenous infusion over a period of time, e.g. over periods 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or approximately 25 minutes.

Before a full dose of mRNA is administered, patients may be given a lower dose, such as a 5% infusion, and monitored for side effects such as an allergic reaction. In another example, undesirable immunostimulatory effects, such as increased levels of cytokines (eg, TNF-alpha or INF-alpha), can be monitored in a patient.

Due to the inhibitory effect on the expression of Serpinc1, the composition according to the invention or a pharmaceutical composition derived therefrom can improve the quality of life.

The mRNA according to the invention can be administered in "naked" form or as "free mRNA". naked mRNA is administered in the absence of a pharmaceutical composition. naked mRNA can be in a suitable buffer solution. the buffer solution may contain acetate, citrate, prolamine, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the mRNA can be adjusted so that it is suitable for administration to the patient.

Alternatively, the mRNA of the invention may be administered as a pharmaceutical composition, such as a liposomal formulation of dsRNA.

Patients who would benefit from a reduction and/or inhibition of Serpinc1 gene expression are those with a bleeding disorder, eg, an inherited bleeding disorder or an acquired bleeding disorder, as described herein. In one embodiment, the patient with an inherited bleeding disorder has hemophilia, e.g., hemophilia A, B, or C. In one embodiment, the patient with an inherited bleeding disorder, e.g., hemophilia, is a patient with an inhibitor ). In one embodiment, the patient with the inhibitor has hemophilia A. In another embodiment, the patient with the inhibitor has hemophilia B. In another embodiment, the patient with the inhibitor has hemophilia C. Treatment of a patient who would benefit from reduction and/or inhibition expression of the Serpinc1 gene, includes therapeutic (for example, on demand, for example, a patient with bleeding (spontaneous bleeding or bleeding due to trauma) and an inability to clot) and prophylactic (for example, a patient without bleeding and / or undergoing surgery) treatment.

The invention further provides methods and uses for using an mRNA or a pharmaceutical composition thereof, for example, to treat a patient that would benefit from reduction and/or inhibition of Serpinc1 expression, for example, a patient with a bleeding disorder, in combination with other pharmaceuticals. drugs and / or other therapeutic methods, for example, with known pharmaceuticals and / or known therapeutic methods, such as, for example, those currently used to treat these disorders.

For example, in some embodiments, the mRNA targeted to Serpinc1 is administered in combination with, for example, a drug useful in the treatment of a bleeding disorder, as described elsewhere herein. For example, additional therapeutic agents and therapeutic methods suitable for treating a patient who would benefit from a reduction in Serpinc1 expression, such as a patient with a bleeding disorder, include fresh frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrates; containing vWF FVIII concentrates with inactivated virus; desensitization treatment, which may include high doses of FVIII or FIX, along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasmapheresis in combination with immunosuppression and infusion of FVIII or FIX with or without antifibriolytic therapy; immune tolerance induction (ITI) with or without immunosuppressive therapy (eg, cyclophosphamide, prednisone, and/or anti-CD20 antibodies); desmopressin acetate [DDAVP]; antifibrinolytics such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilic agents; corticosteroids; immunosuppressive agents; and estrogens.

The mRNA and the additional therapeutic agent and/or treatment may be administered simultaneously and/or in the same combination, e.g. parenterally, or the additional therapeutic agent may be administered as part of a separate composition or at a separate time and/or by another method known herein. area or described in this document.

In one embodiment, the present invention provides methods for treating a patient with a bleeding disorder, such as hemophilia, by subcutaneously administering compound AT3SC-001 (AD-57213-Sense strand: 5'-GfsgsUfuAfaCfaCfCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:13) to the patient, and Antisense strand: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:14) wherein a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G or U; Af, Cf , Gf, or Uf are 2′-fluoro A, C, G, or U; and s is a phosphorothioate bond) at a fixed dose of about 25 mg to about 100 mg, e.g., a fixed dose of about 25 mg, about 50 mg, about 80 mg or about 100 mg (eg, in an amount that reduces Serpinc1 activity in a patient by about 75% or more). In one embodiment, the fixed dose is 50 mg. In another embodiment, the fixed dose is 80 mg.

III. RNA for use in the methods of the invention

This document describes methods of using improved double-stranded RNAi agents that inhibit Serpinc1 gene expression in a cell, e.g., a cell of a patient, e.g., a mammal, such as a human, having a Serpinc1-associated disorder, e.g., a bleeding disorder, e.g., hemophilia.

Accordingly, the invention provides double-stranded RNAi agents with chemical modifications capable of inhibiting the expression of a target gene (ie Serpinc1 gene) in vivo. In some aspects of the invention, essentially all nucleotides of the mRNA according to the invention are modified. In other embodiments of the invention, all mRNA nucleotides according to the invention are modified. mRNAs of the invention in which "substantially all nucleotides are modified" are substantially but not completely modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

The RNAi tool contains a sense strand and an antisense strand. Each strand of the RNAi agent can vary from 12-30 nucleotides in length. For example, each strand may be between 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.

The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as "RNAi agent". The dual region of the RNAi agent may be 12-30 bp in length. For example, a double region may be between 14-30 bp in length, 17-30 bp in length, 27-30 bp in length, 17-23 bp in length, 17-21 bp in length, 17-19 bp long, 19-25 bp long, 19-23 bp long, 19-21 bp long, 21-25 bp long, or 21-23 bp long. In another example, the dual region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangs and/or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang may be 1-6 nucleotides in length, e.g., 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotide in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The protrusions can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang may form a mismatch with the target mRNA, or it may be complementary to the target gene sequences, or may be a different sequence. The first and second strands can also be connected, for example, with additional bases to form a hairpin or other baseless linkers.

In one embodiment, each of the nucleotides in the overhang of the RNAi agent can independently be a modified or unmodified nucleotide containing, but not limited to, a modified 2'-sugar, e.g., 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be a protrusion sequence for any end on any thread. The protrusion may form a mismatch with the target mRNA, or it may be complementary to the target gene sequences, or may be a different sequence.

The 5' or 3' overhangs on the sense strand, the antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang(s) comprise two nucleotides having a phosphorothioate between the two nucleotides, the two nucleotides being the same or different. In one embodiment, the protrusion is at the 3' end of the sense strand, the antisense strand, or both. In one embodiment, this 3'-protrusion is on the antisense strand. In one embodiment, this 3'-protrusion is on the sense strand.

The RNAi agent may contain only one protrusion, which can enhance the RNAi interference activity without affecting its overall stability. For example, the single stranded overhang may be located at the 3'terminal end of the sense strand, or alternatively at the 3'terminal end of the antisense strand. RNAi can also have a blunt end located at the 5' end of the antisense strand (or 3' end of the sense strand) or vice versa. In general, the RNAi antisense strand has a nucleotide overhang at the 3' end and the 5' end is blunt. Without wishing to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the protrusion at the 3' end of the antisense strand contribute to loading the guide strand into the RISC process.

Any of the nucleic acids of the invention can be synthesized and/or modified using methods well known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S.L. et al., (Edrs.), John Wiley & Sons, Inc. New York, NY, USA, which is hereby incorporated by reference into this document. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted bonds) or 3'-terminal modifications (conjugation, DNA nucleotides, inverted bonds, etc.); base modifications, for example, substitution with stabilizing bases, destabilizing bases or bases that form a base pair with an extended repertoire of partners, base deletions (nucleotides lacking a nitrogenous base), or conjugated bases; sugar modifications (eg, at the 2'-position or 4'-position) or sugar substitution; and/or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of mRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or non-natural internucleoside bonds. RNAs having modified backbones include, inter alia, those that do not have a phosphorus atom in their backbone. For the purposes of this description, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In some embodiments, the modified mRNAs will have a phosphorus atom in their internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidate and aminoalkyl phosphoramidates, thionophosphoramidates, thio noalkylphosphonates, thionoalkylphosphotriesters and boranophosphates having normal 3'-5' bonds, 2'-5'-linked analogues thereof, and those having reverse polarity, with adjacent pairs of nucleoside units linked 3'-5' to 5'-3' or 2 '-5' with 5'-2'. Also included are various salts, mixed salts, and free acid forms.

Examples of US patents that teach the preparation of the above phosphorus-containing bonds include, but are not limited to, US Patent Nos. 3,687,808; 4469863; 4476301; 5023243; 5177195; 5188897; 5264423; 5276019; 5278302; 5286717; 5321131; 5399676; 5405939; 5453496; 5455233; 5466677; 5476925; 5519126; 5536821; 5541316; 5550111; 5563253; 5571799; 5587361; 5625050; 6028188; 6124445; 6160109; 6169170; 6172209; 6 239265; 6277603; 6326199; 6346614; 6444423; 6531590; 6534639; 6608035; 6683167; 6858715; 6867294; 6878805; 7015315; 7041816; 7273933; 7321029; and US Patent RE39464, the entire contents of which are hereby incorporated herein by reference.

Modified RNA backbones that do not contain a phosphorus atom in them have backbones that are formed by short chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl internucleoside bonds, or one or more short chain heteroatomic or heterocyclic internucleoside bonds. These include those with morpholine bonds (formed in part from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; backbones of methyleneimino and methylenehydrazino; sulfonate and sulfonamide backbones; amide backbones; and others having mixed portions of the N, O, S and CH ₂ components.

Examples of US patents that teach the preparation of the above oligonucleosides include, but are not limited to, US Pat. Nos. 5,034,506; 5166315; 5185444; 5214134; 5216141; 5235033; 564562; 5264564; 5405938; 5434257; 5466677; 5470967; 5489677; 5541307; 5561225; 5596086; 5602240; 5608046; 5610289; 5618704; 5623070; 5663312; 5633360; 5677437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are provided for use in mRNA in which both the sugar and the internucleoside bond, ie the backbone, of the nucleotide units are replaced with new groups. The base units are saved for hybridization with the appropriate target nucleic acid compound. One such oligomeric compound, an RNA mimetic, which has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the RNA sugar backbone is replaced by an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleic acid bases are retained and linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Examples of US patents that teach the preparation of PNA compounds include, but are not limited to, US Pat. Nos. 5,539,082; 5714331 and 5719262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in mRNA according to the invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments of the invention include RNA with phosphorothioate backbones and oligonucleosides with heteroatomic backbones, and in particular --CH ₂ --NH--CH ₂ -, --CH ₂ --N(CH ₃ )-- O--CH ₂ --[known as 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 --[wherein the native phosphodiester backbone is represented as --O--P--O-- CH ₂ --] of US Pat. No. 5,489,677 cited above, and the amide backbones of US Pat.

Modified RNAs may also contain one or more substituted sugar moieties. RNAs, such as dsRNAs provided herein, may contain at the 2'-position one of the following: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl. Illustrative suitable modifications include O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH 2 ) _n NH ₂ , O(CH ₂ ) _n CH _{3 ,} O(CH ₂ ) _n ONH ₂ and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , where n and m are from 1 to about 10. In other embodiments, the dsRNAs at the 2' position contain one of the following: C ₁ -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 , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of mRNA or group for improving the pharmacodynamic properties of mRNA and other substituents having similar properties. In some embodiments, the modifications include 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), that is, an alkoxy-alkoxy group. Another illustrative modification is 2'-dimethylaminooxyethoxy, i.e. the O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group, also known as 2'-DMAOE, as described in the examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), ie 2'-O--CH ₂ --O--CH ₂ --N(CH ₂ ) ₂ .

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 RNA positions in the mRNA, especially at the 3' sugar position at the 3' end of the nucleotide, or at the 2'-5' linked dsRNAs and at the 5' position of the 5' end of the nucleotide. RNAs can also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugar. Examples of US patents that teach the preparation of such modified sugar structures include, but are not limited to, US Pat. Nos. 4,981,957; 5118800; 5319080; 5359044; 5393878; 5446137; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; and 5700920, some of which in most cases belong to the currently pending application. All contents of each of the foregoing documents are hereby incorporated herein by reference.

mRNA may also contain modifications or substitutions of nucleic bases (often referred to in the art simply as "base"). 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 deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, and others. alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; -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 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Additional nucleobases include those disclosed in US Pat. No. 3,687,808, bases disclosed in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; bases disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, bases disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and bases disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289- 302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful in increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton , 1993, pp. 276-278) and are exemplary base substitutions, even better when combined with 2'-O-methoxyethyl sugar modifications.

Examples of US patents that teach the preparation of some of the above modified nucleobases, as well as other modified nucleobases, include, but are not limited to, US Pat. Nos. 3,687,808 4,845,205 cited above; 513030; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121 5596091; 5614617; 5681941; 5750692; 6015886; 6147200; 6166197; 6222025; 6235887; 6380368; 6528640; 6639062; 6617438; 7045610; 7427672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA in mRNA can also be modified to include one or more bicyclic sugar moieties. "bicyclic sugar" is a furanosyl ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge linking two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the implementation of the bridge links the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agent of the invention may comprise RNA in mRNA, which can also be modified to include one or more blocked nucleic acids (LNAs). A blocked nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety contains an additional bridge linking the 2' and 4' carbons. In other words, LNA is a nucleotide containing a bicyclic sugar fragment containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into the 3'-endo structural conformation. it has been shown that the addition of blocked nucleic acids to siRNA increases the serum stability of siRNA and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843 Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, without limitation, nucleosides containing a bridge between the 4' and 2' atoms of the ribosyl ring. In some embodiments, the antisense polynucleotide agents of the invention comprise one or more bicyclic nucleosides containing a 4'-2' bridge. Examples of such 4'-2' linked bicyclic nucleosides include, but are not limited to, 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ )2-O-2'(ENA);4'-CH(CH ₃ )-O-2' (also called "conformationally restricted ethyl nucleoside" or "cEt") and 4'-CH(CH ₂ OCH ₃ )-O-2' (and its analogs; see , for example, US patent No. 7399845); 4'-C(CH ₃ )(CH ₃ )-O-2' (and its analogues; see, for example, US patent No. 8278283); 4'-CH ₂ -N(OCH ₃ )-2' (and its analogues; see, for example, US patent No. 8278425); 4'-CH ₂ -ON(CH ₃ )-2' (see, for example, US Patent Publication No. 2004/0171570); 4'-CH ₂ -N(R)-O-2', and R represents H, C ₁ -C ₁₂ alkyl or a protective group (see, for example, US patent No. 7427672); 4'-CH ₂ -C(H)(CH ₃ )-2' (see, for example, Chattopadhyaya et al., J. Org. Chem. 2009, 74, 118-134); and 4'-CH ₂ -C(=CH ₂ )-2' (and its analogues; see, for example, US patent No. 8278426). All contents of each of the foregoing documents are hereby incorporated herein by reference.

Additional examples of US patents and US patent publications that teach the production of blocked nucleic acid nucleotides include, but are not limited to, the following: US Patent Nos. 6,268,490; 6525191; 6670461; 6770748; 6794499; 6998484; 7053207; 7034133; 7084125; 7399845; 7427672; 7569686; 7741457; 8022193; 8030467; 8278425; 8278426; 8278283; US 2008/0039618; and US 2009/0012281, the entire contents of which are hereby incorporated herein by reference.

You can get any of the above bicyclic nucleosides having one or more stereochemical configurations of sugars, containing, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA in mRNA can also be modified to include one or more conformationally restricted ethyl nucleotides. In the context of the present invention, a "conformationally restricted ethyl nucleotide" or "cEt" is a blocked nucleic acid containing a bicyclic sugar fragment containing a 4'-CH(CH ₃ )-0-2' bridge. In one embodiment, the conformationally restricted ethyl nucleotide is in the S conformation, referred to herein as "S-cEt".

The mRNA of the invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker linking the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRNs block the ribose ring into a stable conformation and increase the affinity of hybridization for mRNA. The linker is long enough to place the oxygen in the optimal position for stability and affinity, resulting in less folding of the ribose ring.

Examples of publications that outline the production of some of the above CRNs include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT Publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

One or more mRNA nucleotides of the invention may also contain a hydroxymethyl substituted nucleotide. A "hydroxymethyl substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as a "unblocked nucleic acid" ("UNA") modification.

Examples of US publications that describe the preparation of UNA include, but are not limited to, US Pat. No. 8,314,227; and US Patent Publication No. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of which are hereby incorporated herein by reference.

Potentially stabilizing modifications at the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ester), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted base dT( idT), etc. A disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

A. Modified mRNAs containing Motifs according to the Invention

In some aspects of the invention, the double-stranded RNAi agents of the invention include chemically modified agents as disclosed, for example, in U.S. Provisional Application No. 61/561710, filed Nov. 18, 2011, or PCT/US2012/065691, filed Nov. 16, 2012, the entire contents of each of which are incorporated herein by reference.

As shown herein, in Provisional Application No. 61/561710 and in PCT/US2012/065691, an excellent result can be obtained by introducing one or more motifs of three identical modifications of three consecutive nucleotides into the sense strand and/or antisense strand of an RNAi agent, especially at the site splitting or near it. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs violates the nature of the modification, in the presence of a sense and/or antisense strand. The RNAi agent may optionally be conjugated to a GalNAc-derived ligand, for example, on a sense strand. The resulting RNAi tools exhibit excellent gene knockout activity.

More specifically, it has surprisingly been found that when the sense strand and the antisense strand of a double-stranded RNAi agent are modified to have one or more motifs of three identical modifications of three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, the gene knockout activity of the RNAi agent has been greatly improved.

In one embodiment, the RNAi agent is an element with two blunt ends, 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications of three consecutive nucleotides at positions 7, 8, 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

In another embodiment, the RNAi agent is an element with two 20 nucleotides long blunt ends, wherein the sense strand contains at least one motif of three 2'-F modifications of three consecutive nucleotides at positions 8, 9, 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

In yet another embodiment, the RNAi agent is an element with two blunt ends, 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications of three consecutive nucleotides at positions 9, 10, 11 from the 5' end . The antisense strand contains at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications of three consecutive nucleotides at positions 9, 10, 11 from the 5'end; the antisense strand contains at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end, with one end of the RNAi agent being blunt while the other end contains a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3' end of the antisense strand. When a 2 nucleotide overhang is at the 3' end of the antisense strand, there can be two phosphorothioate internucleotide bonds between the three terminal nucleotides, with two of the three nucleotides being overhang nucleotides and the third nucleotide being a paired nucleotide following the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide on the sense strand and antisense strand of the RNAi agent containing nucleotides that are part of the motifs are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, eg in an alternating motif. Optionally, the RNAi agent also contains a ligand (preferably GalNAc ₃ ).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the RNAi agent comprises a first strand having a length that is at least 25 and at most 29 nucleotides and a second strand having a length that is at most 30 nucleotides of at least with one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5-end;, moreover, the 3'-end of the first strand and the 5'-end of the second strand form a blunt end, and the second strand on 1-4 nucleotides longer at its 3' end than the first strand, with a double region that is at least 25 nucleotides in length, and the second strand is sufficiently complementary to the target mRNA at least 19 nucleotides along the length of the second strand to reduce expression of the target gene when the RNAi agent is introduced into a mammalian cell, wherein cleavage of the RNAi agent's dicer preferably results in an siRNA containing the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications of three consecutive nucleotides, with one of the motifs located at a cleavage site on the sense strand.

In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications of three consecutive nucleotides, with one of the motifs located at or near the cleavage site on the antisense strand.

For an RNAi agent having a dual region of 17-23 nucleotides in length, the antisense strand cleavage site is typically approximately 10, 11 and 12 positions from the 5' end. Thus, the motives of three identical modifications can be in 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, with the count starting from the 1st nucleotide from the 5' end of the antisense strand, or the count starting from the 1st paired nucleotide within the double region from the 5' end of the antisense strand. The cleavage site on the antisense strand can also vary according to the length of the RNAi duplex region from the 5' end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications of three consecutive nucleotides at the strand cleavage site; and the antisense strand may have at least one motif of three identical modifications of three consecutive nucleotides at or near the strand cleavage site. When the sense strand and antisense strand form a dsRNA duplex, the sense strand and antisense strand can be aligned such that one three nucleotide motif on the sense strand and one three nucleotide motif on the antisense strand have at least one nucleotide overlap, i.e. at least one of the three nucleotides of the motif on the sense strand forms a base pair with at least one of the three nucleotides of the motif on the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications of three consecutive nucleotides. The first motif may be at or near the cleavage site, and the other motifs may be wing modifications. The term "wing modification" as used herein refers to a motif located in a different part of the strand that is separated from the motif at or near the cleavage site of the same strand. wing modifications are either adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are in close proximity to each other, the composition of the motifs differs from each other, and when the motifs are separated by one or more nucleotides, the compositions can be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each of the wing modifications may be at one end relative to the first motif, which is at or near the cleavage site, or on one side of the front motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motif of three identical modifications of three consecutive nucleotides, with at least one motif located at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications aligned similarly to wing modifications that may be present on the sense strand.

