Serpinc1 irna compositions and methods of use thereof

Abstract

To provide novel therapies for subjects with bleeding disorders, such as hemophilia.SOLUTION: The present invention provides iRNAs, e.g., double-stranded ribonucleic acids (dsRNAs), compositions that target Serpinc1 genes, and methods of inhibiting Serpinc1 expression using the iRNAs, e.g., dsRNAs, compositions, and methods of treating subjects having bleeding disorders, such as hemophiliacs.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 638,952, filed April 26, 2012, U.S. Provisional Patent Application No. 61 / 669,249, filed July 9, 2012, U.S. Provisional Patent Application No. 61 / 734,573, filed December 7, 2012, and U.S. Patent Application Publication No. 13 / 837,129, filed March 15, 2013. Each of the foregoing applications is incorporated herein by reference in its entirety. [Background technology]

[0002] Serpinc1 is a member of the serine proteinase inhibitor (serpin) superfamily. Serpinc1 is a plasma protease inhibitor that inhibits thrombin and other activating serine proteases of the coagulation system, such as factors X, IX, XI, XII, and VII, thus regulating the blood coagulation cascade (see, for example, Figure 1). The anticoagulant activity of Serpinc1 is enhanced by the presence of heparin and other related glycosaminoglycans, which catalyze the formation of the thrombin:antithrombin (TAT) complex.

[0003] Bleeding disorders, whether inherited or acquired, are conditions characterized by insufficient blood clotting. For example, hemophilia is a group of inherited bleeding disorders that impair the body's ability to control blood clotting or coagulation. Hemophilia A is a recessive X-linked genetic disorder involving the absence of functional clotting factor VIII and accounts for 80% of hemophilia cases. Hemophilia B is a recessive X-linked genetic disorder involving the absence of functional clotting factor IX. It comprises approximately 20% of hemophilia cases. Hemophilia C is an autosomal genetic disorder involving the absence of functional clotting factor XI. Hemophilia C is not completely recessive, as heterozygous individuals also exhibit increased bleeding.

[0004] Although there is currently no cure for hemophilia, it can be controlled by regular infusions of the missing clotting factor, such as factor VIII in hemophilia A. However, some hemophilia patients develop antibodies (inhibitors) to the administered replacement factor and therefore become refractory to the replacement clotting factor. Bleeding in such subjects therefore cannot be adequately controlled.

[0005] For example, the development of high-titer inhibitors to factor VIII and other coagulation factors is the most serious complication of hemophilia therapy and makes bleeding extremely difficult to treat. Currently, the only strategies to stop bleeding in such subjects are the use of "bypassing agents," such as factor VIII inhibitor bypassing agents (FEIBA), and activated recombinant factor VII (rFVIIa), plasma exchange, continuous factor replacement, and immune tolerance therapy, none of which are completely effective. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need in the art for alternative therapeutic agents for subjects with bleeding disorders, such as hemophilia. [Means for solving the problem]

[0007] The present invention provides an iRNA composition that induces RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the Serpincl gene. The Serpincl gene may be present in a cell, such as a cell in a subject, such as a human. The present invention also provides methods and uses of the iRNA composition of the present invention for inhibiting Serpincl gene expression and / or treating subjects with disorders that benefit from inhibiting or reducing Serpincl gene expression, such as bleeding disorders such as hemophilia.

[0008] Thus, in one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting Serpinc1 expression, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides.

[0009] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting Serpincl expression. The dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differs from any one of the antisense sequences listed in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 by 3 nucleotides or less.

[0010] In one embodiment, the sense and antisense strands are selected from the group consisting of AD-50487.1, AD-50477.1, AD-50483.1, AD-50475.1, AD-50495.1, AD-50476.1, AD-50499.1, AD-50478.1, AD-50489.1, AD-50501.1, AD-50507.1, AD-50484.1, AD-50515.1, AD-50540.1, AD-50528.1, AD-50549.1, AD-50539.1, AD-50549.1, AD-50550.1, AD-50551.1, AD-50552.1, AD-50553.1, AD-50554.1, AD-50555.1, AD-50556.1, AD-50557.1, AD-50558.1, AD-50559.1, AD-50560.1, AD-50561.1, AD-50562.1, AD-50563.1, AD-50564.1, AD-50565.1, AD-50566.1, AD-50567.1, AD-50568.1, AD-50569.1, AD-50570.1, AD-50571.1, AD-50572.1, AD-50573.1, AD-50574.1, AD-50575.1, AD-50576.1, AD-50577.1, AD-50578.1, AD-50579.1, AD-50580.1, AD-5 0534.1, AD-50527.1, AD-50514.1, AD-50509.1, AD-50529.1, AD-54944, AD-56813, AD-57205, AD-57214, and AD-57213, and any of the sequences listed in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to these sequences. In certain embodiments of the present invention, the dsRNA comprises at least one modified nucleotide. In one embodiment, the at least one modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base-containing nucleotides.

[0011] The complementary region of the dsRNA may be at least 17 nucleotides in length, 19-21 nucleotides in length, or 19 nucleotides in length.

[0012] In one embodiment, each strand of the dsRNA is 30 nucleotides or less in length.

[0013] At least one dsRNA strand may comprise a 3' overhang of at least 1 nucleotide or at least 2 nucleotides.

[0014] In certain embodiments, the dsRNA further comprises a ligand. In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA.

[0015] In some embodiments, the ligand attached through the bivalent or trivalent branched linker is one or more N-acetylgalactosamine (GalNAc) derivatives. In certain embodiments, the ligand is [ka] is.

[0016] In some embodiments, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram: [ka]

[0017] In another embodiment, the RNAi agent is conjugated to a ligand as shown in the following schematic diagram, where X is O or S: [ka]

[0018] In one embodiment, the complementary region of the dsRNA consists of one of the antisense sequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

[0019] In another embodiment, the dsRNA comprises a sense strand consisting of a sense strand sequence selected from any one of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and an antisense strand consisting of an antisense sequence selected from any one of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

[0020] In another aspect, the invention provides a cell containing a dsRNA of the invention.

[0021] In yet another aspect, the invention provides a vector encoding at least one dsRNA strand, wherein the dsRNA comprises a region of complementarity to at least a portion of an mRNA encoding Serpincl, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.

[0022] The region of complementarity may be at least 15 nucleotides in length or 19-21 nucleotides in length.

[0023] In a further aspect, the invention provides a cell comprising a vector encoding at least one dsRNA strand, wherein the dsRNA comprises a region of complementarity to at least a portion of an mRNA encoding Serpinc1, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.

[0024] In one aspect, the present invention provides a pharmaceutical composition for inhibiting Serpinc1 gene expression, comprising a dsRNA or vector of the present invention.

[0025] In one embodiment, the pharmaceutical composition further comprises a lipid formulation, such as an MC3, SNALP, or XTC formulation.

[0026] In another aspect, the present invention provides a method for inhibiting Serpinc1 expression in a cell, the method comprising contacting a cell with a dsRNA or vector of the present invention and maintaining the resulting cell for a time sufficient to achieve degradation of the mRNA transcript of the Serpinc1 gene, thereby inhibiting Serpinc1 gene expression in the cell.

[0027] The cell may be in a subject, such as a human subject, for example, a human subject suffering from a bleeding disorder such as hemophilia.

[0028] In one embodiment of the method of the present invention, Serpincl expression is inhibited by 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%.

[0029] In another aspect, the present invention provides a method for treating a subject with a disorder that would benefit from reduced Serpincl expression, such as a bleeding disorder such as hemophilia. The method comprises administering a therapeutically effective amount of a dsRNA or vector of the present invention to the subject, thereby treating the subject.

[0030] In one aspect, the present invention provides a method for preventing at least one symptom, such as bleeding, in a subject with a disorder that would benefit from reduced Serpincl expression, such as hemophilia. The method includes administering a therapeutically effective amount of an iRNA, such as a dsRNA, or vector of the present invention to the subject, thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced Serpincl expression.

[0031] The disorder may be a bleeding disorder, such as hemophilia.

[0032] In one embodiment, administration of the dsRNA to a subject causes increased blood clotting and / or decreased expression and / or accumulation of Serpinc1 protein.

[0033] In one embodiment, the dsRNA is conjugated to a ligand, for example, at the 3' end of the sense strand of the dsRNA. In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0034] In one embodiment, the dsRNA may be administered in a concentration of, for example, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 10 mg / kg, or about 0. .4mg / kg~about 5mg / kg, about 0.4mg / kg~about 10mg / kg, about 0.5mg / kg~about 5mg / kg, about 0.5mg / kg~about 10mg / kg, about 1mg / kg~about 5mg / kg kg, about 1 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg and about 0.01 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0035] For example, dsRNA may have a molecular weight of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35, 5.36, 5.37, 5.38, 5.39, 5.40, 5.41, 5.42, 5. 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0036] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, or about 25 to about 50 mg / kg. g / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, About 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 4 5 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / m g, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about Approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 30 to approximately 40 mg / kg, approximately 35 to approximately 40 mg / kg, approximately 0.5 to approximately 30 mg / kg, approximately 0.75 to approximately 30 m g / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.The compound may be administered at a dose of 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention. .

[0037] For example, the subject may have approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29 ... .3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21 A therapeutic amount of iRNA may be administered, such as about 0.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0038] For example, the dsRNA conjugated to the ligand may be administered to the subject once a week or twice a month.

[0039] In another aspect, the present invention provides a method for inhibiting Serpincl expression in a subject, the method comprising administering to the subject a therapeutically effective amount of a dsRNA or vector of the present invention, thereby inhibiting Serpincl expression in the subject.

[0040] In one embodiment, the dsRNA is conjugated to a ligand, for example, at the 3' end of the sense strand of the dsRNA. In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0041] In one embodiment, the dsRNA may be administered in a concentration of, for example, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 10 mg / kg, or about 0. .4mg / kg~about 5mg / kg, about 0.4mg / kg~about 10mg / kg, about 0.5mg / kg~about 5mg / kg, about 0.5mg / kg~about 10mg / kg, about 1mg / kg~about 5mg / kg kg, about 1 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg and about 0.01 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0042] For example, dsRNA may have a molecular weight of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35, 5.36, 5.37, 5.38, 5.39, 5.40, 5.41, 5.42, 5. 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0043] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, or about 25 to about 50 mg / kg. g / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, About 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 4 5 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / m g, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about Approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 30 to approximately 40 mg / kg, approximately 35 to approximately 40 mg / kg, approximately 0.5 to approximately 30 mg / kg, approximately 0.75 to approximately 30 m g / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.The compound may be administered at a dose of 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention. .

[0044] For example, the subject may have approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29 ... .3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21 A therapeutic amount of iRNA may be administered, such as about 0.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0045] For example, the dsRNA conjugated to the ligand may be administered to the subject once a week or twice a month.

[0046] In yet another aspect, the present invention provides a kit for carrying out the method of the present invention. In one embodiment, the present invention provides a kit for carrying out the method of inhibiting Serpinc1 expression in a cell by contacting the cell with a double-stranded RNAi agent in an amount effective to inhibit Serpinc1 gene expression in the cell. The kit comprises an RNAi agent and instructions for use, and optionally, a means for administering the RNAi agent to a subject. [Brief explanation of the drawings]