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

In another embodiment, wing modifications on the sense strand or antisense strand of the RNAi agent typically do not contain the first one or two paired nucleotides within the double region at the 3' end, 5' end, or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one lateral modification, the wing modifications may occur at the same end of the duplex region and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be aligned so that the two modifications, each of one strand, are at one end of a duplex region having an overlap of one, two, or three nucleotides; two modifications, each from one strand, fell on the other end of the duplex region having an overlap of one, two or three nucleotides; two modifications of one strand fell on each side of the anterior motif, which had an overlap of one, two, or three nucleotides in the double region.

In one embodiment, each nucleotide on the sense strand and antisense strand of an RNAi agent containing nucleotides that are part of the motifs may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or both non-phosphate-binding oxygens and/or one or more phosphate-binding oxygens; changing the ribose sugar moiety, for example, 2 In one embodiment, when the RNAi hydroxyl agent is on the ribose sugar; complete replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a base existing in nature; and replacement or modification of the ribose phosphate backbone.

Since nucleic acids are polymers of subunits, many modifications occur at a position that is repeated within the nucleic acid, such as modification of a base or phosphate moiety or a non-O-binding phosphate moiety. In some cases, the modification will occur in the patient at all positions in the nucleic acid, but in many cases it will not. As an example, the modification may only occur at the 3' or 5' terminal position, may occur only at the terminal region, for example at a position on the terminal nucleotide, or at the last 2, 3, 4, 5 or 10 nucleotides of the strand. The modification may occur in the double stranded region, in the single stranded region, or in both. The modification may occur only in the double-stranded region of the RNA or may occur only in the single-stranded region of the RNA. For example, the phosphorothioate modification at the non-O-binding position may occur at only one or both ends, may occur only at the terminal region, such as at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may occur at double-stranded and single-stranded regions, especially at the ends. The 5' end or ends may be phosphorylated.

It may be possible, for example, to improve stability, include specific bases in overhangs, or include modified nucleotides or mimic nucleotides in single-stranded overhangs, such as in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhangs. In some embodiments, all or some of the bases in the 3' or 5' ledge can be modified, for example, using the modification described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'- O-methyl instead of the ribosugar of the nucleic base and modifications to the phosphate group, for example, phosphorothioate modifications. Overhangs must not be homologous to the target sequence.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluorine. Threads can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluorine.

Typically, there are at least two different modifications on the sense strand and the antisense strand. These two modifications may be 2'-O-methyl or 2'-fluoro or others.

In one embodiment, N _a and/or N _b contain alternating order modifications. The term "alternating motif" in the context of the present invention refers to a motif having one or more modifications, each modification being on alternating nucleotides on the same strand. an alternating nucleotide may refer to one for every two nucleotides, or one for every three nucleotides, or a similar order. For example, if A, B, and C each represent one type of modification in a nucleotide, the alternating motif could be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…" etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if each of A, B, C, D represents one type of modification per nucleotide, the alternating order, i.e., modifications for every two nucleotides, may be the same, but each of the sense strand or antisense strand may be selected from several modification possibilities within an alternating motive, for example, "ABABAB ...", "ACACAC ..." "BDBDBD ..." or "CDCDCD ...", etc.

In one embodiment, the RNAi agent of the invention comprises shifting the order of modifications for the alternating motif on the sense strand relative to the order of modifications for the alternating motif on the antisense strand. The shift may be such that the modified nucleotide group of the sense strand matches the otherwise modified nucleotide group of the antisense strand, and vice versa. For example, when pairing a sense strand with an antisense strand in a dsRNA duplex, an alternating motif on the sense strand may begin with "ABABAB" from the 5'-3' strand, and an alternating motif on the antisense strand may begin with "BABABA" from the 5'-3' strand. inside the double region. As another example, an alternating motif on the sense strand may begin with "AABBAABB" from the 5'-3' strand, and an alternating motif on the antisense strand may begin with "BBAABBAA" from the 5'-3' strand within the double region, so that there is a complete or partial shift in the orders of modifications between the sense strand and the antisense strand.

In one embodiment, the RNAi agent contains the order of the alternating 2'-O-methyl modification motif and the 2'-F modification on the sense strand is initially offset from the order of the alternating 2'-O-methyl modification motif and the original 2'-F modification on the antisense strand, ie a 2'-O-methyl modified nucleotide on the base pair of the sense strand with a 2'-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may begin with a 2'-F modification, and the 1 position of the antisense strand may begin with a 2'-O-methyl modification.

The introduction of one or more motifs of three identical modifications of three consecutive nucleotides into the sense strand and/or antisense strand disrupts the original order of the modifications present on the sense strand and/or antisense strand. This disruption of the order of modifications of the sense and/or antisense strand by introducing one or more motifs of three identical modifications of three consecutive nucleotides in the sense and/or antisense strand unexpectedly improves gene silencing activity for the target gene.

In one embodiment, when a motif of three identical modifications of three consecutive nucleotides is introduced into any of the strands, the modification of the nucleotide following the motif is a different modification than the motif modification. For example, the sequence region containing the motif is "...N _a YYYN _b ..." where "Y" represents the motif modification of three identical modifications over three consecutive nucleotides, and "N _a " and "N _b " represent the modification of the nucleotide following motif "YYY", which is different from the modification Y, and where N _a and N _b may be the same or different modifications. Alternatively, N _a and/or N _b may or may not be present with the wing modification.

The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. Modification of the phosphorothioate or methylphosphonate internucleotide linkage can occur at any nucleotide of the sense strand or antisense strand, or on both strands, at any position on the strand. For example, modification of the internucleotide bond may occur at each nucleotide on the sense strand and/or antisense strand; each modification of the internucleotide bond may occur in an alternating order on the sense strand and/or antisense strand; or the sense strand or antisense strand may contain both modifications of the internucleotide bond in alternating order. the alternating order of modification of the internucleotide bond on the sense strand may be the same or different from the antisense strand, and the alternating order of modification of the internucleotide bond on the sense strand may have a shift relative to the alternating order of modification of the internucleotide bond on the antisense strand.

In one embodiment, the RNAi contains a modification of the phosphorothioate or methylphosphonate internucleotide bond in the overhang. For example, the overhang may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. Also, an internucleotide bond modification can be made to link overhang nucleotides to terminal paired nucleotides within the double region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked via a phosphorothioate or methylphosphonate internucleotide bond, and optionally there may be additional phosphorothioate or methylphosphonate internucleotide bonds linking the overhang nucleotide to the paired nucleotide that is located next to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between the three terminal nucleotides, in which two of the three nucleotides are overhang nucleotides and the third is a paired nucleotide following the overhang nucleotide. These three terminal nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and/or the 5' end of the antisense strand.

In one embodiment, a 2 nucleotide overhang is at the 3' end of the antisense strand and there are two phosphorothioate internucleotide bonds between the three terminal nucleotides, with two of the three nucleotides being overhang nucleotides and the third nucleotide being a paired nucleotide following the overhang nucleotide. Optionally, the RNAi agent may further have two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

In one embodiment, the RNAi agent has a mismatch(s) with a target within a duplex or combination thereof. A "mismatch" may be a non-canonical base pairing or a non-canonical nucleotide pairing. The mismatch may be in a raised region or a double region. A base pair can be ranked based on their property to promote dissociation or melting (e.g. based on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine pairs based on single pairs, although next neighbors or a similar analysis can also be used. ). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, for example, non-canonical or non-canonical pairings (as described elsewhere in this document) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings that contain a universal base are preferred over canonical pairings. A "universal base" is a base that exhibits the ability to replace any of the four normal bases (G, C, A, and U) without significantly destabilizing neighboring base pair interactions or impairing the expected functional biochemical utility of the modified oligonucleotide. Non-limiting examples of versatile bases include 2'-deoxyinosine (hypoxanthine deoxynucleotide) or derivatives thereof, nitroazole analogs, and hydrophobic aromatic bases without hydrogen bonds.

In one embodiment, the RNAi agent contains at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, independently selected from the group: A:U, G:U, I: C, and mismatched pairs, for example, non-canonical or non-canonical pairings or pairings that contain a universal base that promotes dissociation of the antisense strand at the 5' end of the duplex.

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

In another embodiment, the nucleotide at the 3' end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short deoxy-thymine nucleotide sequence, such as two dT nucleotides at the 3' end of the sense and/or antisense strand.

In one embodiment, the semantic strand sequence can be represented by formula (I):

and:

each of i and j is independently 0 or 1;

p and q are each independently 0-6;

each N _a independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, with each sequence containing at least two differently modified nucleotides;

each N _b independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

each of n _p and n _q independently represents an overhang nucleotide;

moreover, Nb and Y do not have the same modification; A

each of XXX, YYY and ZZZ independently represents one motif of three identical modifications of three consecutive nucleotides. Preferably YYY represents all 2'-F modified nucleotides.

In one embodiment, N _a and/or N _b contain alternating order modifications.

In one embodiment, the YYY motif is located at or near the semantic strand cleavage site. For example, when the RNAi agent has a 17-23 nucleotide long double region, the YYY motif may be at or near the cleavage site (eg: may be at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9 , 10, 11, 10, 11,12, or 11, 12, 13) of the sense strand, with the count starting from the 1st nucleotide from the 5'end;, or optionally counting from the ^1st paired nucleotide within the double region from 5 '-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the semantic thread can be represented by the following formulas:

When the sense strand is represented by formula (Ib), N _b represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a can independently represent an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N _b represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a may independently represent an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N _b independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N _b is 0, 1, 2, 3, 4, 5, or 6. Each N _a may independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

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

In other embodiments, i is 0 and j is 0, and the semantic thread can be represented by the formula:

When the sense strand is represented by formula (Ia), each N _a can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the sequence of an RNAi antisense strand can be represented by formula (II):

and:

k and l are each independently 0 or 1;

p' and q' are each independently 0-6;

each N _a ′ independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, with each sequence containing at least two differently modified nucleotides;

each N _b ′ independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

each N _p ' and n _q ' independently represent a nucleotide overhang;

moreover, N _b ' and Y' do not have the same modification;

A

X'X'X', Y'Y'Y' and Z'Z'Z' each independently represents one motif of three identical modifications of three consecutive nucleotides.

In one embodiment, Na' and/or Nb' contain alternating order modifications.

The Y′Y′Y′ motif is located at or near the antisense strand cleavage site. For example, when the RNAi agent has a 17-23 nucleotide long double region, the Y'Y'Y' motif can be at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the 1st nucleotide from the 5' end; or optionally counting from the 1st paired nucleotide within the double region from the 5' end. Preferably, the Y′Y′Y′ motif is in positions 11, 12, 13.

In one embodiment, the Y'Y'Y' motif is comprised of all 2'-OMe modified nucleotides.

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

Therefore, the antisense strand can be represented by the following formulas:

When the antisense strand is represented by formula (IIb), N _b ^' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a ' independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N _b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides . Each N _a ' independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N _b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a ' independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides. Preferably, N _b is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:

When the antisense strand is represented as formula (IIa), each N _a ' independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

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

Each nucleotide of the sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2' -fluorine. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluorine. Each X, Y, Z, X', Y' and Z', in particular, may be 2'-O-methyl modifications or 2'-fluorine modifications.

In one embodiment, the sense strand of the RNAi agent may contain the YYY motif found at positions 9, 10, and 11 of the strand when the double region is 21 nucleotides, counting from the 1st nucleotide from the 5' end, or optionally counting from 1 -th paired nucleotide inside the double region from the 5'-end; and Y displays modifications 2'-F. The semantic strand may additionally contain the XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represent the 2'-OMe modification or the 2'-F modification.

In one embodiment, the antisense strand may contain a Y′Y′Y′ motif located at positions 11, 12, 13 of the strand, and counting from the 1st nucleotide from the 5' end, or optionally counting from the 1st paired nucleotide inside the double region from the 5' end; and Y' represents 2'-O-methyl modifications. The antisense strand may further comprise the X'X'X' motif or Z'Z'Z' motifs as wing modifications at the opposite end of the duplex region; and X'X'X' and Z'Z'Z' each independently represents the 2'-OMe modification or the 2'-F modification.

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

Accordingly, RNAi tools for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14-30 nucleotides, the RNAi duplex represented by formula (III):

and:

i, j, k and l are each independently 0 or 1;

p, p', q and q' are each independently 0-6;

each N _a and N _a ^' independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, with each sequence containing at least two differently modified nucleotides;

each N _b and N _b ^' independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

and

each N _p ', n _p , n _q ' and n _q , each of which may or may not be present, independently represents an overhang nucleotide; A

XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represents one motif of three identical modifications of three consecutive nucleotides.

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

Exemplary combinations of sense strand and antisense strand to form an RNAi duplex include the formulas below:

When the RNAi agent is represented by formula (IIIa), each N _a independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N _b independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each N _a independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIc), each N _b , N _b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIId), each N _b , N _b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a , N _a ^' independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides. Each of N _a , N _a ', N _b and N _b ^' independently contains alternating order modifications.

When the RNAi agent is represented by formula (IIId), each N _b , N _b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N _a , N _a ^' independently represents an oligonucleotide sequence containing 2-20, 2-15 or 2-10 modified nucleotides. Each of N _a , N _a ', N _b and N _b ^' independently contains alternating order modifications.

When the RNAi agent is represented by formula (IIIe), each N _a and N _a ′ independently represents an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified, or combinations thereof, with each sequence containing at least two differently modified nucleotides.

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

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe), at least one of the Y nucleotides may base pair with one of the Y′ nucleotides. Alternatively, at least two Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z' nucleotides; or all three Z' nucleotides all form base pairs with the corresponding Z' nucleotides.

When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides may base pair with one of the X' nucleotides. Alternatively, at least two X nucleotides form base pairs with the corresponding X' nucleotides; or all three X' nucleotides all form base pairs with the corresponding X' nucleotides.

In one embodiment, the Y nucleotide modification is different from the Y' nucleotide modification, the Z nucleotide modification is different from the Z' nucleotide modification, and/or the X nucleotide modification is different from the X' nucleotide modification.

In one embodiment, when the RNAi agent is represented by formula (IIId), the N _a modifications are 2'-O-methyl or 2'-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N _a modifications are 2′-O-methyl or 2′-fluorine modifications and n _p ′ >0 and at least one n _p ′ is linked to an adjacent nucleotide phosphorothioate bond. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N _a modifications are modifications 2B in one embodiment when the RNAi agent is -O-methyl or 2B in one embodiment when the RNAi agent is -fluoro, n _p ′ >0, and at least one n _p ' is linked to an adjacent nucleotide by a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a monovalent, bivalent, or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (IIId), the N _a modifications are modifications 2B in one embodiment when the RNAi agent is -O-methyl or 2B in one embodiment when the RNAi agent is -fluoro, n _p ′ >0, and at least one n _p ′ is linked to an adjacent nucleotide by a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a monovalent, bivalent, or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the N _a modifications are modifications 2B in one embodiment when the RNAi agent is -O-methyl or 2B in one embodiment when the RNAi agent is -fluoro, n _p ′ >0, and at least one n _p ′ is linked to an adjacent nucleotide by a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a monovalent, bivalent, or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer also contains a ligand. Each of the duplexes can secrete the same gene or two different genes; or each of the duplexes may express the same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe), wherein the duplexes linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer also contains a ligand. Each of the duplexes can secrete the same gene or two different genes; or each of the duplexes may express the same gene at two different target sites.

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

Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO2007/091269, US Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520, all of which are hereby incorporated by reference herein.

An RNAi agent that contains conjugations of one or more carbohydrate moieties to an RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to the modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of the dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier, to which a carbohydrate ligand is attached. a ribonucleotide subunit in which the ribose subunit sugar has been replaced in this way is referred to herein as a ribose replacement modification subunit (RRMS). The cyclic carrier may be a carbocyclic ring system, ie all ring atoms are carbon atoms, or a heterocyclic ring system, ie one or more ring atoms may be a heteroatom, eg nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system or may contain two or more rings, such as fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers contain (i) at least one "core attachment point", preferably two "core attachment points", and (ii) at least one "bonding attachment point". "Point of attachment to the backbone" in the context of the present invention refers to a functional group, for example, a hydroxyl group, or in general, to a bond available for incorporation of a carrier into the backbone, and which is suitable for this, for example, to a phosphate or to a modified phosphate, for example , into the sulfur-containing backbone of ribonucleic acid. A "Tethering Point of Attachment" (TAP) in some embodiments refers to a compound ring atom of a cyclic carrier, such as a carbon atom or a heteroatom (other than the atom that provides the point of attachment to the backbone) that links the selected moiety. The moiety can be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. Optionally, the selected fragment is linked via an intermediate link to a cyclic carrier. Thus, the cyclic carrier will often contain a functional group, such as an amino group, or generally provide a bond that is suitable for incorporation into or bonding to a compound ring of another chemical entity, such as a ligand.

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

In some specific embodiments, the RNAi agent for use in the methods of the invention is AT3SC-001 (AD-57213 - Sense strand: 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:13) and Antisense strand: 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg -3' (SEQ ID NO:14) wherein a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G or U, Af, Cf, Gf or Uf are 2'-fluoro A , C, G or U, and s represents a phosphorothioate bond.

These agents may also contain a ligand.

Ligands

The double-stranded RNA (dsRNA) agents of the invention may optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, a ligand may be conjugated to a sense strand. In preferred embodiments, the ligand is conjugated to the 3' end of the sense strand.

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

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

In one embodiment, GalNAc or a derivative of GalNAc is GalNAc ₃ :

.

In some embodiments, a ligand, such as a GalNAc ligand, is attached to the 3' end of the RNAi agent. In one embodiment, the RNAi agent is conjugated to a ligand, such as a GalNAc ligand, as shown in the following scheme

wherein X is O or S. In one embodiment, X is O.

A large variety of structural units can be linked to the RNAi agents of the present invention. Preferred moieties are ligands that are linked, preferably covalently, either directly or indirectly through an intermediate bond.

In preferred embodiments, the implementation of the ligand changes the distribution, targeting or lifetime of the molecule in which it is included. In preferred embodiments, the ligand provides increased affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor, e.g., cell or organ compartment, tissue, organ, or body region, e.g. ligand. Ligands that provide increased affinity for a selected target are also referred to as targeting ligands.

Some ligands may have endosomolytic properties. Endosomolytic ligands promote lysis of the endosome and/or transport of the compositions of the invention or components thereof from the endosome into the cytoplasm of the cell. The endosomolytic ligand can be a polyanionic peptide or a peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the composition of the invention or its components from the endosome into the cytoplasm of the cell. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586) and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic component may contain a chemical group (eg, an amino acid) that will undergo a charge change or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

Ligands can improve transport, hybridization, and specificity properties, and can also improve nuclease resistance of the resulting natural or modified oligoribonucleotide or polymeric molecule containing any combination of the monomers described herein and/or natural or modified ribonucleotides.

Ligands in General may contain therapeutic modifiers, for example, to improve absorption; diagnostic compounds or reporter groups, for example, to control the distribution; crosslinking agents; and conferring nuclease resistance fragments. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimetics.

The ligands may include a naturally occurring substance such as a protein (eg, human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin); carbohydrate (eg dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The ligand may also be a recombinant or synthetic molecule such as a synthetic polymer, eg a synthetic polyamino acid, an oligonucleotide (eg an aptamer). Examples of polyamino acids include polyamino acid, which is polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic acid copolymer. anhydride, N-(2-hydroxypropyl)methacrylamide (HMPA) copolymer, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimeric polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine quaternary salt, or alpha-helical peptide.

The ligands may also contain targeting groups, eg, a cell or tissue targeting agent, eg, a lectin, glycoprotein, lipid, or protein, eg, an antibody, that binds to said cell type, such as a kidney cell. Target group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose , transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptidomimetic or aptamer.

Other examples of ligands include dyes, intercalators (eg, acridines), crosslinkers (eg, psoralen, mitomycin C), porphyrins (TPPC4, texafirin, sapphirin), polycyclic aromatic carbohydrates (eg, phenazine, dihydrophenazine), artificial endonucleases, or chelator (e.g. EDTA), lipophilic molecules, e.g. cholesterol, cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol , 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (eg PEG-40K), MPEG, [MPEG] ₂ , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (eg biotin), transport/absorption stimulants (eg aspirin, vitamin E, folic acid), synthetic ribonucleases (eg, imidazole, bizimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ tetraazamacrocycle complexes), dinitrophenyl, HRP, or AP.

Ligands can be proteins, eg glycoproteins or peptides, eg molecules having a specific affinity for a coligand, or antibodies, eg an antibody that binds to a specified cell type, eg a cancer cell, endothelial cell or bone cell. Ligands can also be hormones and hormone receptors. They can also be non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or aptamers. The ligand may be, for example, a lipopolysaccharide, a p38 MAP kinase activator, or an NF-κB activator.

The ligand can be a substance, such as a drug, that can increase the uptake of the mRNA agent into the cell, such as by disrupting the cellular cytoskeleton, such as disrupting cellular microtubules, microfilaments, and/or intermediate filaments. The drug may be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swingolide A, indanocin or myoservin.

The ligand can increase uptake of the oligonucleotide into the cell by, for example, activating an anti-inflammatory response. Exemplary ligands that will have such an effect include tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or interferon gamma.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, such as human serum albumin (HSA). The HSA binding ligand provides distribution of the conjugate to target tissues, such as non-renal target tissues of the body. For example, the target tissue may be the liver containing parenchymal liver cells. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to the target cell or cell membrane, and/or (c) it can be used to regulate serum protein binding, e.g. HSA .

A lipid-based ligand can be used to modulate, for example, control the binding of a conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and therefore less likely to be excreted from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid-based ligand binds HSA. Preferably, it binds HSA with sufficient affinity such that the conjugate will preferentially distribute to non-renal tissue. However, it is preferred that the affinity is not so strong that the binding of the HSA ligand cannot be reversible.

In another preferred embodiment, the lipid-based ligand binds HSA weakly or does not bind at all, such that the conjugate will preferably be distributed to the kidney. Instead of or in addition to the lipid-based ligand, other moieties that target kidney cells can also be used.

In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by a target cell, such as a proliferating cell. They are particularly useful in the treatment of disorders characterized by undesired cell proliferation, eg of a malignant or non-cancerous type, eg cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL), and high density lipoprotein (HDL).

In another aspect, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it may be modified, including a peptidyl mimetic, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an alpha helical agent which preferably has a lipophilic and lipophobic phase.

The ligand may be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. a peptide or peptidomimetic fragment may be about 5-50 amino acids in length, eg, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic may be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (eg, consisting primarily of Tyr, Trp, or Phe). The peptide fragment may be a dendrimeric peptide, a conformationally restricted peptide, or a cross-linked peptide. In another alternative, the peptide fragment may contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog (eg, the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) containing a hydrophobic MTS can also be targeted to the fragment. The peptide moiety can be a "delivering" peptide that can carry large polar molecules, including peptides, oligonucleotides and protein, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) were found to be able to function as delivery peptides. The peptide or peptidomimetic may be encoded by an arbitrary DNA sequence, for example, a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic linked to the mRNA agent via an incorporated monomeric unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or an RGD mimetic. The peptide fragment can vary in length from about 5 amino acids to about 40 amino acids. Peptide fragments may have a structural modification, for example, to increase stability or direction of conformational properties. You can use any structural modifications described below. The RGD peptide fragment can be used to target a tumor cell, for example an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res. 62:5139-43, 2002). An RGD peptide can facilitate targeting of an mRNA agent to tumors in a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of the mRNA agent to the kidney. The RGD peptide may be linear or cyclic and may be modified, such as glycosylated or methylated, to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an mRNA agent to a tumor cell expressing α _V β ₃ (Haubner et al., Jour. Nucl. Med. 42:326-336, 2001). You can use peptides that target markers that are rich in proliferating cells. For example, RGD containing peptides and peptidomimetic can target cancer cells, in particular, cells that have integrin. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D-amino acids, as well as synthetic RGD mimetics can be used. In addition to RGD, other moieties that target the integrin ligand can be used. In general, such ligands can be used to regulate cell proliferation and angiogenesis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or another cancer-causing gene, such as the cancer-causing gene described herein.

A "cell-penetrating peptide" is capable of entering a cell, for example a microbial cell such as a bacterial or fungal cell or a mammalian cell such as a human cell. The microbial cell-penetrating peptide can be, for example, an α-helical linear peptide (eg LL-37 or ceropin P1), a disulfide bond-containing peptide (eg α-defensin, β-defensin or bactenecin) or a peptide containing only one or two dominant amino acids (eg PR-39 or indolicidin). The cell-penetrating peptide may also contain a nuclear localization signal (NLS). For example, the cell-penetrating peptide may be a dual amphipathic peptide, such as MPG, which is derived from the HIV-1 gp41 fusion peptide and NLS domain of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724 , 2003).

In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, keratotoxins, S. clava peptides, haxin intestinal antimicrobial peptides (HFIAP), magainins, brevinins-2 , dermaseptins, melitins, pleirocidin, H ₂ A peptides, Xenopus peptides, esculentin-1 and kaerins. Preferably, a number of factors must be considered in order to maintain the stability of the integrity of the helix. For example, the maximum amount of helix-stabilizing residues (eg, leu, ala, or lys) should be used, and the minimum amount of helix-destabilizing residues (eg, proline or cyclic monomer units) should be used. A capping residue must be provided (eg, an exemplary N-capping residue is Gly) and/or a C-terminal amidation may be used to provide an additional H-bond to stabilize the helix. Stability can ensure the formation of salt bridges between residues with opposite charges separated by i ± 3 or i ± 4 positions. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include ligands that are naturally occurring or modified peptides, such as peptides D or L; α, β or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amides, ie a peptide whose moieties have been replaced by one or more urea, thiourea, carbamate or sulfonylurea moieties; or cyclic peptides.

A targeting ligand can be any ligand that is capable of targeting a particular receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters such as GalNAc cluster, mannose cluster, galactose cluster or aptamer. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. Ligands can also be based on a nucleic acid, such as an aptamer. The aptamer may be unmodified or have any combination of the modifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligos or polycations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

PK modulator means a pharmacokinetic modulator. PK modulators include lipophils, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, and the like. Illustrative PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. It is also known that oligonucleotides that contain a number of phosphorothioate linkages bind to whey protein, thus short oligonucleotides, such as oligonucleotides of approximately 5 bases, 10 bases, 15 bases or 20 bases containing many phosphorothioate bonds in the backbone.

In addition, aptamers that bind serum components (eg, serum proteins) also fall within the scope of the present invention as PK modulating ligands.

Other ligand conjugates fall within the scope of the invention described in US Patent Applications USSN: 10/916185, filed August 10, 2004; USSN: 10/946873, filed September 21, 2004; USSN: 10/833934, filed August 3, 2007; USSN: 11/115989 filed April 27, 2005 and USSN: 11/944227 filed November 21, 2007, which are incorporated by reference in their entirety for all purposes.

When there are two or more ligands, all ligands may have the same properties, all may have different properties, or some ligands may have the same properties while others may have different properties. For example, the ligand may have targeting properties, have endosomolytic activity, or have PK modulating properties. In a preferred embodiment, all ligands have different properties.

Ligands can be linked to oligonucleotides at various locations, such as at the 3' end, at the 5' end, and/or at the internal position. In preferred embodiments, the implementation of the ligand is attached to the oligonucleotides through an intermediate connection, for example, the media described in this document. The ligand or bound ligand may be on the monomer when the monomer is incorporated into the growing strand. In some embodiments, the implementation of the ligand can be included by connection with the monomer "predecessor" after the incorporation of the monomer "predecessor" in the growing thread. For example, a monomer having, for example, an amino-terminal bond (ie, no ligand bound), such as TAP-(CH ₂ ) _n NH ₂ , can be inserted into the growing oligonucleotide strand. In a subsequent operation, i.e. after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, for example a pentafluorophenyl ether or an aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the bond of the precursor monomer.

In another example, a monomer having a chemical group suitable for participating in a click chemistry reaction, such as an azide or alkyne end bond/linker, can be included. In a subsequent operation, ie after incorporation of the precursor monomer into the strand, a ligand having a complementary chemical group, such as an alkyne or an azide, can be attached to the precursor monomer by joining together an alkyne and an azide.

For double stranded oligonucleotides, ligands can be attached to one or both strands. In some embodiments, the double-stranded mRNA agent comprises a ligand conjugated to a sense strand. In other embodiments, the double-stranded mRNA agent comprises a ligand conjugated to an antisense strand.

In some embodiments, the ligand may be conjugated to nucleic bases, sugar moieties, or internucleoside bonds of nucleic acid molecules. Conjugation to purine nucleic bases or derivatives thereof can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, 2-, 6-, 7-, or 8-position purine nucleobases are attached to the conjugate moiety. Conjugation to pyrimidine nucleic bases or derivatives thereof can also occur at any position. In some embodiments, the pyrimidine nucleobase at the 2-, 5-, and 6-positions can be replaced with a conjugate moiety. Conjugation to nucleoside sugar moieties can occur at any carbon atom. Exemplary sugar moiety carbons that can be attached to the conjugate moiety include 2', 3', and 5' carbons. The 1' position can also be attached to the conjugate moiety, for example, in a nitrogenous base-free residue. Internucleoside bonds can also carry fragments of conjugates. For phosphorus-containing bonds (eg, phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to the O, N, or S atom bonded to the phosphorus atom. For amino- or amide-containing internucleoside bonds (eg, PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide, or to an adjacent carbon atom.

Any suitable ligand in the RNA interference region can be used, although typically the ligand is a carbohydrate, eg, a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide.

Linkers that connect the ligand to the nucleic acid include the linkers discussed above. For example, the ligand may be one or more derivatives of GalNAc (N-acetylglucosamine) attached through a monovalent, bivalent or trivalent branched linker.

In one embodiment, the dsRNA according to the invention is conjugated to bivalent and trivalent branched linkers containing the structures shown in any of formulas (IV) - (VII):

, , or ;

and:

q ^2A , q ^2B , q ^3A , q ^3B , q4 ^A , q ^4B , q ^5A , q ^5B and q ^5C are presented independently for each occurrence of 0-20, and the repeating unit may be the same or different;

^P2A , ^{P2B , P3A} , ^P3B , ^P4A , P4B ^{, P5A} , ^P5B , ^P5C , T2A, T2B, ^{T3A , T3B , T4A} , ^T4B , ^T4A , ^T5B , T ^5C each independently for each occurrence is CO, NH, O, S, OC(O), NHC(O), CH ₂ , CH ₂ NH or CH ₂ O;

Q ^2A , Q ^2B , Q ^3A , Q ^3B , Q ^4A , Q ^4B , Q ^5A , Q ^5B , Q ^5C independently for each occurrence are alkylene, substituted alkylene, and one or more methylenes may be interrupted or terminated by one or more from O, S, S(O), SO ₂ , N(R ^N ), C(R')=C(R''), C≡C or C(O);

R ^2A , R ^2B , R ^3A , R 3B , R ^4A , R ^4B , R ^5A , R ^5B , ^{R 5C each} independently for each occurrence represents NH, O, S, CH ₂ , C(O)O, C (O)NH, NHCH(R ^a )C(O), -C(O)-CH(R ^a )-NH-, CO, CH=NO, , , , , or heterocyclyl;

L ^2A , L ^2B , L ^3A , L ^3B , L ^4A , L ^4B , L ^5A , L ^5B and L ^5C represent the ligand; that is, each independently for each occurrence is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; And

R ^a represents H or an amino acid side chain.

Trivalent GalNAc conjugating derivatives are particularly useful for use with RNAi agents for inhibiting target gene expression, for example according to formula (VII):

,

wherein L ^5A , L ^5B and L ^5C are a monosaccharide such as a GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups linking GalNAc derivatives include, but are not limited to, the following compounds:

, ,

,

,

, or

.

Examples of US patents that teach the preparation of RNA conjugates include, but are not limited to, US Pat. Nos. 4,828,979; 4948882; 5218105; 5525465; 5541313; 5545730; 5552538; 5578717 5580731; 5591584; 5109124; 5118802; 5138045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5371241 5391723; 5416203 5451463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481; 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941; 6294664; 6320017; 6576752; 6783931; 6900297; 7037646; 8106022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary to uniformly modify all positions of a given compound, and in fact more than one of the aforementioned modifications may be included in a single compound or even a single nucleoside within an mRNA. The present invention also includes mRNA compounds that are chimeric compounds.

"Chimeric" mRNA compounds or "chimeras" in the context of the present invention are mRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each consisting of at least one monomeric unit, i.e. a nucleotide in the case of a dsRNA compound. These mRNAs typically contain at least one region in which the RNA is modified in such a way as to give the mRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. The additional mRNA region can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Therefore, activation of RNase H results in cleavage of the target RNA, thereby greatly improving the efficiency of mRNA inhibition of gene expression. As a result, when using chimeric dsRNAs, comparable results can often be obtained with shorter mRNAs compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the target RNA can usually be detected by gel electrophoresis and, if necessary, by related nucleic acid hybridization techniques known in the art.

In some cases, the RNA in the mRNA can be modified with a non-ligand moiety. a number of non-ligand molecules have been conjugated to the mRNA in order to improve the activity, cellular distribution, or cellular uptake of the mRNA, and methods for performing such conjugations 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), thioether, e.g. hexyl-S-tritylthiol (Manoharan et al. , Ann. N. Y. Acad. Sci. 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20: 533), an aliphatic chain, e.g. dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10:111; Kabanov et al., FEBS Lett. 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g. 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), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantane acetic acid (Manoharan et al. , Tetrahedron Lett. 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229) or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277:923). Examples of US patents that teach the preparation of such RNA conjugates are listed above. Conventional conjugation protocols involve the synthesis of RNAs carrying an amino linker at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using suitable binding or activating reagents. the conjugation reaction can be carried out either with the RNA still bound to the solid support or after the RNA has been digested in the solution phase. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

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

VI. Delivery of mRNA according to the invention

Delivery of the mRNA of the invention into a cell, eg into a cell of a patient, such as a human patient (eg, a patient in need, such as a patient with a bleeding disorder), can be achieved in a number of different ways. For example, delivery can be accomplished by contacting the cell with an mRNA of the invention, either in vitro or in vivo. In vivo delivery can be accomplished by administering a composition containing an mRNA, such as dsRNA, directly to a patient. Alternatively, In vivo delivery can be accomplished indirectly by introducing one or more vectors that encode and direct mRNA expression. These alternatives are discussed further below.

In general, any method for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the mRNA of the invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5): 139-144 and WO94/02595, which are incorporated herein by reference in their entirety). For In vivo delivery, factors to be considered for delivery of an mRNA molecule include, for example, the biological stability of the delivered molecule, the avoidance of non-specific effects, and the accumulation of the delivered molecule in the target tissue. The non-specific effects of the mRNA can be minimized by topical administration, for example by direct injection or implantation into tissue, or by topical administration of the drug. Topical administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may otherwise be damaged by the agent or that may destroy the agent, and ensures that a lower total dose of the mRNA molecule is administered. Several studies have shown successful knockdown of gene products when mRNA is administered topically. For example, it has been shown that intraocular delivery of VEGF dsRNA, both by injection into the vitreous body of the cynomolgus macaque (Tolentino, MJ. et al (2004) Retina 24:132-138) and by subretinal injection in mice (Reich, SJ. et al (2003) Mol Vis 9:210-216) prevents neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA into mice reduces tumor volume (Pille, J. et al (2005) Mol. Ther, 11:267-274) and can prolong the lifespan of tumor-bearing mice (Kim, WJ. et al (2006) Mol. Ther. 14:343-350; Li, S. et al (2007) Mol. Ther. 15:515-523). RNA interference has also been successful when locally delivered to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al (2005) Gene Ther. 12:59-66; Makimura, H et al (2002) BMC Neurosci 3:18 Shishkina GT et al (2004) Neuroscience 129:521-528 Thakker ER et al (2004) Proc Natl Acad Sci U.S.A. 101:17270-17275; Akaneya, Y. et al (2005) J. Neurophysiol. 93:594-602) and into the lungs by intranasal administration (Howard, KA. et al (2006) Mol. Ther. 14:476-484 Zhang, X. et al (2004) J. Biol Chem 279:10677-10684 Bitko, V. et al (2005) Nat Med 11:50-55). To administer the mRNA systemically to treat a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or pharmaceutical carrier can also target the mRNA composition to the target tissue and avoid unwanted off-target effects. mRNA molecules can be modified by chemical conjugation with lipophilic groups such as cholesterol to improve cellular uptake and prevent degradation. For example, mice with mRNA were systemically injected with an anti-ApoB, lipophilic cholesterol-conjugated moiety, resulting in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J. et al (2004) Nature 432:173-178). mRNA conjugation to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, mRNA can be delivered using drug delivery systems such as a nanoparticle, dendrimer, polymer, liposomes, or cationic delivery system. Positively charged cationic delivery systems facilitate binding of the (negatively charged) mRNA molecule and also improve interactions at the negatively charged cell membrane to ensure efficient uptake of the mRNA into the cell. Cationic lipids, dendrimers, or polymers can either be coupled to mRNA or induced to form a vesicle or micelle (see, for example, Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116), which concludes mRNA into itself. The formation of vesicles or micelles also prevents mRNA degradation upon systemic administration. Methods for preparing and introducing cationic-mRNA complexes are within the skill of the art (see e.g. Sorensen, DR. et al (2003) J. Mol. Biol 327:761-766; Verma, UN. et al (2003 ) Clin Cancer Res 9:1291-1300; Arnold, AS et al (2007) J. Hypertens 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems used for systemic delivery of mRNA include DOTAP (Sorensen, DR. et al (2003), supra; Verma, UN. et al (2003), supra), Oligofectamine, "solid lipid particles nucleic acids" (Zimmermann, TS., et al (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al ( 2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME. et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659) , Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487) and polyamidamines (Tomalia, DA. et al (2007) Biochem. Soc. Trans. 35:61- 67; Yoo, H. et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, the mRNA is complexed with a cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of mRNAs and cyclodextrins can be found in US Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

A. Vector encoded by mRNA according to the invention

mRNA targeting the Serpinc1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A. et al., PCT International Publication No. WO 00/22113, Conrad, PCT International Publication No. WO 00/22114 and Conrad, US Patent No. 6054299). Expression can be short term (on the order of hours to weeks) or long term (on the order of weeks to months or longer) depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a round plasmid, or a viral vector, which can be an integrated or non-integrated vector. The transgene can also be engineered to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

A single strand or strands of mRNA can be transcribed from a promoter into an expression vector. When two separate strands need to be expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced (eg, by transfection or infection) into the target cell. Alternatively, each individual strand of dsRNA can be transcribed with promoters that are both on the same expression plasmid. In one embodiment, the dsRNA is expressed as inverted repeat polynucleotides linked by a linker polynucleotide sequence such that the dsRNA has a stem-loop structure.

mRNA expression vectors are typically DNA plasmids or viral vectors. To obtain recombinant mRNA expression constructs as described herein, expression vectors compatible with eukaryotic cells, preferably vectors compatible with vertebrate cells, can be used. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided that contain convenient restriction sites for inserting the desired nucleic acid segment. Delivery of mRNA-expressing vectors can be systemic, for example, by intravenous or intramuscular administration, by introduction into target cells explanted from a patient, followed by repeated administration to the patient, or by any other means that ensures introduction into the desired target cell.

mRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (eg, oligofectamine) or non-cationic lipid-based carriers (eg, Transit-TKO™). The invention also contemplates a plurality of lipid transfections for mRNA-mediated knockdowns targeting different regions of the target RNA over a period of a week or more. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled by a reporter such as a fluorescent marker such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be achieved using markers that confer resistance to specific environmental factors (e.g. antibiotics and drugs) to the transfected cells, such as resistance to hygromycin B.

Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to (a) adenovirus vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated viral vectors; (d) herpes simplex vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors such as orthopox, eg vaccinia or avipoxvirus vectors, eg canary poxvirus or avian poxvirus; and (j) based on helper dependent or eviscerated adenovirus. Viruses with defective replication may also be useful. Different vectors will or will not be included in the genome of the cell. If desired, the constructs may contain viral sequences for transfection. Alternatively, the construct can be inserted into vectors capable of episomal replication, such as the EPV and EBV vectors. Recombinant mRNA expression constructs typically require regulatory elements, such as promoters, enhancers, etc., to allow mRNA expression in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for mRNA delivery will contain regulatory elements (promoter, enhancer, etc.) sufficient for mRNA expression in the desired cell or target tissue. regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

mRNA expression can be precisely controlled, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, such as circulating glucose levels or hormones (DOCHerty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems suitable for regulation of dsRNA expression in cells or mammals include, for example, regulation with ecdysone, with estrogen, progesterone, tetracycline, dimerization chemical inducers, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). One skilled in the art will be able to select an appropriate regulatory/promoter sequence based on the intended use of the mRNA transgene.