[0047] [Figure 1] 1 is a schematic diagram of the blood coagulation cascade. [Figure 2A] 1 is a graph showing inhibition of Serpinc1 expression in Hep3B cells following a single administration of the indicated iRNA. [Figure 2B] 1 is a graph showing inhibition of Serpinc1 expression in Hep3B cells following a single administration of the indicated iRNA. [Figure 3A] Graphs showing inhibition of Serpinc1 mRNA (A) and protein (B) expression in CD-1 mice following a single administration of the indicated LNP formulation of AD-50509 or AD-1955. [Figure 3B] Graphs showing inhibition of Serpinc1 mRNA (A) and protein (B) expression in CD-1 mice following a single administration of the indicated LNP formulation of AD-50509 or AD-1955. [Figure 4A] Graphs showing the duration of inhibition of Serpincl mRNA (A) and protein (B) expression in CD-1 mice following a single administration of 1 mg / kg LNP formulation of AD-50509 or AD-1955. [Figure 4B] Graphs showing the duration of inhibition of Serpincl mRNA (A) and protein (B) expression in CD-1 mice following a single administration of 1 mg / kg LNP formulation of AD-50509 or AD-1955. [Figure 4C] 10 is a graph showing inhibition of Serpinc1 activity and Serpinc1 protein expression in CD1 mice following a single administration of 1 mg / kg LNP formulation of AD-50509 or AD-1955. [Figure 5] Graph showing the percentage knockdown of Serpincl mRNA and protein levels following a single 10 mg / kg dose of the indicated GalNAc-conjugated iRNA. [Figure 6]1 is a graph showing inhibition of Serpinc1 protein expression in C57BL / 6 mice following single doses of GalNAc-conjugated AD-54944 at 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, and 75 mg / kg, and repeated doses of 5 × 5 mg / kg. [Figure 7A] 1 is a graph showing the effect of repeated administration of GalNAc-conjugated AD-54944 on the duration of inhibition of Serpinc1 protein expression in C57BL / 6 mice. [Figure 7B] 1 is a graph showing the effect of repeated administration of GalNAc-conjugated AD-54944 on the duration of inhibition of Serpinc1 protein expression in C57BL / 6 mice. [Figure 8] Graph showing the effect of the indicated fractionated dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-conjugated AD-54944. [Figure 9] Graph showing the effect of the indicated fractionated dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-conjugated AD-54944. [Figure 10] Graphs showing the percentage knockdown of Serpinc1 protein levels following a single dose of 10 mg / kg (A) or 3 mg / kg (B) of the indicated GalNAc-conjugated iRNA. [Figure 11] Graph showing the percentage knockdown of Serpinc1 protein levels following a single dose of 10 mg / kg or 3 mg / kg of the indicated GalNAc-conjugated iRNA. [Figure 12] Graph showing the percentage knockdown of Serpinc1 activity following a single dose of 10 mg / kg or 3 mg / kg of the indicated GalNAc-conjugated iRNA. [Figure 13] 1 is a graph showing the dose-effect response to a single dose of AD-57213. [Figure 14]1 is a graph showing the duration of silencing of Serpinc1 following a single dose of 1 mg / kg, 3 mg / kg, or 10 mg / kg of AD-57213 in hemophilia A mice. [Figure 15] 1 is a graph showing inhibition of Serpinc1 mRNA expression in C57BL / 6 mice following single doses of 30 mg / kg, 10 mg / kg, 3 mg / kg, 1 mg / kg, and 0.3 mg / kg of AD-57213. [Figure 16] Graph showing the duration of silencing of Serpincl following a single dose of AD-57213 (A), AD-57215 (B), and AD-57214 (C) as indicated. [Figure 17] Graph showing the effect of the indicated fractionated dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-conjugated AD-57213. [Figure 18] Graph showing the effect of the indicated fractionated dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-conjugated AD-57213. [Figure 19] Graph showing the effect of the indicated fractionated dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-conjugated AD-57213. [Figure 20] 1 is a graph showing the single-dose screening effect of the indicated compounds on the duration of Serpinc1 protein expression in non-human primates. [Figure 21] 1 is a graph showing the effect of a single dose screening of GalNAc-conjugated AD-57213 on the duration of Serpinc1 protein expression in non-human primates. [Figure 22] 1 is a graph showing the single-dose screening effect of the indicated compounds on the duration of Serpinc1 protein expression in non-human primates. [Figure 23]1 is a graph showing the effect of a single dose of compound AD-57213 on serum antithrombin (Serpincl) levels in non-human primates. [Figure 24] 1 is a graph showing the effect of a single dose of compound AD-57213 at A) 1 mg / kg, B) 3 mg / kg, C) 10 mg / kg, and D) 30 mg / kg on the correlation between serum antithrombin (Serpincl) levels and fold change in peak plasma thrombin levels in non-human primates. The fold change in peak thrombin is depicted on the secondary y-axis (gray), and the relative antithrombin level is depicted on the primary y-axis (black). [Figure 25] Graph showing the effect of AD-57213 as fold change in peak thrombin increase as a function of relative antithrombin (Serpincl) silencing. [Figure 26] Graph showing the effect of multiple doses of Serpinc1 siRNA (0.5 mg / kg qw, 1 mg / kg q2w, 1.5 mg / kg qw, 3 mg / kg q2w) on serum antithrombin levels in non-human primates. Data points represent group means, and error bars represent standard deviations (N=3). (qw = weekly; q2w = biweekly). [Figure 27A] 1 is a graph showing the cumulative effect of Serpinc1 silencing in non-human primates. [Figure 27B] 1 is a graph showing the cumulative effect of Serpinc1 silencing in non-human primates. [Figure 28A] 1 is a graph showing the effect of Serpincl silencing on platelet accumulation following microvascular laser injury. The graph shows the median value from all damaging injuries. [Figure 28B] 1 is a graph showing the effect of Serpincl silencing on fibrin area following microvascular laser injury. The graph shows the median value from all traumatic injuries. [Figure 29] 1 is a graph showing the duration of Serpinc1 silencing following administration of the compound AD-57213 formulated in lipid nucleic acid particles. [Figure 30A]1 shows the nucleotide sequence of human (Homo sapiens) serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) (SEQ ID NO: 1). [Figure 30B] 1 shows the nucleotide sequence of rhesus monkey (Macaca mulatta) serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC2) (SEQ ID NO: 1). [Figure 30C] 1 shows the nucleotide sequence of Mus musculus serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1 (Serpinc1) (SEQ ID NO: 3). [Figure 30D] 1 shows the nucleotide sequence of the brown rat (Rattus norvegicus) serpin peptidase inhibitor, clade C (antithrombin), member 1 (Serpinc1) (SEQ ID NO: 4). [Figure 30E] The reverse complement of SEQ ID NO: 1 (SEQ ID NO: 5) is shown. [Figure 30F] The reverse complement of SEQ ID NO:2 (SEQ ID NO:6) is shown. [Figure 30G] The reverse complement of SEQ ID NO: 3 (SEQ ID NO: 7) is shown. [Figure 30H] The reverse complement of SEQ ID NO: 4 (SEQ ID NO: 8) is shown. [Figure 30I] The amino acid sequence of an exemplary hydrophobic MTS-containing peptide RFGF (SEQ ID NO:9); the amino acid sequence of an exemplary RFGF analog (SEQ ID NO:10); the amino acid sequence of the HIV Tat protein (SEQ ID NO:11); the amino acid sequence of the Drosophila Antennapedia protein (SEQ ID NO:12); and the amino acid sequence of an exemplary peptide-based cleavable tether are shown. [Figure 31A] 1 is a graph depicting that antithrombin reduction increases thrombin generation in factor IX-depleted human plasma in vitro. [Figure 31B] 1 is a graph depicting that antithrombin reduction increases thrombin generation in factor IX-depleted human plasma in vitro. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides an iRNA composition that induces RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the Serpinc1 gene. The Serpinc1 gene may be present in a cell, such as a cell in a subject, such as a human. The present invention also provides a method for using the iRNA composition of the present invention to inhibit Serpinc1 gene expression and / or to treat a subject with a disorder that would benefit from the inhibition or reduction of Serpinc1 gene expression, such as a bleeding disorder, for example, hemophilia. The present invention further provides a method for preventing at least one symptom, such as bleeding, in a subject with a disorder that would benefit from the inhibition or reduction of Serpinc1 gene expression, such as a bleeding disorder, for example, hemophilia.

[0049] Examples of iRNAs of the present invention include those having 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 2 ... These iRNAs include RNA strands (antisense strands) with a region of approximately 30 nucleotides or less in length, such as 9-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which is substantially complementary to at least a portion of the mRNA transcript of the Serpincl gene. The use of these iRNAs enables targeted degradation of the Serpincl gene mRNA in mammals. In particular, very low doses of Serpincl iRNA can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of Serpincl gene expression. The present inventors have demonstrated that iRNAs targeting Serpinc1 can mediate RNAi in vitro and in vivo, resulting in significant inhibition of Serpinc1 gene expression. Thus, methods and compositions comprising these iRNAs are useful for treating subjects who would benefit from reduced Serpinc1 protein levels and / or activity, such as those with bleeding disorders such as hemophilia.

[0050] The following detailed description discloses methods for making and utilizing compositions containing iRNAs that inhibit Serpincl gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from inhibiting and / or reducing the expression of this gene.

[0051] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values ​​for a parameter is listed, values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0052] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or two or more elements, such as, for example, a plurality of elements.

[0053] The term "including" is used herein to mean, and is used interchangeably with, the term "including but not limited to."

[0054] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0055] As used herein, "Serpinc1" refers to a specific polypeptide expressed in cells. Serpinc1 is also known as serpin peptidase inhibitor, clade C (antithrombin), member 1; antithrombin III; AT3; antithrombin; and heparin cofactor 1. The sequence of human Serpinc1 mRNA transcript can be found, for example, in GenBank accession number GI:254588059 (NM_000488; SEQ ID NO:1). The sequence of rhesus monkey Serpinc1 mRNA can be found, for example, in GenBank accession number GI:157167169 (NM_001104583; SEQ ID NO:2). The sequence of mouse Serpinc1 mRNA can be found, for example, in GenBank accession number GI:237874216 (NM_080844; SEQ ID NO:3). The sequence of rat Serpinc1 mRNA is found, for example, in GenBank accession number GI:58865629 (NM_001012027; SEQ ID NO: 4).

[0056] As used herein, the term " Serpinc1 " also refers to the specific polypeptide that is expressed in cells due to the naturally occurring DNA sequence variation of Serpinc1 gene, such as the single nucleotide polymorphism of Serpinc1 gene.A large number of SNPs in Serpinc1 gene have been identified, and are listed in, for example, NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).Non-limiting examples of SNPs in Serpinc1 gene are listed under NCBI dbSNP accession numbers rs677;rs5877;rs5878;rs5879;rs941988;rs941989;rs1799876;rs19637711;rs2008946;and rs2227586.

[0057] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpincl gene, including mRNA, which is an RNA processing product of a primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage at or near the portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpincl gene.

[0058] The target sequence may be about 9-36 nucleotides in length, such as about 15-30 nucleotides in length. For example, the target sequence may be 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, 19-2 The length may be about 15-30 nucleotides, such as 6, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

[0059] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising the nucleotide strand described by the referenced sequence, using standard nucleotide nomenclature.

[0060] "G", "C", "A", "T" and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative replacement moieties, as will be further detailed below (see, for example, Table 2).Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotides comprising nucleotides with such replacement moieties.As a non-limiting example, a nucleotide comprising inosine as a base can base pair with a nucleotide containing adenine, cytosine or uracil.Therefore, a nucleotide containing uracil, guanine or adenine can be replaced with a nucleotide containing inosine, for example, in the nucleotide sequence of the dsRNA of the present invention. In another example, adenine and cytosine can be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured herein.

[0061] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" are used interchangeably herein and refer to an agent that contains RNA, as defined herein, and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, Serpincl expression in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

[0062] In one embodiment, the RNAi agent of the present invention includes a single-stranded RNA that interacts with a target RNA sequence, such as a Serpincl target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding with the appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev.15:188).Therefore, in one aspect, the present invention relates to the single-stranded RNA (siRNA) that is produced in cells, promotes RISC complex formation, and leads to the silencing of target gene, i.e., Serpincl gene.Therefore, the term " siRNA " is also used herein to refer to RNAi as described above.

[0063] In another embodiment, the RNAi agent may be a "single-stranded siRNA" introduced into a cell or organism to inhibit target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA as described herein, or chemically modified as described in Lima et al., (2012) Cell 150;:883-894.

[0064] In another embodiment, the agent is a single-stranded antisense RNA molecule that inhibits a target through an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. The single-stranded antisense RNA molecule can inhibit translation in a stoichiometric manner by base-pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense RNA molecule inhibits the target mRNA by hybridizing (hydriding) and cleaving the target through an RNase H cleavage event. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, a single-stranded antisense RNA molecule may comprise at least about 15, 16, 17, 18, 19, 20 or more contiguous nucleotide sequences from any one of the antisense sequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

[0065] In another embodiment, the "iRNA" used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a ribonucleic acid molecule complex having a double-stranded structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations with respect to the target RNA, i.e., the Serpincl gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) causes target RNA degradation, such as mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0066] The double-stranded region may be any length that allows for specific degradation of the desired target RNA through the RISC pathway, and may range from about 9 to 36 base pairs in length, for example, about 15 to 30 base pairs in length, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18 , 15-17, 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, and 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. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

[0067] The two strands that form a double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules.When the two strands are parts of one larger molecule, and thus the 3'-end of one strand that forms the double-stranded structure and the 5'-end of the other strand are connected by an uninterrupted nucleotide chain, the connected RNA strands are called "hairpin loops".A hairpin loop can comprise at least one unpaired nucleotide; in some embodiments, a hairpin loop can 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 10, at least 20, at least 23 or more unpaired nucleotides.

[0068] When the two substantially complementary strands of dsRNA are constituted by another RNA molecule, these molecules can be covalently linked, but do not necessarily have to be.When the two strands are covalently linked by means other than an uninterrupted nucleotide chain between the 3'-end of one strand that forms a double-stranded structure and the 5'-end of each other strand, the linked structure is called "linker".RNA strands can have the same or different nucleotide numbers.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs that exist in the double strand.In addition to the double-stranded structure, RNAi can also comprise one or more nucleotide overhangs.

[0069] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide can be present on the 5'-end, the 3'-end, or both ends of either the antisense or sense strand of a dsRNA.

[0070] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and / or 5' end. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and / or 5' end. In another embodiment, one or more nucleotides in the overhang are substituted with a thiophosphate nucleoside.

[0071] The term "blunt-ended" or "blunt-ended" used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at a given end of dsRNA, i.e., there is no nucleotide overhang.One or both ends of dsRNA can be blunt-ended.When both ends of dsRNA are blunt-ended, the dsRNA is said to be blunt-ended.For clarity, "blunt-ended" dsRNA is a dsRNA with both ends blunted, i.e., there is no nucleotide overhang at either end of the molecule.In most cases, such molecules are double-stranded throughout their entire length.

[0072] The term "antisense strand" or "guide strand" refers to an iRNA strand, such as a dsRNA, that includes a region that is substantially complementary to a target sequence, such as a Serpincl mRNA. As used herein, the term "region complementary" refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as a Serpincl nucleotide sequence as defined herein. If the complementary region is not completely complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, such as within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the iRNA.

[0073] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

[0074] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions to form a double-stranded structure, as would be understood by one of skill in the art. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. One of skill in the art can determine the optimal set of conditions for testing the complementarity of two sequences depending on the end use of the hybridized nucleotides.