You can use viral vectors that contain nucleic acid sequences encoding mRNA. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into the host cell's DNA. The nucleic acid sequences encoding the mRNA are cloned into one or more vectors that facilitate delivery of the nucleic acid to a patient. More information on retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant. to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al. Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV-based vectors described in US Pat. Nos. 6,143,520; 5665557; and 5981276, which are incorporated herein by reference.

Also contemplated is the use of adenoviruses to deliver mRNA according to the invention. Adenoviruses are particularly attractive means, for example, for delivering genes to the epithelium of the respiratory tract. Adenoviruses naturally infect the epithelium of the respiratory tract and cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscles. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrate the use of adenoviral vectors to transfer genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an mRNA of the invention, a method for constructing a recombinant AV vector, and a method for delivering the vector to target cells are described in Xia H et al., (2002), Nat. Biotech. 20:1006-1010.

Adeno-associated virus (AAV) vectors can also be used to deliver mRNA according to the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); US Pat. No. 5,436,146). In one embodiment, the mRNA can be expressed as two separate complementary single stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoter or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing a recombinant AV vector, and methods for delivering the vectors to target cells are described in Samulski R et al., (1987), J. Virol. 61: 3096-3101; Fisher K J et al., (1996), J. Virol, 70: 520-532; Samulski R et al., (1989), J. Virol. 63: 3822-3826; in US patent No. 5252479; in US patent No. 5139941; in International Patent Application No. WO 94/13788; and in International Patent Application No. WO 93/24641, the entire disclosure of which is incorporated herein by reference.

Another viral vector suitable for delivering mRNA according to the invention is a poxvirus such as vaccinia virus, for example attenuated vaccinia, for example modified Ankara virus (MVA) or NYVAC, avipoxvirus such as avian poxvirus or canary poxvirus.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by replacing different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins of vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. You can get AAV vectors to target different cells by constructing vectors to express different serotypes of capsid proteins; see, for example, Rabinowitz J E et al., (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

The vector pharmaceutical preparation may contain the vector in a suitable diluent or may contain a slow release matrix into which the gene delivery vehicle is embedded. Alternatively, when the entire gene delivery vector can be obtained intact from recombinant cells, such as retroviral vectors, the pharmaceutical preparation may contain one or more cells that produce the gene delivery system.

V. Pharmaceutical compositions according to the invention

The present invention also provides pharmaceutical compositions and formulations that contain the mRNA of the invention. In one embodiment provided herein, pharmaceutical compositions are provided comprising an mRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing mRNA are useful in the treatment of a disease or disorder associated with the expression or activity of the Serpinc1 gene, such as a disease associated with Serpinc1. Such pharmaceutical compositions are formulated based on the mode of delivery. One example are compositions that are formulated for systemic administration via parenteral delivery, for example via subcutaneous (SC) or intravenous (IV) delivery. Another example are compositions that are formulated for direct delivery to the brain parenchyma, eg by infusion into the brain, eg by continuous infusion through a pumping system. Pharmaceutical compositions according to the invention can be administered in dosages sufficient to inhibit the expression of the Serpinc1 gene.

In some embodiments of the invention, for example, when a pharmaceutical composition contains a double-stranded RNAi agent containing one or more motifs of three identical modifications of three consecutive nucleotides, including one such motif at or near the cleavage site of the agent, six phosphorothioate bonds, and a GalNAc ligand, such a composition is administered at a dose of from 0.200 to about 1.825 mg/kg, from 0.200 to about 1.800 mg/kg, from about 0.200 to about 1.700 mg/kg, from about 0.200 to about 1.600 mg/kg, from about 0.200 to about 1.500 mg/kg, from about 0.200 to about 1.400 mg/kg, from about 0.200 to about 1.400 mg/kg, from about 0.200 to about 1.200 mg/kg, from about 0.200 to about 1.100 mg/kg, from about 0.200 to about 1.000 mg/kg, from about 0.200 to about 0.900 mg/kg, from about 0.200 to about 0.800 mg/kg, from about 0.200 to about 0.700 mg/kg, from about 0.200 to about 0.600 mg/kg, from about 0.200 to about 0.500 mg/kg, from about 0.200 to about 0.400 mg/kg, from about 0.225 to about 1.825 mg/kg, from about 0.225 to about 1.800 mg/kg, from about 0.225 to about 1.700 mg/kg, from about 0.225 to about 1.600 mg/kg, from about 0.225 to about 1.500 mg/kg, from about 0.225 to about 1.400 mg/kg, from about 0.225 to about 1.400 mg/kg, from about 0.225 to about 1.200 mg/kg, from about 0.225 to about 1.100 mg/kg, from about 0.225 to about 1.000 mg/kg, from about 0.225 to about 0.900 mg/kg, from about 0.225 to about 0.800 mg/kg, from about 0.225 to about 0.700 mg/kg, from about 0.225 to about 0.600 mg/kg, from about 0.225 to about 0.500 mg/kg, from about 0.225 to about 0.400 mg/kg, from about 0.250 to about 1.825 mg/kg, from about 0.250 to about 1.800 mg/kg, from about 0.250 to about 1.700 mg/kg, from about 0.250 to about 1.600 mg/kg, from about 0.250 to about 1.500 mg/kg, from about 0.250 to about 1.400 mg/kg, from about 0.250 to about 1.400 mg/kg, from about 0.250 to about 1.200 mg/kg, from about 0.250 to about 1.100 mg/kg, from about 0.250 to about 1.000 mg/kg, from about 0.250 to about 0.900 mg/kg, from about 0.250 to about 0.800 mg/kg, from about 0.250 to about 0.700 mg/kg, from about 0.250 to about 0.600 mg/kg, from about 0.250 to about 0.500 mg/kg, from about 0.250 to about 0.400 mg/kg, from about 0.425 to about 1.825 mg/kg, from about 0.425 to about 1.800 mg/kg, from about 0.425 to about 1.700 mg/kg, from about 0.425 to about 1.600 mg/kg, from about 0.425 to about 1.500 mg/kg, from about 0.425 to about 1.400 mg/kg, from about 0.425 to about 1.400 mg/kg, from about 0.425 to about 1.200 mg/kg, from about 0.425 to about 1.100 mg/kg, from about 0.425 to about 1.000 mg/kg, from about 0.425 to about 0.900 mg/kg, from about 0.425 to about 0.800 mg/kg, from about 0.425 to about 0.700 mg/kg, from about 0.425 to about 0.600 mg/kg, from about 0.425 to about 0.500 mg/kg, from about 0.450 to about 1.825 mg/kg, from about 0.450 to about 1.800 mg/kg, from about 0.450 to about 1.700 mg/kg, from about 0.450 to about 1.600 mg/kg, from about 0.450 to about 1.500 mg/kg, from about 0.450 to about 1.400 mg/kg, from about 0.450 to about 1.400 mg/kg, from about 0.450 to about 1.200 mg/kg, from about 0.450 to about 1.100 mg/kg, from about 0.450 to about 1.000 mg/kg, from about 0.450 to about 0.900 mg/kg, from about 0.450 to about 0.800 mg/kg, from about 0.450 to about 0.700 mg/kg, from about 0.450 to about 0.600 mg/kg, from about 0.450 to about 0.500 mg/kg, from about 0.475 to about 1.825 mg/kg, from about 0.475 to about 1.800 mg/kg, from about 0.475 to about 1.700 mg/kg, from about 0.475 to about 1.600 mg/kg, from about 0.475 to about 1.500 mg/kg, from about 0.475 to about 1.400 mg/kg, from about 0.475 to about 1.400 mg/kg, from about 0.475 to about 1.200 mg/kg, from about 0.475 to about 1.100 mg/kg, from about 0.475 to about 1.000 mg/kg, from about 0.475 to about 0.900 mg/kg, from about 0.475 to about 0.800 mg/kg, from about 0.475 to about 0.700 mg/kg, from about 0.475 to about 0.600 mg/kg, from about 0.475 to about 0.500 mg/kg, from about 0.875 to about 1.825 mg/kg, from about 0.875 to about 1.800 mg/kg, from about 0.875 to about 1.700 mg/kg, from about 0.875 to about 1.600 mg/kg, from about 0.875 to about 1.500 mg/kg, from about 0.875 to about 1.400 mg/kg, from about 0.875 to about 1.400 mg/kg, from about 0.875 to about 1.200 mg/kg, from about 0.875 to about 1.100 mg/kg, from about 0.875 to about 1.000 mg/kg, from about 0.875 to about 0.900 mg/kg, from about 0.900 to about 1.825 mg/kg, from about 0.900 to about 1.800 mg/kg, from about 0.900 to about 1.700 mg/kg, from about 0.900 to about 1.600 mg/kg, from about 0.900 to about 1.500 mg/kg, from about 0.900 to about 1.400 mg/kg, from about 0.900 to about 1.400 mg/kg, from about 0.900 to about 1.200 mg/kg, from about 0.900 to about 1.100 mg/kg, from about 0.900 to about 1.000 mg/kg, from about 0.925 to about 1.825 mg/kg, from about 0.925 to about 1.800 mg/kg, from about 0.925 to about 1.700 mg/kg, from about 0.925 to about 1.600 mg/kg, from about 0.925 to about 1.500 mg/kg, from about 0.925 to about 1.400 mg/kg, about 0.925 to about 1.400 mg/kg, about 0.925 to about 1.200 mg/kg, about 0.925 to about 1.100 mg/kg, or about 0.925 to about 1.000 mg/kg. Values and ranges intermediate to the above stated values are also intended to form part of the present invention, for example, an RNAi agent may be administered to a patient at a dose of about 0.015 mg/kg to about 0.45 mg/kg.

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

In some embodiments of the invention, for example, when the double-stranded RNAi agent contains a sense strand and an antisense strand, and the antisense strand contains a region of complementarity to an mRNA encoding Serpinc1 that contains at least 15 adjacent nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 15), wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end, such an agent in a pharmaceutical composition is administered at a dose of about 0.200 to about 1.825 mg/kg, 0.200 to about 1.800 mg/kg, about 0.200 to about 1.700 mg/kg, about 0.200 to about 1.600 mg/kg, about 0.200 to about 1.500 mg/kg, from about 0.200 to about 1.400 mg/kg, from about 0.200 to about 1.400 mg/kg, from about 0.200 to about 1.200 mg/kg, from about 0.200 to about 1.100 mg/kg, from about 0.200 to about 1,000 mg/kg, from about 0.200 to about 0.900 mg/kg, from about 0.200 to about 0.800 mg/kg, from about 0.200 to about 0.700 mg/kg, from about 0.200 to about 0.600 mg/kg, from about 0.200 to about 0.500 mg/kg, from about 0.200 to about 0.400 mg/kg, from about 0.225 to about 1.825 mg/kg, from about 0.225 to about 1.800 mg/kg, from about 0.225 to about 1.700 mg/kg, from about 0.225 to about 1.600 mg/kg, from about 0.225 to about 1.500 mg/kg, from about 0.225 to about 1.400 mg/kg, from about 0.225 to about 1.400 mg/kg, from about 0.225 to about 1.200 mg/kg, from about 0.225 to about 1.100 mg/kg, from about 0.225 to about 1.000 mg/kg, from about 0.225 to about 0.900 mg/kg, from about 0.225 to about 0.800 mg/kg, from about 0.225 to about 0.700 mg/kg, from about 0.225 to about 0.600 mg/kg, from about 0.225 to about 0.500 mg/kg, from about 0.225 to about 0.400 mg/kg, from about 0.250 to about 1.825 mg/kg, from about 0.250 to about 1.800 mg/kg, from about 0.250 to about 1.700 mg/kg, from about 0.250 to about 1.600 mg/kg, from about 0.250 to about 1.500 mg/kg, from about 0.250 to about 1.400 mg/kg, from about 0.250 to about 1.400 mg/kg, from about 0.250 to about 1.200 mg/kg, from about 0.250 to about 1.100 mg/kg, from about 0.250 to about 1.000 mg/kg, from about 0.250 to about 0.900 mg/kg, from about 0.250 to about 0.800 mg/kg, from about 0.250 to about 0.700 mg/kg, from about 0.250 to about 0.600 mg/kg, from about 0.250 to about 0.500 mg/kg, from about 0.250 to about 0.400 mg/kg, from about 0.425 to about 1.825 mg/kg, from about 0.425 to about 1.800 mg/kg, from about 0.425 to about 1.700 mg/kg, from about 0.425 to about 1.600 mg/kg, from about 0.425 to about 1.500 mg/kg, from about 0.425 to about 1.400 mg/kg, from about 0.425 to about 1.400 mg/kg, from about 0.425 to about 1.200 mg/kg, from about 0.425 to about 1.100 mg/kg, from about 0.425 to about 1.000 mg/kg, from about 0.425 to about 0.900 mg/kg, from about 0.425 to about 0.800 mg/kg, from about 0.425 to about 0.700 mg/kg, from about 0.425 to about 0.600 mg/kg, from about 0.425 to about 0.500 mg/kg, from about 0.450 to about 1.825 mg/kg, from about 0.450 to about 1.800 mg/kg, from about 0.450 to about 1.700 mg/kg, from about 0.450 to about 1.600 mg/kg, from about 0.450 to about 1.500 mg/kg, from about 0.450 to about 1.400 mg/kg, from about 0.450 to about 1.400 mg/kg, from about 0.450 to about 1.200 mg/kg, from about 0.450 to about 1.100 mg/kg, from about 0.450 to about 1.000 mg/kg, from about 0.450 to about 0.900 mg/kg, from about 0.450 to about 0.800 mg/kg, from about 0.450 to about 0.700 mg/kg, from about 0.450 to about 0.600 mg/kg, from about 0.450 to about 0.500 mg/kg, from about 0.475 to about 1.825 mg/kg, from about 0.475 to about 1.800 mg/kg, from about 0.475 to about 1.700 mg/kg, from about 0.475 to about 1.600 mg/kg, from about 0.475 to about 1.500 mg/kg, from about 0.475 to about 1.400 mg/kg, from about 0.475 to about 1.400 mg/kg, from about 0.475 to about 1.200 mg/kg, from about 0.475 to about 1.100 mg/kg, from about 0.475 to about 1.000 mg/kg, from about 0.475 to about 0.900 mg/kg, from about 0.475 to about 0.800 mg/kg, from about 0.475 to about 0.700 mg/kg, from about 0.475 to about 0.600 mg/kg, from about 0.475 to about 0.500 mg/kg, from about 0.875 to about 1.825 mg/kg, from about 0.875 to about 1.800 mg/kg, from about 0.875 to about 1.700 mg/kg, from about 0.875 to about 1.600 mg/kg, from about 0.875 to about 1.500 mg/kg, from about 0.875 to about 1.400 mg/kg, from about 0.875 to about 1.400 mg/kg, from about 0.875 to about 1.200 mg/kg, from about 0.875 to about 1.100 mg/kg, from about 0.875 to about 1.000 mg/kg, from about 0.875 to about 0.900 mg/kg, from about 0.900 to about 1.825 mg/kg, from about 0.900 to about 1.800 mg/kg, from about 0.900 to about 1.700 mg/kg, from about 0.900 to about 1.600 mg/kg, from about 0.900 to about 1.500 mg/kg, from about 0.900 to about 1.400 mg/kg, from about 0.900 to about 1.400 mg/kg, from about 0.900 to about 1.200 mg/kg, from about 0.900 to about 1.100 mg/kg, from about 0.900 to about 1.000 mg/kg, from about 0.925 to about 1.825 mg/kg, from about 0.925 to about 1.800 mg/kg, from about 0.925 to about 1.700 mg/kg, from about 0.925 to about 1.600 mg/kg, from about 0.925 to about 1.500 mg/kg, from about 0.925 up to about 1.400 mg/kg, from about 0.925 to about 1.400 mg/kg, from about 0.925 to about 1.200 mg/kg, from about 0.925 to about 1.100 mg/kg, or from about 0.925 to about 1.000 mg/kg. Values and ranges intermediate to the above stated values are also intended to form part of the present invention, for example, an RNAi agent may be administered to a patient at a dose of about 0.015 mg/kg to about 0.45 mg/kg.

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

In some embodiments, a pharmaceutical composition containing an mRNA agent is administered to a patient in a fixed dose. "fixed dose" (eg, dose in mg) means that a single dose of mRNA agent is used for all patients, regardless of any specific patient-related factors such as weight. In one particular embodiment, the fixed dose of an mRNA agent of the invention is based on a given weight or age.

In some embodiments, a pharmaceutical composition containing an mRNA agent is administered at a fixed dose of about 25 mg to about 100 mg, such as about 25 mg to about 95 mg, about 25 mg to about 90 mg, about 25 mg to about 85 mg, about 25 mg to about 80 mg, about 25 mg to about 75 mg, about 25 mg to about 70 mg, about 25 mg to about 65 mg, about 25 mg to about 60 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, about 50 mg to about 95 mg, about 50 mg to about 90 mg, about 50 mg to about 85 mg, about 50 mg to about 80 mg, about 30 mg to about 100 mg, about 30 mg to about 90 mg, about 30 mg to about 80 mg, about 40 mg to about 100 mg, about 40 mg to about 90 mg, about 40 mg to about 80 mg, about 60 mg to about 100 mg, about 60 mg to about 90 mg, about 25 mg to about 55 mg, about 30 mg to about 95 mg, about 30 mg to about 85 mg , about 30 mg to about 75 mg, about 30 mg to about 65 mg, about 30 mg to about 55 mg, about 40 mg to about 95 mg, about 40 mg to about 85 mg, about 40 mg to about 75 mg, from about 40 mg to about 65 mg, from about 40 mg to about 55 mg, or from about 45 mg to about 95 mg.

In some embodiments, a pharmaceutical composition containing an mRNA agent is administered in 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.

The mRNA-containing pharmaceutical composition may be administered to a patient about once a month, about once every five weeks, about once every six weeks, about once every 2 months, or once a quarter.

The pharmaceutical composition containing the mRNA agent may be administered to a patient in one or more doses. In some embodiments, pharmaceutical compositions containing a double-stranded mRNA agent may be administered to a patient as a monthly dose of about 0.200 mg/kg to about 0.250 mg/kg, a monthly dose of about 0.425 mg/kg to about 0.475 mg/kg, a monthly dose of about 0.875 mg/kg to about 0.925 mg/kg, or as a monthly dose of about 1.775 mg/kg to about 1.825 mg/kg. In some embodiments, pharmaceutical compositions containing a double-stranded mRNA agent may be administered to a patient in a fixed dose of about 25 mg to about 100 mg, such as about 25 mg, about 50 mg, about 80 mg, or about 100 mg.

The pharmaceutical composition can be administered by intravenous infusion over a period of time, for example, over a period of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24 or approximately 25 minutes. The administration may be repeated, eg on a regular basis, eg once a week, biweekly (ie every two weeks), monthly, every two months, every three months, every four months or longer. After the initial course of treatment, treatment can be carried out with less frequency. For example, after weekly or biweekly administration for three months, administration may be repeated once a month, for six months or a year, or longer.

The pharmaceutical composition may be administered once a day, or the mRNA may be administered in two, three or more booster doses at suitable intervals throughout the day, or even using continuous infusion or controlled release formulation delivery. In this case, the mRNA contained in each booster dose should be correspondingly less to achieve the total daily dose. The dosage unit can also be formulated for delivery over several days, for example using a conventional sustained release formulation that provides sustained release of the mRNA over a period of several days. sustained release formulations are well known in the art and are particularly useful for delivering agents to a specific site, such as may be used with the agents of the present invention. In this embodiment, the dose unit contains the corresponding amount of the daily dose.

In other embodiments, the implementation of a single dose of pharmaceutical compositions may be long, so that subsequent doses are administered at intervals of no more than 1, 2, 3, 4, 5, 6, 7 or 8 weeks. In some embodiments of the invention, a single dose of pharmaceutical compositions according to the invention is administered once a month.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a patient, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the patient, and other underlying medical conditions. In addition, treating a patient with a therapeutically effective amount of the composition may include a single treatment or a series of treatments. Evaluations of the effectiveness of dosages and in vivo half-lives for human mRNAs covered by the invention can be made using conventional methods or based on in vivo testing using a suitable animal model, as described elsewhere in this document.

Advances in mouse genetics have made it possible to create a number of mouse models for studying various human diseases, such as bleeding disorders, that would benefit from downregulation of Serpinc1. Such models can be used to test iRNA in vivo as well as to determine a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, Hemophilia A mouse models and Hemophilia B mouse models, for example, coagulation factor gene knockout mice, such as those described in Bolliger et al. (2010) Thromb Haemost 103: 1233-1238, Bi L, et al. (1995) Nat Genet 10: 119-21, Lin et al. (1997) Blood 90: 3962-6, Kundu et al. (1998) Blood 92: 168-74, Wang et al. (1997) Proc Natl Acad Sci U S A 94: 11563-6 and Jin et al. (2004) Blood 104:1733.

Pharmaceutical compositions of the present invention may be administered in a variety of ways depending on whether topical or systemic treatment is desired and the area to be treated. Administration can be topical (eg via a transdermal patch), pulmonary, eg via inhalation or insufflation of powders or aerosols, including via a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, by means of an implanted device; or intracranial, eg intraparenchymal, intrathecal or intraventricular administration.

mRNA can be delivered in such a way as to target a specific tissue, such as the liver (eg, liver hepatocytes).

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional aqueous, powder or oil based pharmaceutical carriers, thickeners and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be helpful. Suitable topical formulations include those in which the mRNAs of the invention are in admixture with a topical delivery vehicle such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. substances. Formulations for topical use are detailed in US Pat. No. 6,747,014, which is incorporated herein by reference.

A. Additional preforms

i. emulsions

Compositions according to the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another as droplets typically over 0.1 µm in diameter (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC. , 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often two-phase systems containing two immiscible liquid phases thoroughly mixed and dispersed with each other. In general, emulsions can be of either water-in-oil (w/o) or oil-in-water (o/w) types. When the aqueous phase is finely separated and dispersed as fine droplets into the bulk oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely separated and dispersed as fine droplets into the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. In addition to the dispersed phases, the emulsions may contain additional components and the active drug, which may be present as a solution either in the aqueous phase or in the oil phase, or as a separate phase. Also, if desired, pharmaceutical excipients such as emulsifiers, stabilizers, colorants and antioxidants may be present in the emulsions. Pharmaceutical emulsions can also be multiple emulsions that consist of more than two phases, such as in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide a distinct advantage that simple double emulsions do not. Multiple emulsions in which the individual oil droplets of the o/w emulsion contain small drops of water constitute a w/o/w emulsion. Also, a system of oil droplets enclosed in water beads stabilized in the oil constant phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or dispersed phase of the emulsion is well dispersed into the outer or permanent phase and maintained in that form by means of emulsifiers or by the viscosity of the formulation. Any of the emulsion phases may be semi-solid or solid, as is the case for emulsion-type ointment bases and creams. Other means of stabilizing emulsions include the use of emulsifiers, which can be included in any phase of the emulsion. Broadly speaking, emulsifiers can be divided into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and fine solids (see, for example, HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surfactants, have found widespread use in emulsion formulations and are reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are usually amphiphilic and contain a hydrophilic and hydrophobic part. The ratio of the hydrophilic and hydrophobic nature of a surfactant is referred to as the hydrophilic-lipophilic balance (HLB) and is a valuable tool for classifying and selecting surfactants for formulation purposes. Surfactants can be classified into different classes based on the nature of the hydrophilic group: non-ionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC. , 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations contain lanolin, beeswax, phosphatides, lecithin and gum arabic. Absorbent bases have hydrophilic properties so that they can absorb water to form w/o emulsions while maintaining their semi-solid consistency, such as anhydrous lanolin and hydrophilic vaseline. Finely divided solids are also used as good emulsifiers, especially in combination with surfactants and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

Also included in the emulsion formulations are a wide variety of non-emulsifying materials that contribute to the properties of the emulsions. They include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker , Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p . 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., gum arabic, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (eg carbomers, cellulose ethers and carboxy vinyl polymers). They disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed phase droplets and by increasing the viscosity of the external phase.

Because emulsions often contain a number of ingredients, such as carbohydrates, proteins, sterols, and phosphatides, that can easily promote microbial growth, these formulations often include preservatives. Commonly used preservatives included in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisols, butylated hydroxytoluene or reducing agents such as ascorbic acid and sodium metabisulphite, and antioxidant synergists such as citric acid, tartaric acid and lecithin.

The use of emulsion formulations via dermatological, oral and parenteral routes and methods for their preparation are discussed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199 ). Emulsion formulations for oral delivery are widely used due to ease of formulation as well as efficiency in terms of absorption and bioavailability (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC ., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral oil laxatives, fat-soluble vitamins, and high-fat food preparations are materials that are commonly administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, mRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and amphiphilic material that constitutes a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC ., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are made by first dispersing an oil in an aqueous solution of a surfactant and then adding a sufficient amount of a fourth component, usually an intermediate chain length alcohol, to form a clear system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989 , VCH Publishers, New York, pages 185-215). Microemulsions are typically prepared with a combination of three to five components which include oil, water, surfactant, co-surfactant and electrolyte. Whether the microemulsion is water-in-oil (w/o) or oil-in-water (o/w) depends on the properties of the oil and surfactant used, as well as the structure and geometric packing of the polar heads and hydrocarbon tails. surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach using phase diagrams has been extensively studied and has given those skilled in the art a comprehensive knowledge of how to formulate microemulsions (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions have the advantage of solubilizing water-insoluble drugs into thermodynamically stable droplets that form spontaneously.

Surfactants used to form microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleic ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310) , hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with auxiliary surfactants. The co-surfactant, typically a short chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating the surfactant film and hence creating a disordered film due to void space, produced by surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase typically, but not limited to, can be water, an aqueous drug solution, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and ethylene glycol derivatives. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain mono, di and triglycerides (C ₈ -C ₁₂ ), polyoxyethylated glyceryl esters and fatty acids. acids, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C ₈ -C ₁₀ glycerides, vegetable oils and silicone oil.

Microemulsions are of particular interest from the viewpoint of drug solubilization and drug absorption enhancement. Lipid-based microemulsions (both o/w and w/o) have been proposed to improve the oral bioavailability of drugs containing peptides (see, for example, US Pat. Nos. 6,191,105; 7,063,860; 7,070,802; Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions provide the benefits of improved drug solubilization, drug protection from enzymatic hydrolysis, possible improved drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical activity and reduced toxicity (see, for example, US Pat. , 138-143). Often, microemulsions can form spontaneously when their components are combined at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or mRNAs. Microemulsions are also effective in the transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to enhance systemic uptake of mRNA and nucleic acids from the gastrointestinal tract as well as improve local cellular uptake of mRNA and nucleic acids.

The microemulsions of the present invention may also contain additional ingredients and additives such as sorbitan monostearate (Grill 3), labrasol and penetration enhancers to improve formulation properties and to enhance uptake of the mRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories - surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. microparticles

The RNAi agent of the invention can be incorporated into a particle, eg a microparticle. The microparticles can be obtained using spray drying, but can also be obtained by other methods, including lyophilization, evaporation, fluidized bed drying, vacuum drying, or a combination of these methods.

iv. Penetration enhancers

In one embodiment, the present invention uses different penetration enhancers to effect efficient delivery of nucleic acids, especially mRNA, to the skin of animals. Most drugs are present in solution in both ionized and non-ionized forms. However, usually only fat-soluble or lipophilic drugs easily cross cell membranes. It has been found that even non-lipophilic drugs can permeate cell membranes if the membrane being crossed is treated with a permeation enhancer. In addition to promoting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the penetration of lipophilic drugs.

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

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

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

The physiological role of bile includes facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term "bile salts" includes any naturally occurring components of bile, as well as any synthetic derivatives thereof. Suitable bile salts include, for example, bile acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate) , glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate sodium (STDHF), glycodihydrofusidate sodium and polyoxyethylene-9-lauryl ether (POE) (see e.g. Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from solution by complexing with them, resulting in increased mucosal uptake of mRNA. With respect to their use as penetration enhancers in the present invention, chelating agents have the added benefit of also serving as DNase inhibitors, since most of the characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelating agents (Jarrett, J Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxy salicylate, and homovanylate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of beta-diketones (enamines ) (see, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

For the purposes of the present invention, non-chelating, non-surfactant penetration enhancers can be defined as compounds that exhibit little activity as chelating agents or surfactants but nevertheless increase mRNA uptake through the mucosal lining of the digestive tract (see , For example, Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Also in pharmaceutical and other compositions according to the present invention, you can add agents that improve the uptake of mRNA at the cellular level. For example, cationic lipids such as lipofectin (Junichi et al, US Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT Application WO 97/30731) are also known to improve cellular uptake. dsRNA. Examples of commercially available transfection reagents include, but are not limited to, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA) ), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen ; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 transfection reagent (ROCHe; Grenzacherstrasse, Switzerland), DOTAP liposomal transfection reagent (Grenzacherstrasse, Switzerland), DOSPER liposome transfection reagent (Grenzacherstrasse, Switzerland) or Fugene (Grenzacherstrasse, Switzerland), Transfectam® reagent (Promega; Madison, WI), TransFast™ transfection reagent (Promega; Madison, WI), Tfx™-20 reagent (Promega; Madison, WI), WI), Tfx™-50 reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassª D1 transfection reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, CA, USA), PerFectin transfection reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection Reagent (Genlantis; San Diego, CA , USA), Cytofectin transfection reagent (Genlantis; San Diego, CA, USA), BaculoPORTER transfection reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline ; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect ™ (B-Bridge International, Mountain View, CA, USA).

Other agents can be used to improve the penetration of the introduced nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones and terpenes such as limonene and menthone.

v. carriers

Some of the compositions of the present invention also include carrier compounds in the formulation. Within the scope of the present invention, the term "carrier compound" or "carrier" may refer to a nucleic acid or its analogue that is inert (i.e., has no biological activity per se) but is recognized as a nucleic acid by in vivo processes, thereby reducing bioavailability of the nucleic acid having biological activity, for example, by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of a nucleic acid and a carrier compound, usually with an excess of the latter substance, can result in a significant decrease in the amount of nucleic acid excreted in the liver, kidneys, or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, excretion of partially phosphorothioate dsRNA in liver tissue can be reduced if it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

vi. Excipients

In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable diluent, suspending agent, or any other pharmacologically inert medium for delivery of one or more nucleic acids to an animal. The excipient may be liquid or solid and is chosen with regard to the intended route of administration to provide the desired weight, consistency, etc., in combination with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (eg, pregelatinized cornstarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (eg lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (eg, magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (eg starch, sodium starch glycolate, etc.); and wetting agents (eg sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not adversely affect nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oil bases. Solutions may also contain buffers, diluents, and other suitable additives. You can use pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration, which do not adversely affect the nucleic acids.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

vii. Other Components

Compositions according to the present invention may additionally contain other additional components commonly found in pharmaceutical compositions, at the level of their use established in this field. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, such as, for example, antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful in the physical formulation of various dosage forms of the compositions of the present invention, such as colorants. , flavoring agents, preservatives, antioxidants, transparency agents, thickeners and stabilizers. However, such materials, when added, should not unduly affect the biological activity of the components of the compositions of the present invention. The formulations can be sterilized and optionally mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, osmotic salts, buffers, coloring agents, fragrances and/or fragrances, and the like, which do not have a harmful effect when interacting with the nucleic acid (acids) of the original form.

Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions of the invention comprise (a) one or more mRNA compounds, and (b) one or more agents that do not function through an RNAi mechanism and that are useful in the treatment of a hemolytic disorder. Examples of such agents include, but are not limited to, an anti-inflammatory agent, an anti-steatosis agent, an antiviral agent, and/or an antifibrotic agent. In addition, other substances commonly used for liver protection, such as silymarin, can also be used in combination with the mRNAs described herein. Other agents useful in the treatment of liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and others, as described, for example, in Tung et al. and in Hale et al., US Application Publication No. 2004/0127488.

The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical methods in cell cultures or experimental animals, for example, to determine LD50 (dose lethal in 50% of the population) and ED50 (dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is a therapeutic index and can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic performance are preferred.

Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of the compositions of the present invention is usually in the range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the methods described in the invention, a therapeutically effective dose can initially be estimated from cell culture assays. The dose can be formulated in animal models to achieve a range of circulating plasma concentrations of the compound or, if necessary, of the polypeptide product of the target sequence (e.g., to achieve a reduced concentration of the polypeptide), including ISERPINC10 (i.e., the concentration of the test compound at which half the maximum symptom suppression) as determined in cell culture. This information can be used to more accurately determine useful doses for humans. Plasma levels can be measured, for example, using high performance liquid chromatography.

In addition to their administration as discussed above, the mRNAs of the invention may be administered in combination with other known agents effective in the treatment of pathological processes mediated by SERPINC1 expression. In any case, the attending physician may adjust the amount and timing of iRNA administration based on the results observed using standard efficacy measures known in the art or described herein.

VI. Sets

The present invention also provides kits for performing any of the methods of the invention. Such kits contain one or more RNAi agents and instructions for use, such as instructions for prescribing a prophylactically or therapeutically effective amount of the RNAi agent(s). Optionally, the kits may further comprise a means for administering an RNAi agent (eg, an injection device) or a means for measuring Serpinc1 inhibition (eg, a means for measuring Serpinc1 mRNA inhibition, Serpinc1 protein, and/or Serpinc1 activity). Such means for measuring Serpinc1 inhibition may include means for obtaining a sample from a subject, such as, for example, a plasma sample. Optionally, kits of the invention may further comprise means for determining a therapeutically effective or prophylactically effective amount.

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as generally understood by experts in the field to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the mRNA and methods of the invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in this document are hereby incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

EXAMPLES

Table 1: Abbreviations for nucleotide monomers used to represent nucleic acid sequences. It should be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds. Reduction Nucleotide (nucleotides) A Adenosine-3'-phosphate Af 2'-fluoroadenosine-3'-phosphate Afs 2'-fluoradenosine-3'-phosphorothioate As adenosine-3'-phosphorothioate C cytidine-3'-phosphate cf 2'-fluorocytidine-3'-phosphate cfs 2'-fluorocytidine-3'-phosphorothioate Cs cytidine-3'-phosphorothioate G guanosine-3'-phosphate gf 2'-fluoroguanosine-3'-phosphate gfs 2'-fluoroguanosine-3'-phosphorothioate Gs guanosine-3'-phosphorothioate T 5'-methyluridine-3'-phosphate Tf 2'-fluoro-5-methyluridine-3'-phosphate Tfs 2'-fluoro-5-methyluridine-3'-phosphorothioate Ts 5-methyluridine-3'-phosphorothioate U Uridine 3'-phosphate uf 2'-fluorouridine-3'-phosphate Ufs 2'-fluorouridine-3'-phosphorothioate Us uridine-3'-phosphorothioate N any nucleotide (G, A, C, T or U) a 2'-O-methyladenosine-3'-phosphate as 2'-O-methyladenosine-3'-phosphorothioate c 2'-O-methylcytidine-3'-phosphate cs 2'-O-methylcytidine-3'-phosphorothioate g 2'-O-methylguanosine-3'-phosphate gs 2'-O-methylguanosine-3'-phosphorothioate t 2'-O-methyl-5-methyluridine-3'-phosphate ts 2'-O-methyl-5-methyluridine-3'-phosphorothioate u 2'-O-methyluridine-3'-phosphate us 2'-O-methyluridine-3'-phosphorothioate s phosphorothioate bond L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol
Hyp-(GalNAc-alkyl)3

Example 1 Single Dose Administration of AT3SC-001 to Healthy Human Subjects

Twenty-four healthy human volunteers in 3:1 cohorts (active agent: placebo) received a single dose of 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, or 1. 0 mg/kg AT3SC-001 (sense strand (5'-3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 (SEQ ID NO: 13); antisense strand (5'-3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO:14)). Plasma samples were collected at 0, 1, 2, 3, 7, 10, 14, 21, 28, 42, 56, and 70 days after administration to monitor AT protein levels, AT activity, and duration of AT protein silencing. AT protein levels were monitored using ELISA and AT activity levels were monitored by generating thrombin generation curves using a calibrated automated thrombinoscope (tissue factor=1 pM). The fold change in peak thrombin was calculated relative to the mean peak thrombin for the two pre-dose values for each subject.

There were no serious side effects, there were 3 mild side effects that were probably not related to the administration of the agent, and 1 mild side effect (headache) that could be associated with the administration of the agent. There were also no reactions at the injection site, and medical examinations, vital signs, and electrocardiograms of all subjects were within normal limits. In addition, all liver function tests, total bilirubin levels, international normalized prothrombin time ratio (PT/INR), platelet count, hemoglobin levels, and coagulation tests (i.e. Activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen levels and fibrin D-dimer levels) in all subjects during the study did not change and were within the normal range.

On FIG. 1A-D and 2A-B show that a single dose of 0.03 mg/kg of AT3SC-001 results in a decrease in AT protein levels of approximately 20% to 33% (Fig. 2A and 2B) and a corresponding decrease in AT activity (Fig. .1A-D) with a decline duration of more than 60 days.

On FIG. 3 additionally shows that there is a significant relationship between AT knockdown and peak thrombin generation. In particular, an increase in peak thrombin generation up to 152% was observed with a mean maximum increase in peak thrombin of 138% ± 8.9% (mean ± SEM). In addition, and consistent with the increase in thrombin production with increased AT knockdown, factor VIII or IX levels were normal.

Example 2: Multiple Dose Administration of AT3SC-001 to Human Patients with Hemophilia A or B

Clinical Study Phase I - Parts B, C and D

In part B I phase of the clinical test of AT3SC-001 (semantic thread (5'-3 '): Gfsgsufuafuafacfacfufufufufucfafl96 (Seq ID NO: 13); Antroxy Thread (5'-3'): USUFAFAFUAFUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGUFGU AFACFCSASG (SEQ ID NO: 14)) three patients , suffering from hemophilia A (n=2) or B (n=1), were injected subcutaneously with 0.015 mg/kg weekly for three weeks (15 μg/kg qw x 3; 15 μg/kg) AT3SC-001; six patients suffering from hemophilia A were injected subcutaneously with 0.045 mg/kg weekly for three weeks (45 μg/kg qw x 3; 45 μg/kg) AT3SC-001; and three patients with hemophilia A (n=2) or B (n=1) were injected subcutaneously with 0.075 mg/kg weekly for three weeks (75 μg/kg qw x 3; 75 μg/kg) AT3SC-001.

In Part C, Phase I clinical trial AT3SC-001 (sense strand (5'-3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 (SEQ ID NO:13); antisense strand (5'-3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO:14)), three patients with hemophilia A (n=2) or B (n=1) were dosed subcutaneously once a month with 0.225 mg/kg AT3SC-001 for three months (225 μg/kg qm x 3; 225 μg/kg); three patients with hemophilia A (n=2) or B (n=1) were dosed subcutaneously once a month with 0.450 mg/kg AT3SC-001 for three months (450 µg/kg qm x 3; 450 µg/kg) ; three patients with hemophilia A were dosed subcutaneously once a month with 0.900 mg/kg AT3SC-001 for three months (900 μg/kg qm x 3; 900 μg/kg); three patients with hemophilia A were dosed subcutaneously once a month with 1,800 mg/kg AT3SC-001 for three months (1800 μg/kg qm x 3; 1800 μg/kg); and six patients with hemophilia A (n=3) or B (n=3) received a fixed dose of 80 mg AT3SC-001 subcutaneously once a month for three months (80 mg qM x 3).

In Part D of the Phase I clinical trial AT3SC-001 (sense strand (5'-3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 (SEQ ID NO:13); antisense strand (5'-3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO:14)), six Inhibitor patients with hemophilia A (n=5) or B (n=1) and using bypass agents (BPA) to control bleeding received a fixed dose of 50 mg AT3SC-001 subcutaneously once a month for three months (50 mg qM x 3); and 10 hemophilia A patients using bypass agents (BPA) for bleeding control were given a fixed dose of 80 mg AT3SC-001 subcutaneously once a month for three months (80 mg qM x 3).

after administration of AT3SC-001, plasma samples were collected to monitor AT protein levels, AT activity, and duration of AT protein silencing. AT protein levels were monitored using ELISA, and AT activity levels were monitored by plotting thrombin formation curves using a calibrated automatic thrombinoscope (tissue factor=1 pM). The fold change in peak thrombin was calculated relative to the mean peak thrombin for the two pre-dose values for each patient.

Table 2 lists the demographics and baseline characteristics of patients in Parts B, C, and D of the study.