[0075] For example, a complementary sequence in an iRNA, such as a dsRNA described herein, involves base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most appropriate for their end use, such as inhibiting gene expression through the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, is still referred to as "perfectly complementary" for purposes described herein.

[0076] "Complementary" sequences, as used herein, also include or may be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs.

[0077] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context in which they are used.

[0078] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of a target mRNA (e.g., an mRNA encoding Serpinc1). For example, a polynucleotide is complementary to at least a portion of a Serpinc1 mRNA if its sequence is substantially complementary to a non-interrupted portion of the mRNA encoding Serpinc1.

[0079] Generally, the majority of the nucleotides in each strand are ribonucleotides, but as described in detail herein, one or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, "iRNA" includes ribonucleotides with chemical modifications. Such modifications include all types of modifications disclosed herein or known in the art. For purposes of this specification and claims, any such modifications are encompassed by "iRNA" as used in reference to iRNA molecules.

[0080] As used herein, the term "inhibit" is used synonymously with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0081] As used herein, the phrase "inhibiting Serpinc1 expression" includes inhibition of expression of any Serpinc1 gene (e.g., mouse Serpinc1 gene, rat Serpinc1 gene, monkey Serpinc1 gene, or human Serpinc1 gene), as well as variants or mutants of the Serpinc1 gene that encode the Serpinc1 protein.

[0082] "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 at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition.

[0083] Serpinc1 gene expression may be evaluated based on the level of any variable associated with Serpinc1 gene expression, such as Serpinc1 mRNA level, Serpinc1 protein level, or, for example, thrombin:antithrombin complex level as a measure of thrombin generation capacity, bleeding time, prothrombin time (PT), platelet count, and / or activated partial thromboplastin time (aPTT). Inhibition may be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level may be any type of control level used in the art, such as a pre-administration baseline level or a level measured from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or a non-active agent control).

[0084] In one embodiment, at least partial suppression of Serpincl gene expression is assessed by a decrease in the amount of Serpincl mRNA that can be isolated from or detected in a first cell or group of cells in which the Serpincl gene is transcribed, and that has been treated to inhibit Serpincl gene expression, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been treated accordingly (control cells). The degree of inhibition can be measured by:

number

[0085] As used herein, the phrase "contacting cells with RNAi agent" such as dsRNA includes contacting cells by any possible means.Contacting cells with RNAi agent includes contacting cells with iRNA in vitro or contacting cells with iRNA in vivo.Contacting can be performed directly or indirectly.Therefore, for example, the person who performs the method can physically contact RNAi agent with cells, or alternatively, can place RNAi agent in a situation that allows or causes subsequent contact with cells.

[0086] The contact of cells in vitro can be carried out, for example, by incubating cells with an RNAi agent. The contact of cells in vivo can be carried out, for example, by injecting an RNAi agent into or near the tissue where the cells are located, or by injecting an RNAi agent into another area, such as the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue where the cells to be contacted are located. For example, the RNAi agent can contain and / or be conjugated to a ligand, such as GalNAc3, which directs the RNAi agent to a target site, such as the liver. A combination of in vitro and in vivo contact methods is also possible. For example, cells can be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0087] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering" the iRNA to a cell by facilitating or resulting in uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through unassisted diffusive or active cellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved via β-glucan delivery systems, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Patent Application Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. Ex vivo introduction into cells includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below and / or known in the art.

[0088] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer encapsulating a pharmacologically active molecule, such as a nucleic acid molecule, e.g., an iRNA, or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0089] The term "SNALP" refers to a stable nucleic acid-lipid particle. SNALPs are lipid vesicles with a reducing aqueous interior that contain a nucleic acid, such as an iRNA, or a plasmid from which the iRNA is transcribed. SNALPs are described, for example, in U.S. Patent Application Publication No. 20060240093, U.S. Patent Application Publication No. 20070135372, and International Publication No. 2009082817, the entire contents of which are incorporated herein by reference. Examples of "SNALP" formulations are described below.

[0090] As used herein, a "subject" is an animal such as a mammal, including a primate (such as a human or a non-human primate, e.g., a monkey or chimpanzee), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced Serpincl expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced Serpincl expression; a human being with a disease, disorder, or condition that would benefit from reduced Serpincl expression; and / or a human being treated for a disease, disorder, or condition that would benefit from reduced Serpincl expression as described herein. As used herein, the terms "treat" or "treatment" refer to a beneficial or desired result, including, but not limited to, alleviating or ameliorating one or more symptoms, whether detectable or undetectable, reducing the severity of bleeding, stabilizing (i.e., not worsening) the bleeding condition, or ameliorating or palliating bleeding. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0091] By "reducing," in the context of a disease marker or symptom, is meant a statistically significant decrease in such level, which can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, preferably to a level generally accepted as being within the normal range for individuals without such disease.

[0092] As used herein, "prevention" or "preventing" in relation to a disease, disorder, or condition that would benefit from reduced expression of the Sertpincl gene refers to a reduction in the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as bleeding. The likelihood of developing bleeding is reduced, for example, when an individual with one or more bleeding risk factors does not develop bleeding, or develops less severe bleeding compared to a group with the same risk factors that do not receive the treatment described herein. Not developing a disease, disorder, or condition, or a reduction in the development of symptoms associated with such a disease, disorder, or condition (for example, by at least about 10% in a clinically recognized measure of the disease or disorder), or a delayed onset of symptoms (for example, by several days, weeks, months, or years) is considered effective prevention.

[0093] As used herein, the term "bleeding disorder" refers to 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, such as disseminated intravascular coagulation, pregnancy-associated eclampsia, vitamin K deficiency, autoimmune disorders, inflammatory bowel disease, ulcerative colitis, dermatological disorders (e.g., psoriasis, pemphigus), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease), allergic drug reactions resulting from medications such as aspirin, heparin, or warfarin, diabetes, acute hepatitis B infection, acute hepatitis C infection, malignant lesions or solid tumors (e.g., prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, melanoma, kidney, and / or hematological malignancies). In one embodiment, the inherited bleeding disorder is hemophilia, such as hemophilia A, B, or C. In one embodiment, the subject has an inherited bleeding disorder, e.g., hemophilia, and has developed an inhibitor, e.g., an alloantibody inhibitor, to alternative clotting therapy, and is referred to herein as an "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.

[0094] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent sufficient, when administered to a subject with a bleeding disorder and bleeding, to result in treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more disease symptoms). A "therapeutically effective amount" may vary depending on the RNAi agent, the method of agent administration, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant therapy, if any, and other personal characteristics of the subject being treated.

[0095] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA sufficient to prevent or ameliorate a disease or one or more disease symptoms when administered to a subject with a bleeding disorder who is not bleeding, e.g., a subject with a bleeding disorder scheduled for surgery. Amelioration of disease includes slowing the course of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the iRNA, the method of drug administration, the degree of disease risk, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concomitant therapy, if any, and other individual characteristics.

[0096] A "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 therapeutic agent. The iRNAs used in the methods of the invention may be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatments.

[0097] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0098] As used herein, the phrase "pharmacologically acceptable carrier" refers to a pharmacologically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium, or zinc stearate, or steric acid, etc.), or solvent encapsulating material involved in carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not toxic to the subject being treated. Some examples of materials that can serve as pharmacologically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) magnesium stearate; (8) lubricants such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) magnesium hydroxide. (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0099] As used herein, the term "sample" includes similar fluid, cell, or tissue collections isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples include samples from tissues, organs, or localized areas. For example, samples may be derived from specific organs, parts of organs, or fluids or cells within these organs. In certain embodiments, samples may be derived from the liver (e.g., the entire liver or specific parts of the liver, or specific types of liver cells, e.g., hepatocytes). In some embodiments, a "sample derived from a subject" refers to blood or plasma taken from a subject.

[0100] II. iRNAs of the Invention Described herein is an iRNA that inhibits Serpincl gene expression. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits Serpincl gene expression in cells, such as cells in a subject, such as a mammal, for example, a human, with a bleeding disorder, such as a hereditary bleeding disorder. The dsRNA comprises an antisense strand with a complementary region that is complementary to at least a portion of the mRNA formed during Serpincl gene expression. The complementary region is about 30 nucleotides or less in length (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the Serpincl gene, the iRNA inhibits expression of the Serpincl gene (e.g., a human, primate, non-primate, or avian Serpincl gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as immunofluorescence analysis using, for example, Western blotting or flow cytometry techniques.

[0101] dsRNA comprises two complementary RNA strands, which hybridize to form a double-stranded structure under the conditions in which dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region, which is substantially complementary to the target sequence, and is generally completely complementary.The target sequence can be derived from the sequence of mRNA formed during the expression of Serpincl gene.The other strand (sense strand) comprises a region complementary to the antisense strand, so that when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0102] Generally, the double-stranded structure is, for example, 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. Ranges and lengths intermediate to the recited ranges and lengths are also contemplated as part of the invention.

[0103] Similarly, the complementary region of the target sequence may be, for example, 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, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61 and 15-30 nucleotides in length, such as 7, 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. Ranges and lengths intermediate to the listed ranges and lengths are also contemplated as part of the invention.

[0104] In some embodiments, the dsRNA is about 15 to about 20 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can serve as substrates for Dicer. As those skilled in the art will recognize, the target region of an RNA targeted for cleavage is most often a portion of a larger RNA molecule, which is often an mRNA molecule. Where applicable, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0105] Those skilled in the art will appreciate that the amount of hydroxybenzoates may be, for example, about 10 to 36, 11 to 36, 12 to 36, 13 to 36, 14 to 36, 15 to 36, 9 to 35, 10 to 35, 11 to 35, 12 to 35, 13 to 35, 14 to 35, 15 to 35, 9 to 34, 10 to 34, 11 to 34, 12 to 34, 13 to 34, 14 to 34, 15 to 34, 9 to 33, 10 to 33, 11 to 33, 12 to 33, 13 to 33, 14 to 3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-1 9, 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 It will also be recognized that a double-stranded region, such as a double-stranded region of approximately 9-36 base pairs, such as 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, is the primary functional portion of a dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is a dsRNA, within the range that it is processed into, for example, a 15-30 base pair functional duplex that targets a desired RNA for cleavage. Thus, one skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting Serpincl expression is not generated in the target cell by cleavage of a larger dsRNA.

[0106] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides.Compared with their blunt-end counterparts, dsRNAs with at least one nucleotide overhang can have surprisingly superior inhibitory properties.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides.The overhang can be on the sense strand, the antisense strand, or any combination thereof.Furthermore, the nucleotide of the overhang can be present on the 5'-end, 3'-end, or both ends of either the antisense or sense strand of dsRNA.

[0107] dsRNA can be synthesized by standard methods known in the art using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0108] The iRNA compounds of the present invention can be prepared using a two-step method. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that it can easily prepare oligonucleotide strands that comprise unnatural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0109] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences: sense sequence and antisense sequence.Sense strand is selected from the sequence group provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20 and 21, and the antisense strand corresponding to the sense strand is selected from the sequence group in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20 and 21.In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence that occurs during Serpincl gene expression.Therefore, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide is described as the sense strand in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20 and 21, and the second oligonucleotide is described as the antisense strand that corresponds to the sense strand in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20 and 21. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0110] Although some of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 are described as modified and / or conjugated sequences, it is understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, may comprise any one of the sequences set forth in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 unmodified, unconjugated, and / or modified and / or conjugated differently than described.

[0111] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of approximately 20-23 base pairs, such as 21 base pairs, have been proposed as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures can also be similarly effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, the dsRNAs described herein may contain at least one strand that is at least 21 nucleotides long. It can be reasonably expected that shorter duplexes having one of the sequences in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, which are missing only a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides derived from one of the sequences in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and whose ability to inhibit Serpincl gene expression differs from that of a dsRNA comprising the full-length sequence by about 5, 10, 15, 20, 25, or 30% or less, are intended to be within the scope of the present invention.

[0112] Furthermore, the RNAs provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 identify sites in the Serpincl transcript that are highly susceptible to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target within one of these sequences. As used herein, an iRNA is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript anywhere within that specific site. Such iRNAs generally comprise approximately 15 contiguous nucleotides from one of the sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, linked to additional nucleotide sequences from regions adjacent to the selected sequence in the Serpincl gene.

[0113] Target sequences are generally about 15-30 nucleotides in length, although there is wide variability in the suitability of specific sequences within this range to induce cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken, in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed, either physically or figuratively (e.g., by computer simulation), on the target RNA sequence to identify sequences within a size range that can serve as target sequences. By successively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, while the sequences identified in, for example, any one of Tables 2, 3, 4, 8, 11, 12, 14, 15, 20 and 21 represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by successively "window walking" one nucleotide upstream or downstream of the given sequence to identify sequences with equivalent or better inhibitory properties.

[0114] It is contemplated that further optimization of any sequence identified in, for example, any one of Tables 2, 3, 4, 8, 11, 12, 14, 15, 20, and 21 can be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking through windows of a size longer or shorter than the target RNA from that position. Again, combining this approach of creating new target candidates with testing the effectiveness of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein can lead to further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or modifying overhangs, or other modifications known in the art and / or discussed herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0115] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the range of mismatches is not located in the center of the complementary region. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA agent strand complementary to a region of the SERPINC1 gene, the RNA strand generally does not contain any mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting expression of the SERPINC1 gene. Examining the effectiveness of an iRNA with mismatches in inhibiting expression of the SERPINC1 gene is important, especially when a specific complementary region of the Serpinc1 gene is known to have polymorphic sequence variation within the population.