Table 2. Demographics and Baseline Characteristics of Study Participants Part B
subcutaneously
Once a week × 3 Part C
subcutaneously
Once a month × 3 Part D
subcutaneously
Once a month × 3 15 mcg/kg
N=3 45 mcg/kg
N=6 75 mcg/kg
N=3 225 mcg/kg
N=3 450 mcg/kg
N=3 900 mcg/kg
N=3 1800 mcg/kg
N=3 80
mg
N=6 50
mg
N=6 80
mg
N=10 Age, mean (SD) 28
(9) 42
(14) 40
(4) 37
(21) 37
(15) 38
(16) 46
(12) 32
(12) 32
(7) 37 Hemophilia A
Hemophilia B 2
1 6
0 2
1 2
1 2
1 3
0 3
0 3
3 5
1 10
0 heavy
Medium 3
0 6
0 3
0 2
1 3
0 2
1 3
0 5
1 6
0 10
0 Weight (kg), average (SD) 76
(10) 80
(22) 82
(8) 85
(12) 76
(16) 76
(2) 71
(12) 74
(10) 73
(17) 73 Medical history of Hepatitis C 3 9

For parts B, C and D of the study, there were no serious adverse events, treatment discontinuation, injection site reactions, and physical examinations, vital signs and electrocardiograms of all patients were within normal limits. In addition, all liver function tests and complete blood counts of all patients did not change during the study and were within normal limits. There were also no thromboembolic complications during this study, and no clinically significant increase in fibrin D-dimer levels was observed in any of the patients. Any bleeding events were successfully managed using standard replacement ratio or bypass injection. In addition, there were no cases of anti-drug antibody (ADA) formation.

The knockdown of AT levels in the 15 µg/kg, 45 µg/kg, and 75 µg/kg cohorts, shown as mean AT knockdown relative to baseline, is depicted in FIG. 4. In FIG. 4 shows that weekly doses of 0.015 mg/kg for three weeks of AT3SC-001 resulted in a mean maximal AT knockdown of 29% ± 12% (mean ± SEM). The maximum AT knockdown was up to 53%. In FIG. 4 also shows that weekly doses of 0.045 mg/kg for three weeks of AT3SC-001 resulted in a mean maximum AT knockdown of 55 ± 9% (mean ± SEM) and a maximum AT knockdown of 86%. In addition, in FIG. 4 also shows that weekly doses of 0.075 mg/kg for three weeks of AT3SC-001 resulted in a mean maximum AT knockdown of 61 ± 8% (mean ± SEM) and a maximum AT knockdown of 74%.

The knockdown of AT levels in the 225 µg/kg, 450 µg/kg, 900 µg/kg, 1800 µg/kg, and 80 mg cohorts, shown as mean AT knockdown relative to baseline, is depicted in FIG. 5A. In FIG. 5A shows that monthly doses of 0.225 mg/kg for three months of AT3SC-001 resulted in a mean maximal AT knockdown of 70% ± 9% (mean ± SEM). The maximum AT knockdown was up to 80%. In FIG. 5A also shows that monthly doses of 0.450 mg/kg for three months of AT3SC-001 resulted in a mean maximum AT knockdown of 77 ± 5% (mean ± SEM) and a maximum AT knockdown of 85%. In addition, in FIG. 5A also shows that monthly doses of 0.900 mg/kg for three months of AT3SC-001 resulted in a mean maximal AT knockdown of 78±7% (mean ± SEM) and a maximal AT knockdown of 88%. In addition, in FIG. 5A shows that monthly doses of 1800 mg/kg for three months of AT3SC-001 resulted in a mean maximum AT knockdown of 79 ± 3% (mean ± SEM) and a maximum AT knockdown of 84%. In FIG. 5A also shows that monthly doses of 80 mg for three months of AT3SC-001 result in a mean maximum AT knockdown of 87 ± 1% (mean ± SEM).

As shown in FIG. 5B, which shows AT3SC-001 dose versus relative trough of AT protein levels, administration of AT3SC-001 to human patients reduces AT protein levels in a dose-dependent manner.

Evaluation of thrombin generation in healthy human volunteers (Example 1) and patients with hemophilia A or B showed that weekly doses of AT3SC-001 resulted in an increase of up to 334% (relative to baseline) in thrombin generation in hemophiliac patients with a mean increase in thrombin generation 112 ±38% (p<0.05) relative to baseline with AT knockdown ≥50% (FIG. 6B). In FIG. 6A shows that the maximum thrombin peak achieved in hemophilia A or B patients treated with weekly doses of AT3SC-001 was in the low range of thrombin formation in normal subjects.

Thromboelastometric analysis of ROTEM® (see, e.g., Young, et al., (2013) Blood 121:1944) whole blood from one patient (patient 101-009) shows that administration of AT3SC-001 at 0.045 mg/kg once a week in over three weeks not only resulted in an increase in peak thrombin formation, but also resulted in a marked and lasting improvement in whole blood clot formation, as indicated by a reduction in clot formation time and clotting time (FIG. 7). Patient 101-009 has had no bleeding events since day 2 and is currently bleeding free for 47 days.

Post hoc analysis of thrombin production by AT reduction quartiles (parts B and C) shows that in the largest AT reduction quartile (AT reduction >75%), the increase in mean thrombin production from baseline is 289% (FIG. 9). This level of thrombin formation is in the range of thrombin formation observed in healthy volunteers.

In an additional study of three patients, the equivalence of administration of AT3SC-001 and administration of factor VIII was studied. Briefly, factor VIII was administered to each of the three patients and plasma was collected from the patients at 0.5, 1, 2, 6, 24 and 48 hours post-administration. Samples from each subject were analyzed for factor VIII levels and thrombin production levels and used to determine individual ratios of factor VIII-peak thrombin production. These data were then used for comparison with peak thrombin levels achieved with AT3SC-001 administration. As shown in FIG. 10A-10C, administration of AT3SC-001 is sufficient to achieve peak thrombin production levels in the subject to about the same level as that achieved by administering Factor VIII to the subject, and sufficient to achieve peak thrombin production levels greater than about 40% in the patient.

Post hoc analysis of bleeding events by AT reduction quartile (Parts B and C) shows that there is a reduced propensity to bleed with increased levels of AT reduction with an average estimated annual bleeding rate (ABR) of 5 ± 2 (median=1) in the highest AT reduction quartile (Fig. 11). This analysis includes over 1100 cumulative days with >75% AT reduction in 16 patients.

A post hoc analysis of bleeding events in the Part C cohort was also performed. FIG. 12 shows the patient data used for this analysis. As shown in FIG. 13A, the median history of ABR for all patients enrolled in cohort C and receiving prophylactic (PPx) replacement factors was 2, and the median history of ABR for all patients enrolled in cohort C and receiving replacement factors on demand (OD) was 28. Administration of AT3SC-001 to these patients resulted in a significant reduction in median ABR. Specifically, administration of AT3SC-001 resulted in a median of 53% of patients reporting no bleeding during the follow-up period (day 29 to last study visit or to last dose+56 days, whichever was earlier) and a median of 82% patients who reported no spontaneous bleeding during the follow-up period. In FIG. 13B shows that for patients receiving monthly doses of 80 mg of AT3SC-001 in group C and receiving prophylactic (PPx) replacement factors, the median history of ABR was 6. However, after administration of AT3SC-001, the median ABR during the follow-up period was 0.

The Phase I Part D study evaluated the effect of AT3SC-001 administration in patients with hemophilia A or B who have developed antibodies (inhibitors) against their replacement factors and thus become unresponsive to replacement coagulation factor. Accordingly, to assess the peak thrombin response in these patients, prior to administration of AT3SC-001, patients included in the 50 mg cohort were administered their standard bypass agent (BPA) (e.g., activated prothrombin complex concentrate (APCC) and/or activated recombinant FVII (rFVIIa) ), plasma samples were collected at 1, 2, 6 and 24 hours after BPA administration, and the samples were analyzed for thrombin formation. As shown in FIG. 14A-14F, AT reduction and thrombin formation in patients with inhibitors treated with AT3SC-001 are comparable to AT reduction and thrombin formation observed after administration of AT3SC-001 at similar doses in patients without inhibitors. In addition, figa-14F shows that the formation of thrombin after the introduction of AT3SC-001 consistently exceeds the temporary levels achieved with the introduction of BPA.

As shown in FIG. 15, once-monthly subcutaneous administration of AT3SC-001 at doses of 50 mg and 80 mg provides a dose-dependent reduction in AT of about 80% in hemophiliac patients with inhibitors. In addition, as shown in FIG. 16, the AT lowering effect achieved in patients treated with AT3SC-001 correlates with an increase in thrombin generation.

Also in part D of the study, a post-hoc analysis of bleeding events in patients was performed. In FIG. 17A shows that administration of AT3SC-001 to hemophilia A or B inhibitor patients once a month at a dose of 50 or 80 mg results in a significant reduction in pre-study ABR. In addition, as shown in FIG. 17B, the median annual bleeding rate (ABR) is zero for all inhibitor patients treated with AT3SC-001 in Phase I Part D of this study, and 56% of patients have no bleeding and 69% of patients have zero spontaneous bleeding.

Thus, AT3SC-001 is well tolerated by hemophilia A and B patients with and without inhibitors. There were no study drug related SAEs and no thromboembolic complications. The data indicate the presence of clinical activity and correction of the phenotype of hemophilia in patients without inhibitors. The data also demonstrate that there is a dose-dependent reduction in AT and an increase in thrombin formation with the once-monthly subcutaneous dosing schedule and that administration of a fixed dose of AT3SC-001 50 or 80 mg provides a consistent reduction in AT of approximately 80%.

In addition, the data demonstrate that administration of AT3SC-001 to patients with inhibitors results in a decrease in AT and an increase in thrombin production, which is consistent with patients without inhibitors, and that the consistent increase in thrombin production exceeds the increase achieved short-term with BPA administration.

Example 3: Multiple Dose Administration of AT3SC-001 to Human Patients with Hemophilia A or B

Phase II Open Extended (OLE) Clinical Study

In the Phase II OLE study AT3SC-001 (sense strand (5'-3'): GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 (SEQ ID NO:13); antisense strand (5'-3'): usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg (SEQ ID NO:14)), patients with with or without inhibitors who had previously received AT3SC-001 in Parts B and C of the Phase I clinical trials described above were eligible for participation in a Phase II open-label extension (OLE) study. Fifteen Phase I Part B patients with hemophilia A or B without inhibitors treated with AT3SC-001 0.015 mg/kg once weekly for three weeks (15 µg/kg qw x 3; 15 µg/kg); or subcutaneously administered AT3SC-001 0.045 mg/kg once a week for three weeks (45 μg/kg qw x 3; 45 μg/kg); or subcutaneously administered AT3SC-001 0.075 mg/kg once a week for three weeks (75 μg/kg qw x 3; 75 μg/kg); and 18 Phase I Part C patients with hemophilia A or B without inhibitors who were dosed subcutaneously once a month with AT3SC-001 0.225 mg/kg for three months (225 µg/kg qm x 3; 225 µg/kg); or a monthly dose of AT3SC-001 0.450 mg/kg was administered subcutaneously for three months (450 μg/kg qm x 3; 450 μg/kg); or a monthly dose of AT3SC-001 0.900 mg/kg was administered subcutaneously for three months (900 μg/kg qm x 3; 900 μg/kg); or a monthly dose of AT3SC-001 1,800 mg/kg was administered subcutaneously for three months (1800 μg/kg qm x 3; 1800 μg/kg); or a fixed dose of 80 mg AT3SC-001 administered subcutaneously once a month for three months (80 mg qM x 3); and 16 Phase I Part D patients with hemophilia A or B inhibitors who received a monthly fixed dose of 50 mg AT3SC-001 subcutaneously for three months (50 mg qM x 3); or subcutaneously injected monthly with a fixed dose of 80 mg AT3SC-001 for three months (80 mg qM x 3) were eligible for this study.

Thirty-three patients were included in this study, with 29 patients continuing the study and 5 patients discontinued (4 due to withdrawal of consent and 1 due to AE). Ten patients with hemophilia A (n=7) or hemophilia B (n=3) without inhibitors received a fixed dose of 50 mg AT3SC-001 subcutaneously monthly for three months (50 mg qM x 3); and 9 patients with hemophilia A (n=7) or hemophilia B (n=2) without inhibitors received a fixed dose of 80 mg AT3SC-001 subcutaneously monthly for three months (50 mg × qM 3). Similarly, 3 patients with hemophilia A (n=3) with inhibitors received a fixed dose of 50 mg AT3SC-001 subcutaneously monthly for three months (50 mg qM × 3); and 11 patients with hemophilia A (n=11) or hemophilia B (n=1) with inhibitors received a fixed dose of 80 mg AT3SC-001 subcutaneously monthly for three months (50 mg qM x 3). The demographics, baseline characteristics, and duration of exposure to AT3SC-001 of the patients included in this study are summarized in Table 3 below.

Table 3 Demographics and Baseline Characteristics of Study Participants Patients without inhibitors Patients with inhibitors 50 mg
N=10 80 mg
N=9 50 mg
N=3 80 mg
N=11 Age, years; average value (range) 36 (19-61) 40 (24-58) 31 (22-36) 34 (21-41) Weight, kg; average value (range) 78(58-94) 73 (58-80) 82 (70-100) 72(52-108) Hemophilia A
Hemophilia B 7
3 7
2 3
- 10
1 heavy
Medium 9
1 7
2 3
- eleven
- Positive Medical History for Hepatitis C 8 8 2 9 Impact, months; median value (range) 13 (5-20) 14 (3-18) 11 (5-12) 6 (0-12)

AT3SC-001 was generally well tolerated by patients without inhibitors in phase II OLE. Six patients reported serious adverse events (SAEs), with 2 SAEs considered possibly related to AT3SC-001 administration; one patient with chronic HCV infection experienced an increase in ALT and AST and subsequently discontinued the study, and one patient with a history of a seizure disorder experienced an episode of confusion in the patient. There were no thromboembolic complications or laboratory evidence of abnormal clot formation. The majority of adverse events (AE) were mild or moderate in severity and not associated with AT3SC-001 administration. an increase in asymptomatic alanine aminotransferase (ALT) greater than 3 times the upper limit of normal (ULN) without a simultaneous increase in bilirubin more than 2 times the ULN was observed in 11 patients, all of whom had a history of hepatitis C infection. All bleeding events during treatment were successfully treated with a replacement factor or bypass agent. In addition, there were no cases of anti-drug antibody (ADA) formation.

On FIG. 18, 19A and 19B further show the clinical activity of AT3SC-001 administration. In particular, as shown in FIG. 18, once-monthly subcutaneous administration of AT3SC-001 at a dose of 80 mg consistently produced a reduction in AT. As shown in FIG. 19A, once-monthly subcutaneous administration of AT3SC-001 at 50 mg or 80 mg provides a dose-dependent AT reduction of ~80% with low inter-patient variability and, as shown in FIG. 19B, once-monthly subcutaneous administration of AT3SC-001 at 50 or 80 mg produces thrombin levels approaching the lower end of the normal range.

We also performed a preliminary post hoc analysis of bleeding events in patients with and without inhibitors in the phase II OLE study. In FIG. 20A shows that administration of AT3SC-001 to patients with hemophilia A or B without inhibitors once a month at a dose of 50 or 80 mg leads to a significant decrease in ABR before the study, and in FIG. 20B shows that administration of AT3SC-001 to hemophilia A or B patients with inhibitors once a month at a dose of 50 or 80 mg results in a significant decrease in pre-study ABR. Summing up the median ABR values of all patents participating in the OLE study, 48% (16/33) of patients had no bleeding during the follow-up period, and overall, the median ABR value during the follow-up period was 1. In addition, 67% of patients reported about the absence of spontaneous bleeding during the observation period, and, in general, ABR during the observation period was 0. The characteristics of bleeding events in patients with and without inhibitors during the OLE study are shown in Fig. 21. Bleeding events in patients were assessed with a decrease in antithrombin ≥75%.

In FIG. 22 provides details of the management of bleeding events in hemophilia A or B patients without inhibitors, and FIG. 23 provides details of the management of bleeding events in patients with hemophilia A or B with inhibitors. Surprisingly, patients with hemophilia A or B without inhibitors and patients with hemophilia A or B with inhibitors who were administered AT3SC-001 once a month at a dose of 50 or 80 mg and who had a bleeding event required significantly lower doses of replacement factors or shunting. drug compared to the dose recommended for the treatment of bleeding, for example, a lower dose of factor VIII or a lower dose of aPCC.

Thus, AT3SC-001 was generally well tolerated by hemophilia A and B patients with and without inhibitors. In addition, data demonstrate that AT3SC-001 has clinical activity, with once-monthly subcutaneous administration at doses of 50 mg and 80 mg providing a dose-dependent reduction in AT of ~80% with low inter-patient variability and thrombin production levels approaching lower limit. normal range. In addition, a preliminary post hoc analysis of bleeding events in patients with hemophilia A or B with or without inhibitors shows that administration of AT3SC-001 reduced the median ABR to 1 and the mean annual spontaneous bleeding rate (AsBR) to zero. Sixteen of thirty-three (48%) patients did not bleed, and twenty-two of thirty-three (67%) patients did not have spontaneous bleeding during the follow-up period. All bleeding events were successfully treated with replacement factor or bypass agent.

Example 4: Updated regimen recommendations for managing bleeding: dose reduction of replacement factor/bypass agent

The data from the phase I and phase I/II OLE studies presented above demonstrate that a fixed dose of 50 mg or 80 mg of AT3SC-001 subcutaneously administered once a month provides a permanent decrease in AT by approximately 80% and that there is a dose-dependent decrease in AT and increase in thrombin formation in patients with hemophilia A or B with and without inhibitors.

In silico modeling data supports these observations, predicting an increase in thrombin generation in response to a decrease in AT levels, including in some embodiments reducing AT levels by about 75% or less. Data obtained from plasma samples from patients with hemophilia A and B with and without inhibitors show an increase in the response of thrombin formation to replacement factors and bypass agents after dosing in patients who were administered AT3SC-001. In addition, clinical data show that for patients with hemophilia A or B without inhibitors (Fig. 22) and patients with hemophilia A or B with inhibitors (Fig. 23), who were administered AT3SC-001 once a month at a dose of 50 or 80 mg and who had bleeding episodes used lower doses of replacement factors or a bypass agent compared to the recommended dose for the treatment of bleeding, such as a lower dose of factor VIII or a lower dose of factor IX, or a lower dose with an increased factor half-life IX, or a lower dose of recombinant factor VIIa, or a lower dose of aPCC.

Additional clinical bleeding management data presented in FIG. 24A-24D further demonstrate successful treatment of bleeding in patients treated with AT3SC-001 with lower amounts of replacement factor or bypass agent.

Accordingly, the data presented herein justifies the administration of lower doses of a replacement factor or bypass agent to patients who are administered AT3SC-001, i.e., who are administered a fixed monthly subcutaneous dose of 50 or 80 mg, for example, in which administration of the dsRNA agent to the subject reduces Serpinc1 activity by approximately by 75% or more. Therefore, as outlined in Table 4, bleeding management guidelines have been updated in patients receiving a monthly subcutaneous dose of 50 mg or 80 mg of AT3SC-001, and such bleeding should be managed using lower doses of replacement factor or bypass agent.

For example, the recommended effective amount of replacement factor (Factor VIII) for the treatment of bleeding, eg, moderate or severe bleeding in a hemophilia A patient without inhibitors, is approximately 30-50 IU/kg. However, bleeding, such as moderate bleeding or severe bleeding in a patient with hemophilia A who receives a fixed subcutaneous dose of 50 mg or 80 mg of AT3SC-001 once a month, for example, in which administration of a dsRNA agent to the patient reduces Serpinc1 activity by approximately 75% or more may be treated with a factor VIII dose of about 5 to about 20 IU/kg. For hemophilia B patients without inhibitors, the recommended effective amount of replacement factor (Factor IX or Extended Half-Life Factor IX) for the treatment of bleeding, such as major bleeding, is approximately 65-130 IU/kg. However, bleeding, such as severe bleeding in a patient with hemophilia B who is administered once a month a fixed subcutaneous dose of 50 mg or 80 mg of AT3SC-001, for example, in which administration of a dsRNA agent to the patient reduces Serpinc1 activity by approximately 75% or more, can be treated with a dose of factor IX or Factor IX with an extended half-life of about 10 to about 30 IU/kg. For hemophilia A or B patients with inhibitors, the recommended effective amount of a bypass agent (activated prothrombin complex concentrate; aPCC) for the treatment of bleeding, eg, moderate to severe bleeding, is approximately 100 U/kg. However, bleeding, such as moderate or severe bleeding, in a patient with hemophilia A or B with inhibitors who is given a fixed subcutaneous dose of 50 mg or 80 mg of AT3SC-001 once a month, for example, in which administration of a dsRNA agent to the patient reduces Serpinc1 activity by approximately 75% or more can be treated with an aPCC dose of about 30 to about 50 U/kg. For hemophilia A or B patients with inhibitors, the recommended effective amount of a bypass agent (recombinant factor VIIa; rFVIIa) for the treatment of bleeding, eg, moderate to severe bleeding, is approximately 90 µg/kg. However, bleeding, such as moderate or severe bleeding, in a patient with hemophilia A or B with inhibitors who is given a fixed subcutaneous dose of 50 mg or 80 mg of AT3SC-001 once a month, for example, in which administration of a dsRNA agent to the patient reduces Serpinc1 activity by approximately 75% or more can be treated with a dose of rFVIIa from about 10 to about 45 μg/kg.