[0116] III. Modified iRNAs of the Invention In one embodiment, the RNA of an iRNA of the invention, e.g., dsRNA, is native and does not contain chemical modifications and / or linkages, e.g., those known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial properties. Nucleic acids featured in the invention can be synthesized and / or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. For example, modifications include terminal modifications, such as 5'-end modifications (phosphorylation, conjugated linkage, inverted linkage) or 3'-end modifications (conjugated linkage, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and backbone modifications, including modification or replacement of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or RNAs that do not contain natural internucleoside linkages. RNAs with modified backbones particularly include those that do not have a phosphorus atom in the backbone. For purposes herein, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone are also considered to be oligonucleosides. In some embodiments, modified iRNAs have a phosphorus atom in their internucleoside backbone.

[0117] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and boranophosphates with reverse polarity, in which adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0118] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,717, each of which is hereby incorporated by reference in its entirety. No. 17; U.S. Patent No. 5,321,131; U.S. Patent No. 5,399,676; U.S. Patent No. 5,405,939; U.S. Patent No. 5,453,496; U.S. Patent No. 5,455,233; U.S. Patent No. 5,466,677; U.S. Patent No. 5,476,925; U.S. Patent No. 5,519,126; U.S. Patent No. 5,536,821; U.S. Patent No. 5,541,316; U.S. Patent No. 5,550,111; U.S. Patent No. 5,563,253; U.S. Patent No. 5,57 Nos. 1,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6 ,531,590; U.S. Patent No. 6,534,639; U.S. Patent No. 6,608,035; U.S. Patent No. 6,683,167; U.S. Patent No. 6,858,715; U.S. Patent No. 6,867,294; U.S. Patent No. 6,878,805; U.S. Patent No. 7,015,315; U.S. Patent No. 7,041,816; U.S. Patent No. 7,273,933; U.S. Patent No. 7,321,029; and U.S. Patent No. RE39464, but are not limited thereto.

[0119] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH2 components.

[0120] Representative United States patents that teach the preparation of the above oligonucleosides include U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, each of which is hereby incorporated by reference in its entirety. Nos.; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0121] In another embodiment, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the contents of each of which are incorporated herein by reference in their entirety. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0122] Some embodiments featured in the present invention include RNAs with phosphorothioate backbones, and oligonucleosides with heteroatom backbones that are --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene(methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (natural phosphodiester backbones are represented as --O--P--O--CH2--) of the aforementioned U.S. Pat. No. 5,489,677, and amide backbones of the aforementioned U.S. Pat. No. 5,602,240. In some embodiments, the RNA featured herein has a morpholino backbone structure as described in the aforementioned US Pat. No. 5,034,506.

[0123] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs, such as dsRNAs provided herein, can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl, or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In another embodiment, the dsRNA includes one of the following at the 2' position: C1 to C 10

[0033] In some embodiments, the modification is 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 1996). 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the Examples herein below.

[0124] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, specifically at the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. An iRNA can also have a sugar mimic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of the above modified sugar structures include, certain of which are commonly owned with the present application: U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; and U.S. Pat.

[0010] Examples of patents that may be used include, but are not limited to, U.S. Patent Nos. 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, the contents of each of which are hereby incorporated by reference in their entirety.

[0125] iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil (pseudouracil); 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine (5-hydroxymethylcytosine); xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil (pseudouracil); 4-thiouracil (pseudouracil); 4-thiouracil (pseudouracil); 4-thiouracil (pseudouracil); 4- Other synthetic and natural nucleobases include uracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, specifically 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-daazaadenine; and 3-deazaguanine and 3-deazaadenine.Further, the nucleobase can include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0126] Representative United States patents that teach the preparation of the above-mentioned specific modified nucleobases, as well as other modified nucleobases, include the above-mentioned U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469, each of which is hereby incorporated by reference in its entirety. Nos.; U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent No. 5,614,617; U.S. Patent No. 5,681,941; U.S. Patent No. 5,750,692; U.S. Patent No. 6,015,886; U.S. Patent No. 6,147,200; U.S. Patent No. 6,166,197; U.S. Patent No. 6,222,025; U.S. Patent No. 6,235,887; U.S. Patent No. 6,380,368; U.S. Patent No. 6,528,640; U.S. Patent No. 6,639,062; U.S. Patent No. 6,617,438; U.S. Patent No. 7,045,610; U.S. Patent No. 7,427,672; and U.S. Patent No. 7,495,088.

[0127] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce nonspecific 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).

[0128] Representative United States patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the contents of each of which are incorporated herein by reference in their entirety.

[0129] Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, and inverted base dT (idT). Disclosure of this modification is found in WO 2011 / 005861.

[0130] IV. Ligand-conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or complexes that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, for example, di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0131] In one embodiment, the ligand changes the distribution, targeting or life span of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity to a selected target, such as a molecule, a cell or cell type, a compartment, such as a subcellular or organ compartment, a tissue or organ or region of the body, for example, compared to a chemical species in the absence of such a ligand. Preferred ligands do not participate in double-stranded pairing in duplexed nucleic acid.

[0132] Ligands can include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines are polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or alpha helical peptides.

[0133] The ligand can also include a targeting group such as a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a kidney cell. The targeting group can be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetylglucoseamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0134] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include acetylcholinesterases (e.g., acetylcholinesterase ...

[0135] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for co-ligands; or antibodies, such as antibodies that bind to specific cell types, such as liver cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0136] The ligand can be a substance, such as a drug, that can increase uptake of an iRNA agent into a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0137] In some embodiments, the ligand attached to the iRNA described herein refers to a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides comprising several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, such as, for example, about 5-, 10-, 15-, or 20-base oligonucleotides comprising multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0138] Ligand-conjugated oligonucleotides of the invention may be synthesized by using oligonucleotides bearing pendant reactive functional groups, such as those derived from the addition of a binding molecule onto an oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing an attached binding moiety.

[0139] The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely produced through well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems (Foster City, Calif.). Additionally or alternatively, any other means for such synthesis known in the art may be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0140] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule, or building blocks bearing a non-nucleoside ligand.

[0141] When using a nucleotide conjugate precursor that already has a binding moiety, synthesis of the sequence-specific linked nucleoside is typically completed, and then a ligand molecule is reacted with the binding moiety to produce the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0142] A. Lipid Complex In one embodiment, the ligand or complex is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of complex to target tissue, for example, non-renal target tissue of the body.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of complex, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) be used to regulate the binding of serum protein, for example, HSA.

[0143] For example, lipid-based ligand can be used for inhibition, such as controlling the binding of complex to target tissue.For example, the 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 removed from the body.The lipid or lipid-based ligand that binds weaker to HSA can be used to target complex to the kidney.

[0144] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

[0145] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells may also be used in place of or in addition to the lipid-based ligand.

[0146] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, e.g., liver cells. Also included are HSA and low-density lipoprotein (LDL).

[0147] B. Cell-penetrating agents In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the cell-penetrating agent is amphipathic. An exemplary cell-penetrating agent is a peptide such as tat or antennopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidylmimetic, invertomer, non-peptide or pseudo-peptide bond, and D-amino acid use. The helical agent is preferably an α-helical agent with a lipophilic and lipophobic phase.

[0148] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. The addition of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by facilitating cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0149] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include 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 containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting numerous polar molecules, including peptides, oligonucleotides, and proteins, 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)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, an example of a peptide or peptidomimetic tethered to a dsRNA agent through an incorporated monomer unit is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications to increase stability or induce conformational properties. Any of the structural modifications described below can be used.

[0150] The RGD peptide used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues.RGD-containing peptides and peptidomimetics include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties that target integrin ligands can be used.Preferred complexes of this ligand target PECAM-1 or VEGF.

[0151] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or cecropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell-penetrating peptides can be bisected amphipathic peptides, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0152] C. Carbohydrate Complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, "carbohydrate" refers to either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or a compound having a carbohydrate moiety as part of its structure, composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 and above (e.g., C5, C6, C7, or C8); di- and trisaccharides include sugars with two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0153] In one embodiment, the carbohydrate complex used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] and other N-acetylgalactosamines.

[0154] In another embodiment, the carbohydrate complexes used in the compositions and methods of the present invention comprise: [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0155] Other exemplary carbohydrate conjugates for use in the embodiments described herein include: [ka] (In the formula, wherein one of X or Y is an oligonucleotide and the other is hydrogen).

[0156] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0157] D. Linker In some embodiments, the conjugates or ligands described herein may be attached to the iRNA oligonucleotide by various linkers, which may be cleavable or non-cleavable.

[0158] The term "linker" or "linking group" means an organic moiety that joins two parts of a compound, for example, by covalently bonding the two parts of the compound. Linkers are typically a direct bond, or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, aryl and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (wherein R 8 is hydrogen, acyl, aliphatic, or substituted aliphatic). In one embodiment, the linker is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-16 atoms, or 8-16 atoms.

[0159] A cleavable tether is one that is sufficiently stable outside the cell but is cleaved upon entry into a target cell to release the two moieties tethered by the linker. In preferred embodiments, the cleavable tether is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold more rapidly in the target cell, or under first standard conditions (e.g., which may be selected to mimic or correspond to intracellular conditions), than in the subject's blood, or under second standard conditions (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

[0160] Cleavable linking groups are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidizing or reducing enzymes or reducing agents such as mercaptans present in cells, which can degrade redox-cleavable linking groups by reduction, and are selective for specific substrates or do not have substrate specificity; esterases; agents that can create an acidic environment, such as endosomes or those that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific), and phosphatases.

[0161] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or to a desired compartment of the cell.

[0162] Linker can contain cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, the ligand for targeting liver can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, therefore linker is more efficiently cleaved in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0163] Linkers containing peptide bonds may be used in targeting peptidase-rich cell types such as hepatocytes and synoviocytes.

[0164] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradable agent (condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, with the first condition selected to indicate cleavage in target cells and the second condition selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0165] i. Redox-cleavable linker In one embodiment, the cleavable linker is a redox-cleavable linker that is cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can rely on the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. A candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0166] ii. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. A phosphate-based cleavable linker can be cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0167] iii. Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or below) or by an agent such as an enzyme that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon is attached to the oxygen of the ester (alkoxy group), an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above.

[0168] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0169] v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable tether. Peptide-based cleavable tethers are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable tethers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, but do not include the entire amide functionality. Peptide-based cleavable tethers have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates may be evaluated using methods similar to those described above.

[0170] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrates conjugated to linkers in the compositions and methods of the present invention include: [ka] [ka] (In the formula, wherein one of X or Y is an oligonucleotide and the other is hydrogen).

[0171] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

[0172] In one embodiment, the dsRNA of the invention is Formulas (XXXI) to (XXXIV), [ka] (In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent each occurrence from 0 to 20, and the repeat units may be identical or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is, each independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently for each occurrence absent, alkylene, or substituted alkylene, and one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Cis independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., independently for each occurrence, a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain). The trivalent conjugated GalNAc derivative is conjugated to a bivalent or trivalent branched linker selected from the group of structures represented by: Formula (XXXV), [ka] (In the formula, L 5A , L 5B and L 5C It is particularly useful for use with RNAi agents to inhibit the expression of target genes such as ribonucleotides (wherein represents a monosaccharide, such as a GalNAc derivative).

[0173] Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII.

[0174] Representative United States patents that teach the preparation of RNA complexes include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; and 5,591,584, the contents of each of which are hereby incorporated by reference in their entirety. Details; U.S. Patent Nos. 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737 Nos.; U.S. Patent Nos. 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469 ;U.S. Patent No. 5,258,506;U.S. Patent No. 5,262,536;U.S. Patent No. 5,272,250;U.S. Patent No. 5,292,873;U.S. Patent No. 5,317,098;U.S. Patent No. 5,371,241, U.S. Patent No. 5,391,723;U.S. Patent No. 5,416,203, U.S. Patent No. 5,451,463;U.S. Patent No. 5,510,475;U.S. Patent No. 5,512,667;U.S. Patent No. 5,514,785;U.S. Patent No. 5,565,552;Examples of such patents include, but are not limited to, U.S. Patent Nos. 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022.

[0175] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications may be incorporated in a single compound, or even in a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0176] "Chimeric" iRNA compounds or "chimeras," in the context of the present invention, are iRNA compounds, preferably dsRNA, that contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., nucleotides in the case of dsRNA compounds. These iRNAs typically contain at least one region in which the RNA has been modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates 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. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. As a result, comparable results are often obtained with shorter iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0177] In some cases, the RNA of an iRNA may be modified by a non-ligand group. To enhance the activity, cellular distribution, or intracellular uptake of an iRNA, several non-ligand molecules have been conjugated to the iRNA, and procedures for performing such conjugation are available in the scientific literature.Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantaneacetic 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).Representative U.S. patents that teach the preparation of such RNA complexes are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activation reagent. The conjugation reaction can be carried out in solution phase while the RNA is still bound to the solid support, or following RNA cleavage. Purification of the RNA complex by HPLC typically yields a pure complex.

[0178] IV. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, such as a cell in a subject, e.g., a human subject (e.g., a subject in need thereof, e.g., a subject with a bleeding disorder), can be achieved in several different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention, either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and induce expression of the iRNA. These alternatives are discussed further below.

[0179] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. WO 94 / 02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to consider for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into tissue or local administration of the formulation. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure to the agent in systemic tissues that may otherwise be harmed by or degrade the agent, and allows for administration of a lower total dose of the iRNA molecule. Several studies have demonstrated successful gene product knockdown when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and by subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and prolonged the survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference can be delivered 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. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lung by intranasal administration (Howard, KA., et (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) have demonstrated successful localized delivery. To administer iRNA systemically to treat disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endogenous endo- and exonucleases. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical attachment of lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNAs directed against ApoB conjugated to lipophilic cholesterol moieties into mice resulted in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al. (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to suppress tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J.O., et al. (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNAs can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of iRNA molecules (which are negatively charged) and also enhance their interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNAs or induced to form vesicles or micelles that encapsulate iRNAs (see, for example, Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs upon systemic administration. Methods for making and administering cationic iRNA complexes are well within the capabilities of one of ordinary skill in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N., et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al. (2007) J. Hypertens. 25:197-205, the contents of which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N., et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y., et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al. (2005) Int J. Oncol. 26:1087-1091), and PEG-1 (Polymerase Chain Receptor Blockers). al (2008) Pharm. Res. August 16, advance online publication; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA is complexed with cyclodextrin. Methods and pharmaceutical compositions for administering iRNA and cyclodextrin are described in U.S. Patent No. 7,427,605, the entire contents of which are incorporated herein by reference.

[0180] A. Vectors Encoding iRNAs of the Invention Serpincl gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A., et al., TIG. (1996), 12:5-10; Skillern, A., et al., WO 00 / 22113; Conrad, WO 00 / 22114; and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (hours to weeks) or persistent (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0181] Each iRNA strand or strands can be transcribed from the promoter on the expression vector.When expressing two separate strands to produce, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection).Alternatively, each of the promoters can be transcribed from the individual strands of dsRNA by being located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide that is linked by a linker polynucleotide sequence, so that dsRNA has a stem-loop structure.

[0182] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors can be by systemic administration, such as intravenous or intramuscular administration, administration to target cells explanted from a patient and then reintroduced into the patient, or any other means that allows for introduction into desired target cells.

[0183] iRNA expression plasmids can be transfected into target cells as complexes with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown, targeting different regions of a target RNA over a period of one week or more, are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells in vitro can be ensured using markers that confer resistance to specific environmental factors (e.g., antibiotics and drugs) on transfected cells, such as hygromycin B resistance.

[0184] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may contain viral sequences for transfection, if desired. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in target cells. Other contemplated aspects of vectors and constructs are described in more detail below.

[0185] Vectors useful for delivering iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression.

[0186] The expression of iRNA can be precisely regulated using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24).Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG).Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0187] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to patients. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells, for example, to generate stem cells that are more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors that may be used include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; U.S. Patent No. 5,665,557; and U.S. Patent No. 5,981,276, which are incorporated herein by reference.

[0188] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of 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); WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0189] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., with either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol, 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; WO 94 / 13788; and WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0190] Another viral vector suitable for delivering the iRNA of the invention is a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or a poxvirus, e.g., an avipox, e.g., fowlpox or canarypox.

[0191] The tropism of viral vectors can be modified, if necessary, by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0192] The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0193] V. PHARMACEUTICAL COMPOSITIONS OF THE INVENTION The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one embodiment, the present invention provides a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with Serpincl gene expression or activity, such as bleeding disorders. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma, e.g., by intracerebral infusion, such as continuous pump infusion. Pharmaceutical compositions of the present invention may be administered at a dosage sufficient to inhibit Serpincl gene expression. Generally, appropriate doses of the iRNA of the present invention range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose.

[0194] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, The compound may be administered at a dose of 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0195] In another embodiment, the dsRNA is about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, or about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0. 5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / mg, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m g / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present invention.

[0196] For example, dsRNA has approximately 0..01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0197] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, or about 25 to about 50 mg / kg. g / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, About 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 4 5 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / m g, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about Approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 30 to approximately 40 mg / kg, approximately 35 to approximately 40 mg / kg, approximately 0.5 to approximately 30 mg / kg, approximately 0.75 to approximately 30 m g / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered at a dose of 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. In one embodiment, the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0198] For example, the subject may have approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29 ... .3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21 A therapeutic amount of iRNA may be administered, such as about 0.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0199] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or controlled-release delivery. In this case, the amount of iRNA contained in each subdose must be correspondingly smaller 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 iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for site-specific agent delivery, such as those used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0200] In another embodiment, a single dose of the pharmaceutical composition can be administered over an extended period of time, such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered twice a month.

[0201] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other diseases present, may influence the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. The effective dosage and in vivo half-life of the individual iRNAs encompassed by the present invention may be estimated using conventional procedures or based on in vivo studies using appropriate animal models, as described elsewhere herein.

[0202] Advances in mouse genetics have produced several mouse models for studying various human diseases, such as bleeding disorders, that benefit from reduced Serpincl expression. These models can be used for in vivo testing of iRNAs and to determine therapeutically effective doses. Suitable mouse models are known in the art, such as hemophilia A and B mouse models, for example, mice with clotting factor gene knockouts 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 USA 94:11563-6, and Jin, et al. (2004) Blood 104:1733.

[0203] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary, for example, via inhalation or insufflation of powders or aerosols, including nebulizers; intratracheal, intranasal, transepidermal, and transdermal; oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal administration, for example, via an implanted device; or intracranial administration, for example, intracerebral parenchymal, intrathecal, or intraventricular. iRNA can be delivered in a manner that targets specific tissues, such as the liver (e.g., liver parenchymal cells). Pharmaceutical compositions and formulations for local administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like, may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is in admixture with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA can be complexed with lipids, particularly cationic lipids.Suitable fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C. 1~20 Topical formulations include, but are not limited to, alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0204] A. iRNA Formulations Comprising Membrane-Like Molecular Assemblies The iRNAs used in the compositions and methods of the present invention may be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar or multilamellar vesicles, with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition, but may in some cases. Liposomes are useful for transporting and delivering active ingredients to their site of action. Because the liposomal membrane is structurally similar to biological membranes, upon application of the liposome to a tissue, the liposomal bilayer fuses with the bilayer of the cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the iRNA, are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate RNAi. In some cases, liposomes are also specifically targeted, for example, to direct iRNA to a particular cell type.

[0205] Liposomes containing RNAi agents can be prepared by a variety of methods. In one example, the lipid components of the liposomes are dissolved in detergent so that micelles are formed without the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid complexes. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the RNAi agent, condensing around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the RNAi agent.

[0206] If necessary, a carrier compound, e.g., to aid in condensation, can be added during the condensation reaction by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0207] Methods for generating stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga, et al. The present invention may also include one or more embodiments of the exemplary method described in [Endocrinol. 115:757, 1984]. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and a combination of freeze-thawing and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). If consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are easily adapted to the packaging of RNAi agent preparations within liposomes.

[0208] Liposomes are divided into two broad classes: cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes; the positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized inside endosomes; the acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0209] pH-sensitive or negatively charged liposomes do not complex with nucleic acids but rather encapsulate them. Because both nucleic acids and lipids have similar charges, repulsion occurs rather than complexation. Nevertheless, some nucleic acid is encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected within the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0210] One major type of liposome composition contains phospholipids in addition to naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0211] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0212] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their efficacy in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A into the dermis of mouse skin. The results suggested that such nonionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0213] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that the improved circulation half-life of sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0214] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe the use of monosialoganglioside G M1 reported the ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G M1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0215] In one embodiment, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with plasma membrane effectively, but it can be taken up by macrophage in vivo and can be used to deliver RNAi agent to macrophage.

[0216] Additional advantages of liposomes include: Liposomes derived from natural phospholipids are biocompatible and biodegradable; Liposomes can incorporate a wide range of water- and lipid-soluble drugs; Liposomes can protect the RNAi agents encapsulated in their internal compartments from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size and aqueous volume of liposomes.

[0217] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the plasma membrane of tissue culture cells, resulting in RNAi agent delivery (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355, for a description of DOTMA and its use in combination with DNA).

[0218] The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells. It comprises positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complexes is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0219] Other reported cationic lipid compounds include those conjugated to a variety of moieties, including carboxyspermine conjugated to one of two lipid types, such as compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0220] Another cationic lipid complex involves derivatizing lipids with cholesterol ("DC-Chol") in combination with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0221] Liposome formulations are particularly suitable for topical administration, and liposomes have several advantages over other formulations.These advantages include reducing the side effects associated with high systemic absorption of administered drugs, increasing the accumulation of administered drugs in desired targets, and the ability to administer RNAi agents into the skin.In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and enhance the penetration of RNAi agents into skin tissues, such as the skin.For example, liposomes can be applied topically. Topical delivery of therapeutic agents formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0222] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their utility in delivering drugs to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing RNAi agents are useful for treating skin diseases.

[0223] Liposomes containing iRNA can be highly deformable. Such deformability can allow liposomes to penetrate pores smaller than the average diameter of the liposome. For example, transferosomes are a type of deformable liposome. Transferosomes can be created by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing RNAi agents can be delivered subcutaneously, for example, by infection, to deliver the RNAi agent to keratinocytes within the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and are often self-loading.

[0224] Other formulations to which the present invention may be applied are described in U.S. Provisional Patent Application Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT application PCT / US2007 / 080331, filed October 3, 2007, also describes formulations to which the present invention may be applied.

[0225] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to the shape of skin pores), self-repair, often reach their targets without fragmentation, and are often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to a standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0226] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the properties of the many different surfactant types, both natural and synthetic, is by using the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0227] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used over a wide pH range. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0228] If surfactant molecule carries negative charge when dissolved or dispersed in water, surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylate, acyl amide of amino acid, sulfate esters such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate.The most important members of anionic surfactant class are alkyl sulfate and soap.

[0229] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0230] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

[0231] The use of surfactants in pharmaceutical preparations and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0232] The iRNA used in the methods of the present invention can also be provided as a micellar formulation. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure so that the hydrophobic portions of the molecules all face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. If the environment is hydrophobic, the opposite arrangement will exist.

[0233] Mixed micelle formulations suitable for transdermal delivery include siRNA compositions, alkali metal C8-C6 22 The mixed micelle may be prepared by mixing an aqueous solution of alkyl sulfate and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salts, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polidocanol alkyl ether and its analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles can be formed regardless of the mixing of the components, but to provide smaller micelles, vigorously mixing is required.

[0234] In one method, a first micelle composition is prepared, containing an siRNA composition and at least an alkali metal alkyl sulfate.The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition.In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by adding the remaining micelle-forming compounds with vigorous mixing.

[0235] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added along with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0236] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispensing device, which can then be loaded with the propellant. The propellant, under pressure, is in liquid form within the dispensing device. The ratio of the components is adjusted so that the aqueous phase and the propellant phase are one, i.e., one phase. If there are two phases, the dispensing device must be shaken before dispensing a portion of the contents, for example, through a metered valve. The dispensed dose of the pharmaceutical product is expelled from the metered valve in a fine spray.

[0237] Propellants include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0238] The specific concentrations of the essential ingredients can be determined by relatively simple experimentation. For absorption through the oral cavity, it is often desirable to increase the dose, e.g., at least two or three times, that for administration through injection or through the gastrointestinal tract.

[0239] B. Lipid particles For example, an iRNA, such as a dsRNA, of the invention may be fully encapsulated in a lipid formulation, such as an LNP, to form, for example, an SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle.

[0240] As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLPs. As used herein, the term "SPLP" refers to nucleic acid-lipid particles comprising plasmid DNA encapsulated within lipid vesicles. SNALPs and SPLPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs exhibit long circulatory life following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration), making them extremely useful for systemic applications. SPLPs include "pSPLPs," which contain encapsulated condensing agent-nucleic acid conjugates, as described in WO 00 / 03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, when present in the nucleic acid-lipid particle of the present invention, nucleic acid is resistant to nuclease degradation in aqueous solution.Nucleic acid-lipid particle and the method for preparing them are disclosed in, for example, United States Patent No. 5,976,567; United States Patent No. 5,981,501; United States Patent No. 6,534,484; United States Patent No. 6,586,410; United States Patent No. 6,815,432; United States Patent Application Publication No. 2010 / 0324120; and International Publication No. 96 / 40964.

[0241] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above-cited ranges are also considered part of the invention.

[0242] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA). 1,2-Dilinoleyl-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

[0243] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0244] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0245] Ionic / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-2-methylpropional (DOPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-2-methylpropional (DOPE). The lipid may be an anionic or neutral lipid, including, but not limited to, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0246] The conjugated lipid that inhibits particle aggregation can be, for example, without limitation, a polyethylene glycol (PEG)-lipid, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (Ci8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particles.

[0247] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0248] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be created using lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, the contents of which are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can then be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) to achieve a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously when mixed.Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc.).In some cases, the extrusion step can be omitted.Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.The buffer can be exchanged with phosphate buffered saline (PBS) at about pH 7, for example, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

[0249] LNP01 formulations are described, for example, in WO 2008 / 042973, which is incorporated herein by reference.

[0250] Additional exemplary lipid dsRNA formulations are described in Table 1.

[0251] [Table 1-1]

[0252] [Table 1-2]

[0253] [Table 1-3]

[0254] Formulations comprising SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0255] Formulations comprising XTC are described, for example, in U.S. Provisional Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Application No. 61 / 239,686, filed September 3, 2009, and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0256] Formulations comprising MC3 are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed Jun. 10, 2010, the entire contents of which are incorporated herein by reference.