Table 4 Updated Bleeding Management Guidelines Factor VIII Factor IX standard half-life Factor IX extended half-life aPCC Recombinant Factor VIIa Recommended single dose 10 IU/kg 20 IU/kg 20 IU/kg 30 U/kg ≤45mcg/kg Single dose should not exceed 20 IU/kg 30 IU/kg 30 IU/kg 50 U/kg 45 mcg/kg Repeat Dose Instructions Before the second dose, be sure to contact the clinical research center. Consider assessment and treatment at the clinical trial site (see section 5.4) Before the third dose, be sure to contact the site Should not be repeated in less than 24 hours Should not be repeated in less than 24 hours Should not be repeated in less than 5-7 days Should not be repeated in less than 24 hours Should not be repeated in less than 24 hours Must see site within 48-72 hours if more than 2 doses needed (see SOA for evaluation) Must see site within 48-72 hours if more than 3 doses needed For situations requiring higher doses, more frequent administration, multiple repeat doses, discussion with the study's medical supervisor and clinical consultant is recommended, and AT substitution should be considered.
Do not use antifibrinolytic agents in combination with BPA

Note: Doses of rFVIIa and aPCC are included for completeness. Additional management of bleeding episodes should be carried out according to the standard of care. It is expected that such patients without inhibitors should be routinely treated with FVIII and FIX.

Example 5 Thrombin Formation Reaction Added Shunt Agents in Plasma of Human Patients Treated with AT3SC-001 for Treatment of Hemophilia A

As indicated in Example 4, data obtained from plasma samples of patients with hemophilia A with and without inhibitors show an improved response of thrombin formation to replacement factors and bypass agents (BPA) after administration of patients with a dose of AT3SC-001. In particular, data from the Phase II OLE study demonstrate that subcutaneous dosing of AT3SC-001 provides improved thrombin production in post-dose plasma samples compared to pre-dose plasma samples supplemented with BPA; suggesting that lower doses of BPA could potentially be used to achieve a similar hemostatic effect.

To investigate the response of thrombin formation to BPA in selected plasma samples from patients with AT3SC-001 mediated AT reduction, platelet-poor plasma was collected from 8 patients with hemophilia A (7 without inhibitors, 1 with inhibitors) both before and after AT3SC treatment. -001. AT activity in the patient's plasma was measured and normalized to normal pool plasma using a chromogenic assay based on FXa activity (SIEMENS INNOVANCE® Antithrombin). Different doses of BPA were added to plasma ex vivo: aPCC (0.5 or 1 U/ml; corresponding to doses of 37.5 and 75 U/kg, respectively) or rFVIIa (0.75, 1.75 or 2.5 µg/ml ; corresponds to doses of 27, 63 and 90 mcg/kg). Thrombin formation response and AT activity levels were monitored by plotting thrombin formation curves using a calibrated automatic thrombinoscope (tissue factor=1 pM). All reagents, including the thrombin calibrator, FluCa kit, and PPP-Reagent-Low (1 pM tissue factor [TF] and 4 µM phospholipid) were obtained from Thrombinoscope BV.

The data presented in Figures 25A-26N demonstrate enhanced thrombin formation in plasma samples obtained from patients after achieving a decrease in AT with the introduction of AT3SC-001. Indeed, only a decrease in AT led to an increase in peak thrombin formation. Both aPCC and rFVIIa caused a linear increase in peak thrombin formation when added to patient plasma treated with AT3SC-001. Lower doses of aPCC were sufficient to produce peak levels of thrombin formation equivalent to those achieved with a full dose of aPCC in plasma prior to AT3SC-001 in hemophilia A patients with inhibitors (FIGS. 25A and 25B) or without inhibitors (FIG. 26A). -26N). In the plasma of all patients, the level of thrombin formation approached, but remained below the lower limit of normal (LLN) range when all amounts of rFVIIa were added to AT-reduced plasma.

Thus, enhanced thrombin production was achieved with AT3SC-001 administration in post-dose plasma samples compared to pre-dose plasma samples with the addition of bypass agents. These results also support the use of lower doses of bypass agents compared to recommended doses to achieve hemostasis in patients who bleed during treatment with AT3SC-001 as described in Example 4 above, including, for example, reduced initial doses of replacement factor or bypass. agent and a reduced maximum dose of replacement factor or bypass agent, a minimum interval between doses, and no repeat dose of replacement factor or bypass agent less than 24 hours apart (with the exception of rFVIIa).

Example 6 Quantitative Systemic Pharmacology (QSP) for Predicting Thrombin Generation with Concomitant AT Decrease and Factor Replacement.

Hemophilia A and B are bleeding disorders characterized by insufficient thrombin production due to a lack of factors VIII and IX, respectively. As described above, AT3SC-001 is a once-monthly subcutaneous investigational RNAi therapeutic targeted antithrombin (AT) as a means to improve thrombin formation and promote hemostasis in hemophilia A and hemophilia B patients with or without inhibitors. Patients receiving AT3SC-001, however, may experience bleeding during treatment, which may require replacement factor treatment. As described above, clinical and ex vivo data support the use of lower doses of bypass agents and replacement factors compared to the recommended doses to achieve hemostasis in bleeding patients during AT3SC-001 treatment.

To further justify the use of lower doses of bypass agents and replacement factors compared to the recommended doses to achieve hemostasis in patients with bleeding during treatment with AT3SC-001, as well as to provide a better understanding of the relationship between AT levels, factor dose administration, and thrombin formation ( TG), the Quantitative Systems Pharmacology (QSP) in silico kinetic model describing the coagulation cascade (Nayak, et al. (2015) CPT: pharmacometrics & systems pharmacology 4.7: 396-405) was used to model the ex vivo (TG) thrombin generation assay. In particular, the model was described in terms of 66 reactions and 106 parameters, and the initial plasma concentrations of factors were varied to simulate hemophilia and rare bleeding disorders. In addition, since plasma concentrations of factors in different individuals vary ± 50%, Monte Carlo methods were used to model the normal range of TG: mean - 140 nM; Range 50-250 nM.

The table below shows the input factor concentrations used for the model in different disease conditions.

ATIII 3400 On Fitusiran 1-100 VIII 0.7 (100 IU/dl) Hemophilia A 0,1,0,5,1,2,5,10,100 IX 90 (100 IU/dl) Hemophilia B 0,1,0,5,1,2,5,10,100 V 20 (100 U/dl) Factor V deficiency 0,1,0,5,1,2,5,10,100 VII 10 (100 U/dl) Factor VII deficiency 0,1,0,5,1,2,5,10,100 X 160 (100 U/dl) Factor X Deficiency 0,1,0,5,1,2,5,10,100

As shown in FIG. 27A-27C, data obtained using the QSP model predicted thrombin (TG) formation with a decrease in AT. In particular, the clinical normal range for TG was consistent with QSP predictions, and a correlation was observed between clinically measured and model predicted TG for patients with AT depression. In FIG. 27A shows the measured TG values from Phase I results (Parts A-C) and FIG. 27B depicts simulated TG values (based on AT knockdown), each represented as AT reduction quartiles. In FIG. 27C is a scatterplot depicting a strong correlation between simulated TG data and measured TG data.

The QSP in silico model was also used to quantify the non-linear relationship between TG, AT reduction, and factor VIII dose in severe hemophilia A (FIG. 28A). The presentation of the TG intensity map with AT reduction and factor VIII dosing demonstrates that 5-10 IU/kg factor VIII may be sufficient to normalize TG (FIG. 28B) at AT levels seen with AT3SC-001 (10-25%). Similarly, the presentation of the TG intensity map with AT reduction and factor IX dose demonstrates that 10-20 IU/kg factor IX at 10-25% AT may be sufficient to normalize TG (FIG. 28C).

Factor pharmacokinetics (single chamber model) and QSP model were combined to simulate thrombin peak potential (PTP) as a function of time after factor dose. In FIG. 29B (20% base AT) with respect to FIG. 29A (100% AT) shows the effect of factor dosing over time in patients with severe hemophilia A and demonstrates that lowering AT increases thrombin production both at peak times and at times after FVIII administration for bleeding during treatment.