[0257] ALNY-100-containing formulations are described, for example, in International Application PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0258] C12-200-containing formulations are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0259] Synthesis of ionic / cationic lipids Any of the compounds, such as the cationic lipids used in the nucleic acid-lipid particles of the present invention, can be prepared by known organic synthesis techniques, including those methods described in more detail in the Examples. Unless otherwise indicated, all substituents are as defined below.

[0260] "Alkyl" means a straight-chain or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0261] Alkenyl refers to the alkyl as defined above, containing at least one double bond between adjacent carbon atoms.Alkenyl includes both cis and trans isomers.Representative straight-chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc.

[0262] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight-chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0263] "Acyl" means any alkyl, alkenyl, or alkynyl, as defined below, where the carbon at the point of attachment is replaced by an oxo group. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0264] "Heterocycle" means a saturated, unsaturated, or aromatic 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, including bicycles in which any of the heterocycles below are fused to a benzene ring; the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls, as defined below. Examples of heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0265] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle" mean that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, the substituents include oxo, halogen, heterocycle, -CN, -OR. x , -NR x R y , -NR x C(=O)R y , -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x , and -SO n NR x R y n is 0, 1 or 2; R x and R y are the same or different and independently represent hydrogen, alkyl, or heterocycle, and each of said alkyl and heterocycle substituents may be selected from one or more of oxo, halogen, —OH, —CN, alkyl, —OR x , heterocycle, -NR x R y , -NR x C(=O)R y , -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x , and -SO n NR x R y It can be further substituted by:

[0266] "Halogen" means fluoro, chloro, bromo, and iodo.

[0267] In some embodiments, the methods of the present invention may require the use of protecting groups. Protecting group procedures are well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, in the context of the present invention, a protecting group is any group that reduces or eliminates the unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a specific reaction and then removed to expose the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0268] Synthesis of Formula A In some embodiments, the nucleic acid-lipid particles of the present invention have Formula A: [ka] (wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be substituted or unsubstituted; R3 and R4 are independently lower alkyl; or R3 and R4 can be joined together to form a heterocycle, which may be substituted or unsubstituted). In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, lipids of the above formula A can be prepared according to the following reaction schemes 1 or 2, in which all substituents are as defined above unless otherwise specified.

[0269] [ka] Lipid A, in which R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be substituted or unsubstituted, and R3 and R4 are independently lower alkyl, or R3 and R4 can be taken together to form a heterocycle, which may be substituted or unsubstituted, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 results in ketal 3. Treatment of ketal 3 with amine 4 yields lipids of Formula A. Lipids of Formula A can be converted to the corresponding ammonium salts with an organic salt of Formula 5, where X is an anionic counterion selected from halogens, hydroxides, phosphates, sulfates, and the like.

[0270] [ka] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 provides ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is as described in Scheme 1.

[0271] Synthesis of MC3 DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid) was prepared as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed to give a colorless oil (0.54 g).

[0272] Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out using Scheme 3 below. [ka]

[0273] Synthesis of 515 To a stirred suspension of LiAlH (3.74 g, 0.09852 mol) in 200 mL of anhydrous THF in a two-necked RBF (1 L) was slowly added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0°C under a nitrogen atmosphere. After the addition was complete, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After the reaction was complete (by TLC), the mixture was cooled to 0°C and quenched by the careful addition of saturated NaSO solution. The reaction mixture was stirred at room temperature for 4 h and filtered. The residue was washed well with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. Volatilities were stripped under vacuum to give the hydrochloride salt of 515 as a white solid. Yield: 7.12g 1H-NMR (DMSO, 400MHz): δ=9.34(broad,2H),5.68(s,2H),3.74(m,1H),2.66-2.60(m,2H),2.50-2.45(m,5H).

[0274] Synthesis of 516 To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-necked RBF, NEt (37.2 mL, 0.2669 mol) was added and cooled to 0°C under a nitrogen atmosphere. After the slow addition of N-(benzyloxycarbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was allowed to warm to room temperature. After the reaction was complete (2-3 h by TLC), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO solution (1 × 50 mL). The organic layer was then dried over anhydrous NaSO, and the solvent was evaporated to give the crude product, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11g (89%).1H-NMR (CDCl3, 400MHz): δ=7.36-7.27(m,5H),5.69(s,2H),5. 12(s,2H),4.96(br.,1H)2.74(s,3H),2.60(m,2H),2.30-2.25(m,2H).LC-MS [M+H]-232.3(96.94%).

[0275] Synthesis of 517A and 517B Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of a 10:1 acetone / water mixture in a 500 mL one-neck RBF at room temperature, to which N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added, followed by 4.2 mL of a 7.6% solution of OsO (0.275 g, 0.00108 mol) in tert-butanol. After completion of the reaction (approximately 3 h), the mixture was quenched by the addition of solid NaSO, and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous NaSO, and the solvent was removed in vacuo. Silica gel column chromatography purification of the crude material gave a diastereomeric mixture which was separated by preparative HPLC. Yield: 6 g crude

[0276] 517A-Peak-1 (white solid), 5.13 g (96%). H-NMR (DMSO, 400 MHz): δ = 7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS - [M+H] - 266.3, [M+NH] - 283.5 present, HPLC - 97.86%. Stereochemistry confirmed by X-ray.

[0277] Synthesis of 518 Using a procedure similar to that described for the synthesis of compound 505, compound 518 was obtained as a colorless oil (1.2 g, 41%). H-NMR (CDCl, 400 MHz): δ = 7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC - 98.65%.

[0278] General procedure for the synthesis of compound 519 A solution of compound 518 (1 eq) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH (1 M, 2 eq) in THF. After the addition was complete, the mixture was heated at 40 °C for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous NaSO, then filtered through Celite and concentrated to an oil. Column chromatography afforded pure 519 as a colorless oil (1.3 g, 68%). 13C NMR = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight calculated for C44H80NO2 (M+H)+, 654.6, found 654.6.

[0279] Formulations prepared by either standard or non-extrusion methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be a whitish, translucent solution without aggregates or sediment. The particle size and size distribution of lipid-nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particle size should be approximately 20-300 nm, such as 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation and encapsulated fraction was estimated using a dye exclusion assay. Samples of formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting detergent, such as 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the detergent-containing sample compared to a standard curve. Encapsulation fraction is calculated by subtracting "free" dsRNA content (measured by signal in the absence of surfactant) from total dsRNA content. The percentage of encapsulated dsRNA is typically >85%. In SNALP formulations, particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120nm. Suitable ranges are typically at least about 50nm to at least about 110nm, at least about 60nm to at least about 100nm, or at least about 80nm to at least about 90nm.

[0280] Compositions and preparations for oral administration include powder or granule, microparticle, nanoparticle, suspension or solution in water or non-aqueous medium, capsule, gel capsule, sachet, tablet or mini-tablet.Thickener, flavoring agent, diluent, emulsifier, dispersing aid or binder may be required.In some embodiments, oral preparations are those in which the DsRNA of the present invention is administered in combination with one or more penetration-promoting surfactants and chelating agents.Suitable surfactants include fatty acid and / or ester or their salt, bile acid and / or their salt. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acid / salts combined with bile acids / salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA featured in the present invention can be orally delivered in granular form, including spray-dried particles, or can be complexed to form micro- or nanoparticles.DsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulans, cellulose, and starch.Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate. acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0281] Compositions and formulations for parenteral, intraparenchymal (intracerebral), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0282] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. When treating liver disorders, such as liver cancer, liver-targeted formulations are particularly preferred.

[0283] The pharmaceutical preparation of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining active ingredients with pharmaceutical carriers or excipients. Generally, the preparation is prepared by uniformly and intimately combining active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0284] The compositions of the present invention can be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers.

[0285] C. Additional Formulations i. Emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and (See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed within one another. In general, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed and dispersed as minute droplets within the bulk oily phase, the resulting composition is referred to as a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed and dispersed as minute droplets within the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o / w) emulsion.In addition to the dispersed phase and the active agent, which may be present as a solution in either the aqueous or oily phase or as a separate phase, emulsions may contain additional components. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions comprising more than two phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages not offered by simple binary emulsions. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, oil droplet systems encapsulated in globules of water and stabilized within an oily continuous phase provide o / w / o emulsions.

[0286] Emulsions are characterized by little or no thermodynamic stability. Frequently, the dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase and is maintained in this form through the use of emulsifiers or formulation viscosity. Either of the emulsion phases can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Another means of stabilizing emulsions involves the use of emulsifiers, which can be incorporated into either of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0287] Synthetic surfactants, also known as surface active agents, have a wide range of uses in emulsion formulations and have been reviewed in 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, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199).Surfactants are typically amphiphilic and comprise hydrophilic and hydrophobic moieties. The ratio of hydrophilicity to hydrophobicity is called the hydrophilic / lipophilic balance (HLB) of surfactant, and is a useful tool for classifying and selecting surfactant in the preparation of formulation.Surfactant can be classified into different classes based on the nature of hydrophilic group: nonionic, anionic, cationic and amphoteric (see for example Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285).

[0288] Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases with hydrophilic properties, such as anhydrous lanolin and hydrophilic petrolatum, can absorb water to form water-in-oil emulsions while still maintaining their semi-solid consistency. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, especially in combination with surfactants. 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.

[0289] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion, including fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0290] Hydrophilic colloids, or hydrocolloids, include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around dispersed phase droplets and by increasing the viscosity of the external phase.

[0291] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can easily support the growth of microorganisms, so preservatives are often incorporated into these preparations.The commonly used preservatives contained in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also generally added to emulsion preparations to prevent the preparation from deteriorating.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene; or reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0292] The application of emulsion formulations via dermal, oral, and parenteral routes and methods for preparing them have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of preparation and efficiency in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are among the materials commonly administered orally as o / w emulsions.

[0293] ii. Microemulsions In one embodiment of the present invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single optically isotropic and thermodynamically stable 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, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are typically prepared through the combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0294] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with comprehensive knowledge of microemulsion formulation (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, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into a formulation of spontaneously formed, thermodynamically stable droplets.

[0295] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, 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), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. Cosurfactants, which are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant film, resulting in irregular coatings due to the gaps between surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, aqueous pharmaceutical solutions, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0296] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential drug absorption enhancement due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides, or iRNAs. Microemulsions have been effective in transdermal delivery of active ingredients for both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of iRNA and nucleic acids.

[0297] 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 the properties of the formulation and enhance the absorption of the iRNA 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.

[0298] iii. Particulates The RNAi agents of the present invention may be incorporated into particles, such as microparticles. Microparticles may be produced by spray drying, but may also be produced by other methods, including freeze-drying, evaporation, fluidized-bed drying, vacuum drying, or a combination of these techniques.

[0299] iv. Penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve efficient delivery of nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, usually, only lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been discovered that even non-lipophilic drugs can pass through cell membranes if the membrane they pass through is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0300] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the aforementioned classes of penetration enhancers is described in more detail below.

[0301] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved iRNA absorption through mucosal membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0302] 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-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1~20 These include alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.). (See, for example, 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).

[0303] The physiological role of bile includes promoting 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, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycolic 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 (STDHF), sodium glycodihydrofusidate, 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, 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).

[0304] Chelating agents used in the context of the present invention can be defined as compounds that complex with metal ions, removing them from solution and resulting in improved iRNA absorption through mucous membranes. Regarding their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as deoxyribonuclease inhibitors, since most DNA nucleases require divalent metal ions for catalysis and are 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-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-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).

[0305] As used herein, a non-chelating, non-surfactant penetration enhancer may be defined as a compound that demonstrates insignificant activity as a chelating agent or as a surfactant, but still enhances the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancer includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0306] Agents that enhance cellular level iRNA uptake can also be added to the pharmaceutical and other compositions of the present invention.For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97 / 30731 to Lollo et al.) are also known to enhance cellular uptake of dsRNA.Examples of commercially available transfection reagents include, for example, 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, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), RNAiMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent(Roche;Grenzacherstrasse,Switzerland), DOTAP Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), DOSPER Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), or Fugene(Grenzacherstrasse,Switzerland), Transfectam(R) Reagent(Promega;Madison,WI), TransFast(TM)Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass.a D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM) (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(TM) transfection Reagent (Genlantis; San Examples of suitable fusion proteins include RiboFect (Bioline; Taunton, MA, 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).

[0307] Other agents can be utilized to enhance the penetration of the administered nucleic acid, including glycols, such as ethylene glycol and propylene glycol; pyrroles, such as 2-pyrrole; azone; and terpenes, such as limonene and menthone.

[0308] v. Carrier Certain compositions of the present invention also incorporate a carrier compound into their formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or an analog thereof, that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter substance, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory reservoirs, likely due to competition between the carrier compound and the nucleic acid for their normal receptors. For example, recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic, 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).

[0309] vi. excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected with the intended mode of administration in mind to provide the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0310] The composition of the present invention can be prepared using pharmaceutically acceptable organic or inorganic excipients that do not adversely react with nucleic acid and are suitable for oral administration.Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone etc.

[0311] The preparation for topical administration of nucleic acid can include sterile and non-sterile aqueous solution, non-aqueous solution in common solvent such as alcohol, or nucleic acid solution in liquid or solid oil base.The solution can also contain buffer, diluent and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable for oral administration that do not adversely react with nucleic acid can be used.

[0312] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0313] vii. Other ingredients The compositions of the present invention may further contain other auxiliary ingredients conventionally found in pharmaceutical compositions at their technically established usage levels.Thus, for example, the compositions may contain additional compatible pharmacologically active ingredients, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, when added, such materials should not excessively interfere with the biological activity of the components of the compositions of the present invention.The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0314] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0315] In some embodiments, pharmaceutical compositions featured herein include (a) one or more iRNA compounds and (b) one or more agents that function via a non-RNAi mechanism and are useful for treating bleeding disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-lipid agents, antiviral agents, and / or anti-fibrotic agents. In addition, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the iRNAs described herein. Other agents useful for treating liver disease include telbivudine, entecavir, telaprevir, and protease inhibitors, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217 to Tung et al.; and U.S. Patent Application Publication No. 2004 / 0127488 to Hale et al.

[0316] The toxicity and therapeutic effect of such compounds can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indices are preferred.

[0317] 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 featured herein generally lies within a range of circulating concentrations, including the ED50, with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods featured herein, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range (e.g., achieve a reduction in polypeptide concentrations) of the compound, or, if appropriate, of the polypeptide product of the target sequence, including the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms), as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0318] In addition to the administrations discussed above, the iRNAs described herein can be administered in combination with other known agents effective in treating pathological processes mediated by Serpincl expression. In any case, the treating physician can adjust the amount and timing of iRNA administration based on the results observed using standard measures of effectiveness known in the art or described herein.

[0319] VI. Method of Invention The present invention also provides methods for reducing and / or inhibiting Serpincl expression in cells using iRNAs of the present invention and / or compositions containing iRNAs of the present invention. In other aspects, the present invention provides iRNAs of the present invention and / or compositions comprising iRNAs of the present invention for use in reducing and / or inhibiting Serpincl expression in cells. In yet another aspect, the present invention provides use of iRNAs of the present invention and / or compositions comprising iRNAs of the present invention for manufacturing a drug that reduces and / or inhibits Serpincl expression in a cell.

[0320] The methods and uses include contacting a cell with an iRNA, such as a dsRNA of the present invention, and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the Serpinc1 gene, thereby inhibiting Serpinc1 gene expression in the cell.

[0321] The reduction of gene expression can be evaluated by any method known in the art. For example, the reduction of Serpinc1 expression can be determined by measuring the mRNA expression level of Serpinc1 using methods familiar to those skilled in the art, such as Northern blotting or qRT-PCR, by measuring the protein level of Serpinc1 using methods familiar to those skilled in the art, such as Western blotting or immunological techniques, and / or by measuring the biological activity of Serpinc1 that affects one or more molecules involved in the cellular blood coagulation mechanism (or blood coagulation itself in an in vivo situation).

[0322] In the methods and uses of the present invention, the cells may be contacted in vitro or in vivo, i.e. the cells may be in a subject.

[0323] Cells suitable for treatment using the methods of the present invention may be any cell that expresses the Serpincl gene. Cells suitable for use in the methods and uses of the present invention may be mammalian cells, such as primate cells (such as human cells or non-human primate cells, for example, monkey cells or chimpanzee cells), non-primate cells (such as cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (such as duck cells or goose cells), or whale cells. In one embodiment, the cells are human cells, such as human hepatocytes.

[0324] Serpinc1 expression is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100% inhibition.

[0325] The in vivo methods and uses of the present invention may include administering a composition containing an iRNA to a subject, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the Serpincl gene of the mammal being treated. When treating an organism such as a mammal, such as a human, the composition may be administered by any means known in the art, including, but not limited to, oral; intraperitoneal; or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and other parenteral routes, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection.

[0326] In some embodiments, administration is by depot injection. Depot injections may release iRNA consistently over a long period of time. Thus, depot injections may reduce the frequency of dosing required to achieve a desired effect, such as the desired Serpincl inhibition, or a therapeutic or prophylactic effect. Depot injections may also provide a more consistent serum concentration. Depot injections include subcutaneous or intramuscular injections. In a preferred embodiment, the depot injection is a subcutaneous injection.

[0327] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In another embodiment, the pump is an infusion pump. Infusion pumps may be used for intravenous, subcutaneous, arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In another embodiment, the pump is a surgically implanted pump that delivers iRNA to the liver.

[0328] The mode of administration may be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration may be selected to improve targeting.

[0329] In one aspect, the present invention also provides a method for inhibiting Serpinc1 gene expression in a mammal, e.g., a human. The present invention also provides a composition comprising an iRNA, e.g., a dsRNA, that targets the Serpinc1 gene in a mammalian cell, for use in inhibiting Serpinc1 gene expression in a mammal. In another aspect, the present invention provides the use of an iRNA, e.g., a dsRNA, that targets the Serpinc1 gene in a mammalian cell, for the manufacture of a medicament for inhibiting Serpinc1 gene expression in a mammal.

[0330] The methods and uses include administering to a mammal, e.g., a human, a composition comprising an iRNA, e.g., a dsRNA, that targets the Serpinc1 gene in a mammalian cell, and maintaining the mammal for a time sufficient to result in degradation of mRNA transcripts of the Serpinc1 gene, thereby inhibiting Serpinc1 gene expression in the mammal.

[0331] Decreased gene expression can be assessed by any method known in the art, including methods described herein, such as qRT-PCR. Decreased protein production can be assessed by any method known in the art, including methods described herein, such as ELISA. In one embodiment, a puncture liver biopsy sample serves as tissue material for monitoring decreased Serpinc1 gene and / or protein expression. In another embodiment, a blood sample serves as tissue material for monitoring decreased Serpinc1 gene and / or protein expression. In another embodiment, inhibition of Serpinc1 gene expression is monitored indirectly, for example, by determining the expression and / or activity of genes in the Serpinc1 pathway (see, for example, Figure 1). For example, factor Xa activity can be monitored to determine inhibition of Serpinc1 gene expression. Antithrombin levels, clot formation, and / or endogenous thrombin generation capacity in a sample, such as a blood or liver sample, can also be assessed. Suitable assays are further described in the Examples section below.

[0332] The present invention further provides methods for treating a subject having a disorder that would benefit from reduced expression of Serpinc1, such as hemophilia. The treatment methods (and uses) of the present invention comprise administering to a subject, e.g., a human, a therapeutically effective amount of an iRNA that targets the Serpinc1 gene, or a pharmaceutical composition comprising an iRNA that targets the Serpinc1 gene, thereby treating the subject having a disorder that would benefit from reduced expression of Serpinc1.

[0333] In one aspect, the present invention provides a method for preventing at least one symptom in the subject with the disorder that is beneficial from reducing Serpinc1 expression.Method comprises administering to the subject a therapeutically effective amount of iRNA or vector, such as the dsRNA of the present invention, thereby preventing at least one symptom in the subject with the disorder that is beneficial from reducing Serpinc1 expression.For example, the present invention provides a method for preventing bleeding in the subject with the disorder that is beneficial from reducing Serpinc1 expression, such as hemophilia.

[0334] In another aspect, the invention provides the use of a therapeutically effective amount of an iRNA of the invention to treat a subject, e.g., a subject who would benefit from reduced and / or inhibited expression of Serpincl, including an iRNA that targets the Serpincl gene or a pharmaceutical composition comprising an iRNA that targets the Serpincl gene.

[0335] In yet another aspect, the invention provides the use of an iRNA of the invention that targets the Serpinc1 gene, or a pharmaceutical composition comprising an iRNA that targets the Serpinc1 gene, in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from reduced and / or inhibited Serpinc1 expression.

[0336] In another aspect, the invention provides the use of an iRNA, e.g., a dsRNA of the invention, to prevent at least one symptom in a subject suffering from a disorder that would benefit from reduced and / or inhibited Serpincl expression, e.g., a bleeding disorder such as hemophilia.

[0337] In a further aspect, the present invention provides use of an iRNA of the present invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced and / or inhibited Serpinc1 expression, such as a bleeding disorder such as hemophilia. The iRNA of the present invention may be administered in a "naked" or "free iRNA" form. Naked iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmolality of the buffer solution containing the iRNA may be adjusted to suit administration to a subject.

[0338] Alternatively, the iRNA of the present invention may be administered in pharmaceutical compositions, such as dsRNA liposome formulations.

[0339] A subject who would benefit from reduced and / or inhibited Serpincl gene expression is a subject with a bleeding disorder, such as, for example, an inherited or acquired bleeding disorder described herein. In one embodiment, the subject with a genetic bleeding disorder is afflicted with hemophilia, such as hemophilia A, B, or C. In one embodiment, the subject with a genetic bleeding disorder, such as hemophilia, is an inhibitor subject. In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C. Treatments for subjects who would benefit from reduced and / or inhibited Serpincl gene expression include therapeutic treatments (e.g., on-demand, such as when the subject is bleeding (spontaneous bleeding or bleeding as a result of trauma) and is unable to form a blood clot) and prophylactic treatments (e.g., when the subject is not bleeding and / or is scheduled to undergo surgery).

[0340] The present invention further provides methods and uses for the use of iRNAs or pharmaceutical compositions thereof to treat subjects who would benefit from reduced and / or inhibited Serpincl expression, such as, for example, subjects with bleeding disorders, in combination with other pharmaceuticals and / or other therapies, such as known pharmaceuticals and / or known therapies, e.g., those currently used to treat these disorders. For example, in certain embodiments, iRNAs targeting Serpincl are administered in combination with agents useful for treating bleeding disorders, e.g., as described elsewhere herein. Additional therapeutic agents and therapies suitable for treating subjects who would benefit from a reduction in Serpincl expression, such as those with bleeding disorders, include fresh frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrate; inactivated virus, vWF-containing FVIII concentrate; desensitization therapy, which may include high doses of FVIII or FIX along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasma exchange therapy combined with immunosuppression and FVIII or FIX infusion with or without antifibrinolytic therapy; immune tolerance induction (ITI) with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and / or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytic drugs such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilic drugs; corticosteroids; immunosuppressants; and estrogen. The iRNA and the additional therapeutic agent(s) and / or therapy(ies) may be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent(s) may be administered as part of separate compositions, or at separate times, and / or by separate methods known in the art or described herein.

[0341] In one embodiment, the methods and uses comprise administering a composition featured herein such that expression of the target Serpincl gene is reduced, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target Serpincl gene is reduced for an extended period of time, such as at least about 2, 3, 4, 5, 6, 7 days or more, such as about 1 week, 2 weeks, 3 weeks, or about 4 weeks or more.

[0342] Preferably, the iRNAs useful in the methods, uses, and compositions featured herein specifically target the RNA (primary or processed) of the target Serpincl gene. Compositions, uses, and methods using iRNAs to inhibit expression of these genes can be prepared and performed as described herein.

[0343] The administration of dsRNA according to the method and use of the present invention can cause the severity, signs, symptoms and / or markers of such disease or disorder to be reduced in patients with bleeding disorder.In this context, " reduction " means the statistically significant reduction of such level.The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100%.

[0344] Efficacy of disease treatment or prevention may be assessed by measuring, for example, disease progression, disease remission, symptom severity, bleeding frequency, pain reduction, quality of life, drug dose required to maintain therapeutic efficacy, disease marker levels, or any other measurable parameter levels appropriate for the given disease being treated or targeted for prevention. It is well within the capabilities of one of ordinary skill in the art to monitor therapeutic or prophylactic efficacy by measuring any one or any combination of such parameters. For example, efficacy of a bleeding disorder treatment may be assessed by periodically monitoring, for example, thrombin:antithrombin levels. Comparison of initial readings with subsequent readings provides the physician with an indication of whether the treatment is effective. It is well within the capabilities of one of ordinary skill in the art to monitor therapeutic or prophylactic efficacy by measuring any one or any combination of such parameters. In the context of administration of an iRNA or pharmaceutical composition thereof targeting Serpinc1, "effective against" a bleeding disorder means that administration in a clinically relevant manner will result in a beneficial effect in at least a statistically significant proportion of patients, such as symptom improvement, cure, disease reduction, prolongation of life, improved quality of life, or other effect generally recognized as favorable by physicians familiar with the treatment of bleeding disorders and related causes.

[0345] A therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is a lack of worsening or onset of symptoms that would be expected in the absence of treatment. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more, in a measurable parameter of the disease may indicate effective treatment. The efficacy of a given iRNA agent or combination of agents may also be determined using an experimental animal model for a given disease known in the art. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0346] Alternatively, efficacy can be assessed by a reduction in disease severity, as determined by one skilled in the art of diagnostics, based on a clinically accepted disease severity assessment scale, such as the Child-Pugh score (sometimes also referred to as the Child-Turcotte-Pugh score). Any favorable change resulting in a reduction in disease severity, e.g., as assessed using a suitable scale, indicates appropriate treatment with an iRNA or iRNA formulation described herein.