--->

SEQUENCE LIST

<110> GENZYME CORPORATION

<120> METHODS AND COMPOSITIONS FOR TREATMENT OF BLEEDING IN A PATIENT

HEMOPHILIA

<130> 117811-02720

<140>

<141>

<150> 62/530.518

<151> 2017-07-10

<150> 62/599.223

<151> 2017-12-15

<150> 62/614,111

<151> 2018-01-05

<150> 62/673.424

<151> 2018-05-18

<160> 16

<170>PatentIn version 3.5

<210> 1

<211> 1599

<212> DNA

<213> Homo sapiens

<400> 1

tctgccccac cctgtcctct ggaacctctg cgagatttag aggaaagaac cagttttcag 60

gcggattgcc tcagatcaca ctatctccac ttgcccagcc ctgtggaaga ttagcggcca 120

tgtattccaa tgtgatagga actgtaacct ctggaaaaag gaaggtttat cttttgtcct 180

tgctgctcat tggcttctgg gactgcgtga cctgtcacgg gagccctgtg gacatctgca 240

cagccaagcc gcgggacatt cccatgaatc ccatgtgcat ttaccgctcc ccggagaaga 300

aggcaactga ggatgagggc tcagaacaga agatcccggga ggccaccaac cggcgtgtct 360

gggaactgtc caaggccaat tcccgctttg ctaccacttt ctatcagcac ctggcagatt 420

ccaagaatga caatgataac attttcctgt cacccctgag tatctccacg gcttttgcta 480

tgaccaagct gggtgcctgt aatgacaccc tccagcaact gatggaggta tttaagtttg 540

acaccatatc tgagaaaaca tctgatcaga tccacttctt ctttgccaaa ctgaactgcc 600

gactctatcg aaaagccaac aaatcctcca agttagtatc agccaatcgc ctttttggag 660

acaaatccct taccttcaat gagacctacc aggacatcag tgagttggta tatggagcca 720

agctccagcc cctggacttc aaggaaaatg cagagcaatc cagagcggcc atcaacaaat 780

gggtgtccaa taagaccgaa ggccgaatca ccgatgtcat tccctcggaa gccatcaatg 840

agctcactgt tctggtgctg gttaacacca tttacttcaa gggcctgtgg aagtcaaagt 900

tcagccctga gaacacaagg aaggaactgt tctacaaggc tgatggagag tcgtgttcag 960

catctatgat gtacggaa ggcaagttcc gttatcggcg cgtggctgaa ggcacccagg 1020

tgcttgagtt gcccttcaaa ggtgatgaca tcaccatggt cctcatcttg cccaagcctg 1080

agaagagcct ggccaaggta gagaaggaac tcaccccaga ggtgctgcaa gagtggctgg 1140

atgaattgga ggagatgatg ctggtggtcc acatgccccg cttccgcatt gaggacggct 1200

tcagtttgaa ggagcagctg caagacatgg gccttgtcga tctgttcagc cctgaaaagt 1260

ccaaactccc aggtattgtt gcagaaggcc gagatgacct ctatgtctca gatgcattcc 1320

ataaggcatt tcttgaggta aatgaagaag gcagtgaagc agctgcaagt accgctgttg 1380

tgattgctgg ccgttcgcta aaccccaaca gggtgacttt caaggccaac aggcctttcc 1440

tggtttttat aagagaagtt cctctgaaca ctattatctt catgggcaga gtagccaacc 1500

cttgtgttaa gtaaaatgtt cttattcttt gcacctcttc ctatttttgg tttgtgaaca 1560

gaagtaaaaa taaatacaaa ctacttccat ctcacatta 1599

<210> 2

<211> 1545

<212> DNA

<213> Macaca mulatta

<400> 2

ggcacgagga ccatctccac ttgcccagcc ctgtggaaga ttagcgacca tgtattccaa 60

tgtgatagga accgtagcct ctggaaaaag gaaggtttat cttctgtcct tgctgctcat 120

tggcctctgg gactgtatga cctgtcacgg gagccctgtg gacatctgca cagccaagcc 180

gcgggacatt cccatgaatc ccatgtgcat ttaccgctcc ccggagaaga aggcaactga 240

ggatgagggc tcagaacaga agatccccga ggccaccaac cggcgcgtct gggaactgtc 300

caaggccaat tcccgctttg ctaccacttt ctatcagcac ctggcagatt ccaagaacga 360

caaggataac attttcctgt cacccctgag tgtctccacg gcttttgcta tgaccaagct 420

gggtgcctgt aatgacaccc tcaagcaact gatggaggta tttaagtttg acaccatatc 480

tgagaaaaca tctgatcaga tccacttctt ctttgccaaa ctgaactgcc gactctatcg 540

aaaagccaac aaatcctcca agttagtatc agccaatcgc ctttttggag acaaatccct 600

taccttcaat gagacctacc aggacatcag tgagttggta tacggagcca agctccagcc 660

cctggacttc aaggaaaatg cagagcaatc cagagcggcc atcaacaaat gggtgtccaa 720

taagaccgaa ggccgaatca ccgatgtcat tcccccggaa gccatcaacg agctcactgt 780

tctggtgctg gttaacacca tttacttcaa gggcctgtgg aagtcaaagt ttagccctga 840

gaacacaagg atggaaccgt tctacaaggc tgatggagag tcgtgttcag cgtctatgat 900

gtacggaa ggcaagttct gttatcggcg cgtggctgaa ggcacccagg tgcttgagtt 960

gcccttcaag ggtgatgaca tcaccatggt gctcatcctg cccaagcctg agaagagcct 1020

gaccaaggtg gagcaggaac tcaccccaga ggtgctgcag gagtggctgg atgagttgga 1080

ggagatgatg ctggtggttc acatgccccg cttccgcatt gaggacggct tcagtttgaa 1140

ggagcagctg caagacatgg gccttgtcga tctgttcagc cctgaaaagt ccaaactccc 1200

aggtattgtt gcagaaggcc gggatgacct ctatgtctcc gatgcattcc ataaggcatt 1260

tcttgaggta aatgaagaag gcagtgaagc agctgcaagt accgccattg ggattgctgg 1320

ccgttcgcta aaccccaaca gggtgacctt caaggccaac aggcctttcc tggtttttat 1380

aagagaagtt cctctgaaca ctattatctt catgggcaga gtagccaacc cttgtgtgag 1440

ctaaactgtt cttattcttt gtacctcttc ctattttggt ttgtgaatag aagtaaaaat 1500

aaatacaact actcccatct tacattaaaa aaaaaaaaaa aaaaa 1545

<210> 3

<211> 2171

<212> DNA

<213> Mus musculus

<400> 3

attaggtaatt ttagaaatag atctgatttg tatctgagac attttagtga agtggtgaga 60

tataagacat aatcagaaga catatctacc tgaagacttt aaggggagag ctccctcccc 120

cacctggcct ctggacctct cagatttagg ggaaagaacc agttttcgga gtgatcgtct 180

cagtcagcac catctctgta ggagcatcgg ccatgtattc ccctggggca ggaagtgggg 240

ctgctggtga gaggaagctt tgtctcctct ctctgctcct catcggtgcc ttgggctgtg 300

ctatctgtca cggaaaccct gtggacgaca tctgcatagc gaagccccga gacatccccg 360

tgaatccctt gtgcatttac cgctcccctg ggaagaaggc caccgaggag gatggctcag 420

agcagaaggt tccagaagcc accaaccggc gggtctggga actgtccaag gccaattcgc 480

gatttgccac taacttctac cagcacctgg cagactccaa gaatgacaac gacaacattt 540

tcctgtcacc cttgagcatc tccactgctt ttgctatgac caagctgggt gcctgtaacg 600

acactctcaa gcagctgatg gaggttttta aatttgatac catctccgag aagacatccg 660

accagatcca cttcttcttt gccaaactga actgccgact ctatcgaaaa gccaacaagt 720

cctctgactt ggtatcagcc aaccgccttt ttggagacaa atccctcacc ttcaacgaga 780

gctatcaaga tgttagtgag gttgtctatg gagccaagct ccagcccctg gacttcaagg 840

agaatccgga gcaatccaga gtgaccatca acaactgggt agctaataag actgaaggcc 900

gcatcaaaga tgtcatccca cagggcgcca ttaacgagct cactgccctg gttctggtta 960

acaccattta cttcaagggc ctgtggaagt caaagttcag ccctgagaac acaaggaagg 1020

aaccgttcta taaggtcgat gggcagtcat gcccagtgcc tatgatgtac caggaaggca 1080

1140

atgacatcac catggtgctc atcctgccca agcctgagaa gagcctggcc aaggtggagc 1200

aggagctcac cccagagctg ctgcaggagt ggctggatga gctgtcagag actatgcttg 1260

tggtccacat gccccgcttc cgcaccgagg atggcttcag tctgaaggag cagctgcaag 1320

acatggggcct cattgatctc ttcagccctg aaaagtccca actcccaggg atcgttgctg 1380

gaggcaggga cgacctctat gtctccgacg cattccacaa agcatttctt gaggtaaatg 1440

aggaaggcag tgaagcagca gcgagtactt ctgtcgtgat tactggccgg tcactgaacc 1500

ccaatagggt gaccttcaag gccaacaggc ccttcctggt tcttataagg gaagttgcac 1560

tgaacactat tatattcatg gggagagtgg ctaatccttg tgtgaactaa aatattctta 1620

atctttgcac cttttcctac tttggtgttt gtgaatagaa gtaaaaataa atacgactgc 1680

cacctcacga gaatggactt ttccacttga agacgagaga ctggagtaca gatgctacac 1740

cacttttggg caagtgaagg gggagcagcc agccacggtg gcacaaacct atatcctggt 1800

gcttttgaag gtagaagcag ggcggtcagg agttaaggcc agttgaggct gggctgcaga 1860

gtgaaagacc atgtctcaag atggtctttc tcctccccaa agtagaaaag aaaaccataa 1920

aaacaagagg taaatatatt actatttcat cttagagggat agcaggcatc ttgaaagggt 1980

agagggacct taaattctca ttattgcccc catactacaa actaaaaaac aaacccgaat 2040

caatctccca taaagacaga gattcaaata agagtattaa acgttttatt tctcaaacca 2100

ctcacatgca taatgttctt atacacagtg tcaaaataaa gagaaatgca tttttataca 2160

aaaaaaaaaa 2171

<210> 4

<211> 1561

<212> DNA

<213> Rattus norvegicus

<400> 4

cggagggatt gctcagcact gtctcccgg cttctctgca gaagcgtcca ccatgtattc 60

cccgggaata ggaagtgcgg ttgctggaga gaggaagctt tgtctcctct ctctgctact 120

cattggtgcc ttgggctgtg ctgtctgtca tggaaaccct gtggacgaca tctgcatagc 180

gaagccccga gacatccccg tgaaccccat gtgcatttac cgctcccctg cgaagaaggc 240

cacggaggag gatgtcctag agcagaaggt tccggaagcc accaaccggc gggtctggga 300

actgtccaag gccaattctc gatttgccac taacttctat cagcacctgg cagactccaa 360

gaacgacaac gacaacattt tcctgtcacc cttgagcatc tccacggcgt ttgctatgac 420

caagctgggt gcttgtaata acaccctcaa gcagctgatg gaggttttta aatttgatac 480

catctccgag aagacatccg accagatcca cttcttcttt gccaaactga actgccgact 540

ctatcgaaaa gccaacaagt cctctaactt ggtgtcagcc aaccgccttt ttggagacaa 600

atcccttacc ttcaatgaga gctatcaaga cgttagtgag attgtctatg gagccaagct 660

tcagcccctg gacttcaagg agaatccgga gcaatccaga gtgaccatca acaactgggt 720

agctaataag actgaaggcc gcatcaaaga cgtcatcccc caaggagcca ttgatgagct 780

cactgccctg gtgctggtta acaccattta cttcaagggc ctgtggaagt caaagttcag 840

ccctgagaac acaaggaagg aaccattcca caaagttgat gggcagtcat gcctggtgcc 900

catgatgtac caggaaggca aattcaaata caggcgtgtg ggagagggta cccaggtgct 960

agagatgccc ttcaaggggg acgacatcac catggtgctc atcctgccca agcctgagaa 1020

gagcctggct aaggtggagc aggaactcac cccggagctg ctgcaggagt ggctggatga 1080

gctgtcggag gtcatgcttg tggtccacgt gccccgcttc cgcatcgagg acagcttcag 1140

tctgaagggag cagctgcaag acatgggcct tgttgatctc ttcagccctg agaagtccca 1200

actcccaggg atcattgctg aaggcaggga cgacctcttt gtctccgatg cattccacaa 1260

1320

1380

tcttataagg gaagtcgcac tgaacactat tatattcatg gggagagtgt ctaatccttg 1440

tgtgaactaa aatattctta atctttgcac cttttcctat ctcggtgttt gttaatggaa 1500

1560

a 1561

<210> 5

<211> 1599

<212> DNA

<213> Homo sapiens

<400> 5

taatgtgaga tggaagtagt ttgtatttat ttttacttct gttcacaaac caaaaatagg 60

aagaggtgca aagaataaga acattttact taacacaagg gttggctact ctgcccatga 120

agataatagt gttcagagga acttctctta taaaaaccag gaaaggcctg ttggccttga 180

aagtcaccct gttggggttt agcgaacggc cagcaatcac aacagcggta cttgcagctg 240

cttcactgcc ttcttcattt acctcaagaa atgccttatg gaatgcatct gagacataga 300

ggtcatctcg gccttctgca acaatacctg ggagtttgga cttttcaggg ctgaacagat 360

cgacaaggcc catgtcttgc agctgctcct tcaaactgaa gccgtcctca atgcggaagc 420

ggggcatgtg gaccaccagc atcatctcct ccaattcatc cagccactct tgcagcacct 480

ctggggtgag ttccttctct accttggcca ggctcttctc aggcttgggc aagatgagga 540

ccatggtgat gtcatcacct ttgaagggca actcaagcac ctgggtgcct tcagccacgc 600

gccgataacg gaacttgcct tcctggtaca tcatagatgc tgaacacgac tctccatcag 660

ccttgtagaa cagttccttc cttgtgttct cagggctgaa ctttgacttc cacaggccct 720

tgaagtaaat ggtgttaacc agcaccagaa cagtgagctc attgatggct tccgagggaa 780

tgacatcggt gattcggcct tcggtcttat tggacaccca tttgttgatg gccgctctgg 840

attgctctgc attttccttg aagtccaggg gctggagctt ggctccatat accaactcac 900

tgatgtcctg gtaggtctca ttgaaggtaa gggatttgtc tccaaaaagg cgattggctg 960

atactaactt ggaggatttg ttggcttttc gatagagtcg gcagttcagt ttggcaaaga 1020

agaagtggat ctgatcagat gttttctcag atatggtgtc aaacttaaat acctccatca 1080

gttgctggag ggtgtcatta caggcaccca gcttggtcat agcaaaagcc gtggagatac 1140

tcaggggtga caggaaaatg ttatcattgt cattcttgga atctgccagg tgctgataga 1200

aagtggtagc aaagcgggaa ttggccttgg acagttccca gacacgccgg ttggtggcct 1260

ccgggatctt ctgttctgag ccctcatcct cagttgcctt cttctccggg gagcggtaaa 1320

tgcacatggg attcatggga atgtcccgcg gcttggctgt gcagatgtcc acagggctcc 1380

cgtgacaggt cacgcagtcc cagaagccaa tgagcagcaa ggacaaaaga taaaccttcc 1440

tttttccaga ggttacagtt cctatcacat tggaatacat ggccgctaat cttccacagg 1500

gctgggcaag tggagatagt gtgatctgag gcaatccgcc tgaaaactgg ttctttcctc 1560

taaatctcgc agaggttcca gaggacaggg tggggcaga 1599

<210> 6

<211> 1545

<212> DNA

<213> Macaca mulatta

<400> 6

tttttttttt ttttttttta atgtaagatg ggagtagttg tatttatttt tacttctatt 60

cacaaaccaa aataggaaga ggtacaaaga ataagaacag tttagctcac acaagggttg 120

gctactctgc ccatgaagat aatagtgttc agaggaactt ctcttataaa aaccaggaaa 180

ggcctgttgg ccttgaaggt caccctgttg gggtttagcg aacggccagc aatcccaatg 240

gcggtacttg cagctgcttc actgccttct tcatttacct caagaaatgc cttatggaat 300

gcatcggaga catagaggtc atcccggcct tctgcaacaa tacctgggag tttggacttt 360

tcagggctga acagatcgac aaggcccatg tcttgcagct gctccttcaa actgaagccg 420

tcctcaatgc ggaagcgggg catgtgaacc accagcatca tctcctccaa ctcatccagc 480

cactcctgca gcacctctgg ggtgagttcc tgctccacct tggtcaggct cttctcaggc 540

ttgggcagga tgagcaccat ggtgatgtca tcacccttga agggcaactc aagcacctgg 600

gtgccttcag ccacgcgccg ataacagaac ttgccttcct ggtacatcat agacgctgaa 660

cacgactctc catcagcctt gtagaacggt tccatccttg tgttctcagg gctaaacttt 720

gacttccaca ggcccttgaa gtaaatggtg ttaaccagca ccagaacagt gagctcgttg 780

atggcttccg ggggaatgac atcggtgatt cggccttcgg tcttattggga cacccatttg 840

ttgatggccg ctctggattg ctctgcattt tccttgaagt ccaggggctg gagcttggct 900

ccgtatacca actcactgat gtcctggtag gtctcattga aggtaaggga tttgtctcca 960

aaaaggcgat tggctgatac taacttggag gatttgttgg cttttcgata gagtcggcag 1020

ttcagtttgg caaagaagaa gtggatctga tcagatgttt tctcagatat ggtgtcaaac 1080

ttaaatacct ccatcagttg cttgagggtg tcattacagg cacccagctt ggtcatagca 1140

aaagccgtgg agacactcag gggtgacagg aaaatgttat ccttgtcgtt cttggaatct 1200

gccaggtgct gatagaaagt ggtagcaaag cgggaattgg ccttggacag ttcccagacg 1260

cgccggttgg tggcctcggg gatcttctgt tctgagccct catcctcagt tgccttcttc 1320

tccggggagc ggtaaatgca catgggattc atgggaatgt cccgcggctt ggctgtgcag 1380

atgtccacag ggctcccgtg acaggtcata cagtcccaga ggccaatgag cagcaaggac 1440

agaagataaa ccttcctttt tccagaggct acggttccta tcacattgga atacatggtc 1500

gctaatcttc cacagggctg ggcaagtgga gatggtcctc gtgcc 1545

<210> 7

<211> 2171

<212> DNA

<213> Mus musculus

<400> 7

tttttttttt ttgtataaaa atgcatttct ctttattttg acactgtgta taagaacatt 60

atgcatgtga gtggtttgag aaataaaacg tttaatactc ttatttgaat ctctgtcttt 120

180

aaggtccctc taccctttca agatgcctgc tatcctctaa gatgaaatag taatatattt 240

300

tggtctttca ctctgcagcc cagcctcaac tggccttaac tcctgaccgc cctgcttcta 360

ccttcaaaag caccaggata taggtttgtg ccaccgtggc tggctgctcc cccttcactt 420

gcccaaaagt ggtgtagcat ctgtactcca gtctctcgtc ttcaagtgga aaagtccatt 480

ctcgtgaggt ggcagtcgta tttattttta cttctattca caaacaccaa agtaggaaaa 540

ggtgcaaaga ttaagaatat tttagttcac acaaggatta gccactctcc ccatgaatat 600

aatagtgttc agtgcaactt cccttataag aaccaggaag ggcctgttgg ccttgaaggt 660

caccctattg gggttcagtg accggccagt aatcacgaca gaagtactcg ctgctgcttc 720

actgccttcc tcatttacct caagaaatgc tttgtggaat gcgtcggaga catagaggtc 780

840

gaggcccatg tcttgcagct gctccttcag actgaagcca tcctcggtgc ggaagcgggg 900

catgtggacc acaagcatag tctctgacag ctcatccagc cactcctgca gcagctctgg 960

ggtgagctcc tgctccacct tggccaggct cttctcaggc ttgggcagga tgagcaccat 1020

ggtgatgtca tcccccttga agggcagctc tagcacctgg gtgccctctg ccacgcgccg 1080

gtatttgaat ttgccttcct ggtacatcat aggcactggg catgactgcc catcgacctt 1140

atagaacggt tccttccttg tgttctcagg gctgaacttt gacttccaca ggcccttgaa 1200

gtaaatggtg ttaaccagaa ccagggcagt gagctcgtta atggcgccct gtgggatgac 1260

atctttgatg cggccttcag tcttattagc tacccagttg ttgatggtca ctctggattg 1320

ctccggattc tccttgaagt ccaggggctg gagcttggct ccatagacaa cctcactaac 1380

atcttgatag ctctcgttga aggtgaggga tttgtctcca aaaaggcggt tggctgatac 1440

caagtcagag gacttgttgg cttttcgata gagtcggcag ttcagtttgg caaagaagaa 1500

gtggatctgg tcggatgtct tctcggagat ggtatcaaat ttaaaaacct ccatcagctg 1560

cttgagagtg tcgttacagg cacccagctt ggtcatagca aaagcagtgg agatgctcaa 1620

gggtgacagg aaaatgttgt cgttgtcatt cttggagtct gccaggtgct ggtagaagtt 1680

agtggcaaat cgcgaattgg ccttggacag ttcccagacc cgccggttgg tggcttctgg 1740

aaccttctgc tctgagccat ccctcctcggt ggccttcttc ccaggggagc ggtaaatgca 1800

caagggattc acggggatgt ctcggggctt cgctatgcag atgtcgtcca cagggtttcc 1860

gtgacagata gcacagccca aggcaccgat gaggagcaga gagaggagac aaagcttcct 1920

ctcaccagca gccccacttc ctgccccagg ggaatacatg gccgatgctc ctacagagat 1980

ggtgctgact gagacgatca ctccgaaaac tggttctttc ccctaaatct gagaggtcca 2040

gaggccaggt gggggaggga gctctcccct taaagtcttc aggtagatat gtcttctgat 2100

tgtctcagat acaaatcaga tctatttcta 2160

aattaccta t 2171

<210> 8

<211> 1561

<212> DNA

<213> Rattus norvegicus

<400> 8

tttttttttt tttttttttt tttttttttt tttttgaggt ggcagtcata tttattttta 60

cttccattaa caaacaccga gataggaaaa ggtgcaaaga ttaagaatat tttagttcac 120

acaaggatta gacactctcc ccatgaatat aatagtgttc agtgcgactt cccttataag 180

aaccaggaag ggcctgttgg ccttgaaggt caccctactg gggttcagtg accggccagt 240

aatcacgaca gaagtactcg ctgctgcttc actgccttcc tcatttacct caagaaacgc 300

tttgtggaat gcatcggaga caaagaggtc gtccctgcct tcagcaatga tccctgggag 360

ttgggacttc tcagggctga agagatcaac aaggcccatg tcttgcagct gctccttcag 420

actgaagctg tcctcgatgc ggaagcgggg cacgtggacc acaagcatga cctccgacag 480

ctcatccagc cactcctgca gcagctccgg ggtgagttcc tgctccacct tagccaggct 540

cttctcaggc ttgggcagga tgagcaccat ggtgatgtcg tcccccttga agggcatctc 600

tagcacctgg gtaccctctc ccacacgcct gtatttgaat ttgccttcct ggtacatcat 660

gggcaccagg catgactgcc catcaacttt gtggaatggt tccttccttg tgttctcagg 720

gctgaacttt gacttccaca ggcccttgaa gtaaatggtg ttaaccagca ccagggcagt 780

gagctcatca atggctcctt gggggatgac gtctttgatg cggccttcag tcttattagc 840

tacccagttg ttgatggtca ctctggattg ctccggattc tccttgaagt ccaggggctg 900

aagcttggct ccatagacaa tctcactaac gtcttgatag ctctcattga aggtaaggga 960

tttgtctcca aaaaggcggt tggctgacac caagttagag gacttgttgg cttttcgata 1020

gagtcggcag ttcagtttgg caaagaagaa gtggatctgg tcggatgtct tctcggagat 1080

ggtatcaaat ttaaaaacct ccatcagctg cttgaggggtg ttattacaag cacccagctt 1140

ggtcatagca aacgccgtgg agatgctcaa gggtgacagg aaaatgttgt cgttgtcgtt 1200

cttggagtct gccaggtgct gatagaagtt agtggcaaat cgagaattgg ccttggacag 1260

ttcccagacc cgccggttgg tggcttccgg aaccttctgc tctaggacat cctcctccgt 1320

ggccttcttc gcaggggagc ggtaaatgca catggggttc acggggatgt ctcggggctt 1380

cgctatgcag atgtcgtcca cagggtttcc atgacagaca gcacagccca aggcaccaat 1440

gagtagcaga gagaggagac aaagcttcct ctctccagca accgcacttc ctattcccgg 1500

ggaatacatg gtggacgctt ctgcagagaa gccgtggaga cagtgctgag caatccctcc 1560

g 1561

<210> 9

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of unknown: RFGF peptide"

<400> 9

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

<210> 10

<211> 11

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of unknown: RFGF analogue peptide"

<400> 10

Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro

1 5 10

<210> 11

<211> 13

<212> PRT

<213> human immunodeficiency virus

<400> 11

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln

1 5 10

<210> 12

<211> 16

<212> PRT

<213> Drosophila sp.

<400> 12

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210> 13

<211> 21

<212> RNA

<213> Faux Sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

oligonucleotide"

<400> 13

gguuaacacc auuuacuuca a 21

<210> 14

<211> 23

<212> RNA

<213> Faux Sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

oligonucleotide"

<400> 14

uugaaguaaa ugguguuaac cag 23

<210> 15

<211> 23

<212> RNA

<213> Faux Sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

oligonucleotide"

<400> 15

uugaaguaaa ugguguuaac cag 23

<210> 16

<211> 21

<212> RNA

<213> Faux Sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

oligonucleotide"

<400> 16

gguuaacacc auuuacuuca a 21

<---

Claims (92)

1. A method of treating a bleeding episode in a human hemophilia A or B patient with inhibitors who is undergoing routine hemophilia prophylaxis with a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of an activated prothrombin complex concentrate (aPCC), wherein the therapeutically effective amount of aPCC is not more than about 50 U/kg per dose. 2. A method for treating a bleeding episode in a patient with hemophilia A or B with inhibitors who is undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of an activated prothrombin complex concentrate (aPCC), wherein the therapeutically effective amount of aPCC is approximately 30 U/kg per dose. 3. The method of claim 1 wherein the therapeutically effective amount of aPCC is about 10 U/kg per dose. 4. The method of any one of claims 1 to 3, wherein the administration of aPCC is repeated, if necessary, at least after about 24 hours. 5. The method of claim 4, insofar as it depends on claim 1, wherein the therapeutically effective amount of aPCC is approximately 50 U/kg per dose. 6. The method of claim 1, wherein the therapeutically effective amount of aPCC is no more than about 50 U/kg per 24 hours. 7. A method for treating a bleeding episode in a patient with hemophilia A or B with inhibitors who is undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of recombinant factor VIIa (rFVIIa), wherein the therapeutically effective amount of rFVIIa is not more than about 45 μg/kg. 8. A method of treating a bleeding episode in a patient with hemophilia A or B with inhibitors who is undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of recombinant factor VIIa (rFVIIa), wherein the therapeutically effective amount of rFVIIa is approximately 45 μg/kg. 9. The method of claim 7, wherein the therapeutically effective dose of rFVIIa is approximately 10 μg/kg per dose. 10. The method of any one of claims 7-9, wherein the administration of rFVIIa is repeated, if necessary, after at least about 2 hours. 11. The method of claim 7, wherein the therapeutically effective amount of rFVIIa is no more than about 45 μg/kg for 2 hours. 12. The method according to any one of claims 1-11, wherein the nucleotide sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) and the nucleotide sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' ( SEQ ID NO: 14). 13. The method according to any one of paragraphs. 1-12, wherein routine prophylaxis comprises administering a dose of about 30 mg to about 90 mg of the dsRNA molecule by subcutaneous injection at about once a month or about once every two months intervals. 14. The method of any one of claims 1-12, wherein the routine prophylaxis comprises administering a dose of about 50 mg of the dsRNA molecule by subcutaneous injection at an interval of about once a month or about once every two months. 15. The method of any one of claims 1 to 12, wherein the routine prophylaxis comprises administering a dose of about 80 mg of the dsRNA molecule by subcutaneous injection at an interval of about once a month or about once every two months. 16. A method of treating a bleeding episode in a patient with hemophilia A or B with inhibitors who is undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of an activated prothrombin complex (aPCC), wherein the therapeutically effective amount of aPCC is not more than about 50 U/kg per dose. 17. A method for treating a bleeding episode in a patient with hemophilia A or B with inhibitors who is undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of an activated prothrombin complex (aPCC), wherein the therapeutically effective amount of aPCC is approximately 50 U/kg per dose. 18. A method for treating a bleeding episode in a patient with hemophilia A or B with inhibitors who is undergoing routine hemophilia prophylaxis using a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand; где смысловая нить содержит нуклеотидную последовательность 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13) и антисмысловая нить содержит нуклеотидную последовательность 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), где a, c, g и u представляют is 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, C, G and U, respectively; and s is a phosphorothioate bond; And where the ligand is conjugated to the 3' end of the sense strand as shown in the following diagram: , where X is O, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of an activated prothrombin complex (aPCC), wherein the therapeutically effective amount of aPCC is approximately 30 U/kg per dose. 19. The method according to any one of paragraphs. 16-18, further comprising repeating the administration step at least 24 hours later. 20. A method of treating a bleeding episode in a human hemophilia A or B patient with inhibitors who is undergoing routine hemophilia prophylaxis with a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand, moreover, the nucleotide sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf represent 2'-fluoro A, C, G and U, respectively; and s represents a phosphorothioate bond; And while the 3'-end of the sense strand is conjugated to the ligand, as shown in the following diagram: , where X is O; the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of recombinant factor VIIa (rFVIIa), wherein the therapeutically effective amount of rFVIIa is not more than about 45 μg/kg. 21. A method of treating a bleeding episode in a human hemophilia A or B patient with inhibitors who is undergoing routine hemophilia prophylaxis with a double-stranded ribonucleic acid (dsRNA) molecule, where the dsRNA molecule contains a sense strand and an antisense strand, moreover, the nucleotide sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 14), where a, c, g and u are 2'-O-methyl (2'-OMe) A, C, G and U, respectively; Af, Cf, Gf and Uf represent 2'-fluoro A, C, G and U, respectively; and s represents a phosphorothioate bond; And while the 3'-end of the sense strand is conjugated to the ligand, as shown in the following diagram: , where X is O; And the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of recombinant factor VIIa (rFVIIa), wherein the therapeutically effective amount of rFVIIa is approximately 45 μg/kg. 22. The method of claim 20 or 21, further comprising repeating the administration step after at least 2 hours. 23. The method according to any one of paragraphs. 1-22, in which the bleeding episode is not associated with surgery. 24. The method of any one of claims 1 to 23, wherein the bleeding episode is spontaneous. 25. The method of any one of claims 1 to 23, wherein the bleeding episode results from an injury. 26. The method according to any one of paragraphs. 1-22, in which the patient is at risk of a bleeding episode as a result of surgery and where the patient is treated with BPA in the perioperative period. 27. The method of any one of claims 1 to 26, wherein the bleeding episode occurs in or occurs internally in a joint, muscle, skin, or mucosa. 28. The method according to any one of claims 1 to 27, wherein the dsRNA molecule is administered to the patient in the form of a pharmaceutical composition. 29. The method of claim 28 wherein the pharmaceutical composition comprises phosphate buffered saline (PBS). 30. The method of any one of claims 1-29 further comprising measuring thrombin levels in the patient. 31. The method of any one of claims 1-30, further comprising measuring levels of the replacement factor or bypass agent in the patient. 32. The method according to any one of paragraphs. 1-31, where the patient is a hemophilia A patient with inhibitors. 33. The method according to any one of claims 1 to 31, wherein the patient is a hemophilia B patient with inhibitors. 34. The method of any one of claims 1 to 33, wherein the bleeding episode is a minor bleeding episode. 35. The method of any one of claims 1-33, wherein the bleeding episode is a moderate bleeding episode. 36. The method of any one of claims 1-33, wherein the bleeding episode is a severe bleeding episode.

Images