[0347] Subjects received approximately 0.01mg / kg, 0.02mg / kg, 0.03mg / kg, 0.04mg / kg, 0.05mg / kg, 0.1mg / kg, 0.15mg / kg, 0.2mg / kg, 0.25mg / kg, 0.3mg / kg, 0.35mg / kg, 0.4mg / kg, 0.45mg / kg, 0.5mg / kg, 0.55mg / kg, 0.6mg / kg, 0.65mg / kg, 0.7mg / kg, 0.75mg / kg, 0.8mg / kg, 0.85mg / kg, 0.9mg / kg, 0.95mg / kg, 1.0mg / kg, 1.1mg / kg, 1.2mg / kg, 1.4mg / kg, 1.6mg / kg, 1.8mg / kg, 1.9mg / kg, 1.1mg / kg, 1.2mg / kg, 1.3mg / kg, 1.4mg / kg, 1.5mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9 ...9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg / kg, 1.9mg mg / kg, 1.3mg / kg, 1.4mg / kg, 1.5mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9mg / kg, 2.0mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg dsRNA, 2.6mg / kg dsRNA, 2.7mg / kg dsRNA, 2.8mg / kg dsRNA, 2.9mg / kg dsRNA, 3.0mg / kg dsRNA, 3.1mg / kg dsRNA, 3.2mg / kg dsRNA, 3.3mg / kg dsRNA, 3.4mg / kg dsRNA, 3.5mg / kg dsRNA, 3.6mg / kg dsRNA, 3.7mg / kg dsRNA, 3.8mg / kg dsRNA, 3.9mg / kg dsRNA, 4.0mg / kg dsRNA, 4.1mg / kg dsRNA, 4.2mg / kg dsRNA, 4.3mg / kg dsRNA, 4.4mg / kg dsRNA, 4.5mg / kg dsRNA, 4.6mg / kg dsRNA, 4.7mg / kg dsRNA, 4.8mg / kg dsRNA, 4.9mg / kg dsRNA, 5.0mg / kg dsRNA, 5.1mg / kg dsRNA, 5.2mg / kg dsRNA, 5.3mg / kg dsRNA, 5.4mg / kg dsRNA, 5.5mg / kg dsRNA, 5.6mg / kg dsRNA, 5.7mg / kg dsRNA, 5.8mg / kg dsRNA, 5.9mg / kg dsRNA, 6.0mg / kg dsRNA, 6.1mg / kg dsRNA, 6.2mg / kg dsRNA, 6.3mg / kg dsRNA, 6.4mg / kg dsRNA, 6.5mg / kg dsRNA, 6.6mg / kg dsRNA, 6.7mg / kg dsRNA, 6.8mg / kg dsRNA, 6.9mg / kg dsRNA, 7.0mg / kg dsRNA, 7.1mg / kg dsRNA, 7.2mg / kg dsRNA, 7.3mg / kg dsRNA, 7.4mg / kg dsRNA, 7.5mg / kg dsRNA, 7.6mg / kg dsRNA, 7.7mg / kg dsRNA, 7.8mg / kg dsRNA, 7.9mg / kg dsRNA, 8.0mg / kg dsRNA, 8.1mg / kg dsRNA, 8.2mg / kg dsRNA, 8.3mg / kg dsRNA, 8.4mg / kg dsRNA, 8.5mg / kg dsRNA, 8.6mg / kg dsRNA, 8.7mg / kg dsRNA, 8.8mg / kg dsRNA , 8.9 mg / kg dsRNA, 9.0 mg / kg dsRNA, 9.1 mg / kg dsRNA, 9.2 mg / kg dsRNA, 9.3 mg / kg dsRNA, 9.4 mg / kg dsRNA, 9.5 mg / kg dsRNA, 9.6 mg / kg dsRNA, 9.7 mg / kg dsRNA, 9.8 mg / kg dsRNA, 9.9 mg / kg dsRNA, 9.0 mg / kg dsRNA, 10 mg / kg dsRNA, 15 mg / kg dsRNA, 20 mg / kg dsRNA, 25 mg / kg dsRNA, 30 mg / kg dsRNA, 35 mg / kg dsRNA, 40 mg / kg dsRNA, 45 mg / kg dsRNA, or about 50 mg / kg dsRNA may be administered. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of this invention.

[0348] For example, in certain embodiments in which the compositions of the invention comprise a dsRNA described herein and a lipid, a subject is administered about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, Approximately 0.2 mg / kg to approximately 10 mg / kg, approximately 0.3 mg / kg to approximately 5 mg / kg, approximately 0.3 mg / kg to approximately 10 mg / kg, approximately 0.4 mg / kg to approximately 5 mg / kg, approximately 0.4 mg / kg to approximately 10 mg / kg, Approximately 0.5mg / kg to approximately 5mg / kg, approximately 0.5mg / kg to approximately 10mg / kg, approximately 1mg / kg to approximately 5mg / kg, approximately 1mg / kg to approximately 10mg / kg, approximately 1.5mg / kg to approximately 5mg / kg, approximately 1.5m g / kg~about 10mg / kg, about 2mg / kg~about 2.5mg / kg, about 2mg / kg~about 10mg / kg, about 3mg / kg~about 5mg / kg, about 3mg / kg~about 10mg / kg, about 3.5mg / kg ~5mg / kg, approximately 4mg / kg~5mg / kg, approximately 4.5mg / kg~5mg / kg, approximately 4mg / kg~10mg / kg, approximately 4.5mg / kg~10mg / kg, approximately 5mg / kg~10mg / kg A therapeutic amount of iRNA may be administered, such as about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0349] For example, dsRNA may have a molecular weight of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0350] For example, in another embodiment in which the composition of the present invention comprises a dsRNA described herein and N-acetylgalactosamine, a subject is administered about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, or about 7.5 to about 50 mg / kb. g, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50mg / kg, about 0.1 to about 45mg / kg, about 0.25 to about 45mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / mg, about 1.5 to about 45mg / kb, about 2 to about 45mg / kg, about 2.5 to about 45mg / kg, about 3 to about 45m g / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 ~40mg / mg, 1.5~40mg / kb, 2~40mg / kg, 2.5~40mg / kg, 3~40mg / kg, 3.5~40mg / kg, 4~40mg / kg, 4.5~40mg / kg, 5~40mg / kg, 7.5~40mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg , about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, Therapeutic doses of iRNA may be administered, such as doses of about 0.1 to about 20 mg / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. In one embodiment, when the compositions of the present invention comprise a dsRNA and N-acetylgalactosamine as described herein, a therapeutic dose of about 10 to about 30 mg / kg of dsRNA may be administered to a subject. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of this invention.

[0351] For example, the subject may have a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 ,4,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21 Therapeutic amounts of iRNA such as 1, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0352] The iRNA can be administered by intravenous infusion 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. Administration can be repeated periodically, for example, weekly or biweekly (i.e., every two weeks) for one month, two months, three months, four months, or more. After the initial treatment regimen, treatment can be administered less frequently. For example, after weekly or biweekly administration for three months, monthly administration can be repeated for six months or a year or more.

[0353] In one embodiment, the present invention provides a method of treating a subject suffering from a bleeding disorder, e.g., hemophilia, by subcutaneously administering to said subject compound AD-57213 at a cumulative weekly dose of about 0.5 mg / kg to about 5 mg / kg, or about 1 mg / kg to about 3 mg / kg.

[0354] In one embodiment, the method may include subcutaneously administering to the subject a cumulative weekly dose of about 0.5 mg / kg. For example, in one embodiment, the method may include administering to the subject a cumulative weekly dose of 0.5 mg / kg at about 0.5 mg / kg every week. In another embodiment, the method may include administering to the subject a cumulative weekly dose of 0.5 mg / kg at 1 mg / kg every other week.

[0355] In another embodiment, the method may comprise subcutaneously administering to the subject a cumulative weekly dose of about 1.5 mg / kg. For example, in one embodiment, the method may comprise administering to the subject a cumulative weekly dose of 1.5 mg / kg at about 1.5 mg / kg every week. In another embodiment, the method may comprise administering to the subject a cumulative weekly dose of 1.5 mg / kg at 3 mg / kg every other week.

[0356] In another embodiment, the method may include subcutaneously administering to the subject a cumulative weekly dose of about 2 mg / kg. For example, in one embodiment, the method may include administering to the subject a cumulative weekly dose of 2 mg / kg at about 2 mg / kg every week. In another embodiment, the method may include administering to the subject a cumulative weekly dose of 2 mg / kg at 4 mg / kg every other week.

[0357] In yet another embodiment, the method may include subcutaneously administering to the subject a cumulative weekly dose of about 3 mg / kg. For example, in one embodiment, the method may include administering to the subject a cumulative weekly dose of 3 mg / kg at about 3 mg / kg every week. In another embodiment, the method may include administering to the subject a cumulative weekly dose of 3 mg / kg at 6 mg / kg every other week.

[0358] In another embodiment, the present invention provides a method of preventing at least one symptom of a bleeding disorder, e.g., hemophilia, in a subject by subcutaneously administering to the subject compound AD-57213 at a cumulative weekly dose of about 0.5 mg / kg to about 5 mg / kg or about 1 mg / kg to about 3 mg / kg.

[0359] In one embodiment, the method may include subcutaneously administering to the subject a cumulative weekly dose of about 0.5 mg / kg. For example, in one embodiment, the method may include administering to the subject a cumulative weekly dose of 0.5 mg / kg at about 0.5 mg / kg every week. In another embodiment, the method may include administering to the subject a cumulative weekly dose of 0.5 mg / kg at 1 mg / kg every other week.

[0360] In another embodiment, the method may comprise subcutaneously administering to the subject a cumulative weekly dose of about 1.5 mg / kg. For example, in one embodiment, the method may comprise administering to the subject a cumulative weekly dose of 1.5 mg / kg at about 1.5 mg / kg every week. In another embodiment, the method may comprise administering to the subject a cumulative weekly dose of 1.5 mg / kg at 3 mg / kg every other week.

[0361] In another embodiment, the method may include subcutaneously administering to the subject a cumulative weekly dose of about 2 mg / kg. For example, in one embodiment, the method may include administering to the subject a cumulative weekly dose of 2 mg / kg at about 2 mg / kg every week. In another embodiment, the method may include administering to the subject a cumulative weekly dose of 2 mg / kg at 4 mg / kg every other week.

[0362] In yet another embodiment, the method may include subcutaneously administering to the subject a cumulative weekly dose of about 3 mg / kg. For example, in one embodiment, the method may include administering to the subject a cumulative weekly dose of 3 mg / kg at about 3 mg / kg every week. In another embodiment, the method may include administering to the subject a cumulative weekly dose of 3 mg / kg at 6 mg / kg every other week.

[0363] iRNA administration can reduce Serpincl levels by at least about 5%, for example, in the patient's cells, tissues, blood, urine, or other compartments. , 46, 47, 48, 39, 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.

[0364] In one embodiment, a therapeutic and / or prophylactic method includes subcutaneously administering to a subject the compound AD-57213, e.g., at a dose sufficient to inhibit reduce Serpincl levels in the patient's cells, tissues, blood, urine, or other compartment by at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 69, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80%.

[0365] Prior to administration of the full dose of iRNA, the patient may be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as allergic reactions. In another example, the patient may be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-α or INF-α) levels.

[0366] Due to their inhibitory effect on Serpinc1 expression, compositions according to the present invention or pharmaceutical compositions prepared therefrom may improve quality of life.

[0367] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the iRNA and methods featured in this invention, and suitable methods and materials are listed below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, shall prevail.In addition, materials, methods and examples are intended to be illustrative only and are not intended to be limiting. [Example]

[0368] Example 1: iRNA synthesis Reagent Supplier Unless the source of a reagent is specifically indicated herein, such reagents may be obtained from any supplier of molecular biology reagents of quality / purity standards for molecular biology applications.

[0369] Transcripts siRNA design was performed to identify siRNAs targeting human, rhesus macaque (Macacamulatta), dog, mouse, and rat SERPINC1 transcripts annotated in the NCBI gene database ( http: / / www.ncbi.nlm.nih.gov / gene / ). The design used the following transcripts from the NCBI RefSeq collection: Human-NM_000488.2, NM_000488.3; Rhesus-NM_001104583.1; Dog-XM_856414.1; Mouse-NM_080844.4; Rat-NM_001012027.1. Due to the high degree of primate / canine / rodent sequence diversity, siRNA duplexes were designed in several separate batches, including, but not limited to, batches containing only duplex-matched human and rhesus transcripts; only human, rhesus, and dog transcripts; only human, rhesus, mouse, and rat transcripts; and only mouse and rat transcripts. All siRNA duplexes were designed to share 100% identity with the listed human transcripts and transcripts of other species discussed in each design batch (above).

[0370] siRNA design, specificity, and efficacy prediction The predicted specificity of all possible 19-mers was estimated from each sequence. Candidate 19-mers lacking repeats longer than 7 nucleotides were then selected. These 874 candidate human / rhesus, 67 human / rhesus / dog, 103 human / rhesus / mouse / rat, and 569 mouse / rat siRNAs were used in a comprehensive search against the appropriate transcriptome (defined as the NM_ and XM_records sets within the human, rhesus, dog, mouse, or rat NCBI Refseq set) using an exhaustive "brute force" algorithm implemented in the Python script "BruteForce.py." The script then parsed the transcript oligo alignment and generated a score based on the placement and number of mismatches between the siRNA and any possible "off-target" transcripts. The off-target score was weighted to emphasize differences within the siRNA's "seed" region, between positions 2 and 9 from the 5' end of the molecule.

[0371] Each oligo-transcript pair from the brute force search was assigned a mismatch score by adding the individual mismatch scores; a mismatch at positions 2–9 was assigned a score of 2.8, a mismatch at the cleavage site at positions 10–11 was assigned a score of 1.2, and a mismatch at region 12–19 was assigned a score of 1.0. Off-target prediction was further performed by comparing the frequency of heptamers and octamers derived from three different seed-derived hexamers for each oligo. Two heptamers and one octamers were generated using hexamers from positions 2–7 relative to the 5′ start. "Heptamer 1" was generated by adding a 3′-A to the hexamer; heptamer 2 was generated by adding a 5′-A to the hexamer; and the octamers were generated by adding A to both the 5′ and 3′ ends of the hexamer. The frequencies of octamers and heptamers in the human, rhesus monkey, mouse, or rat 3'UTRome (defined as a partial transcriptome sequence from NCBI's Refseq database, with clearly defined coding sequence ends, or "CDS") were pre-calculated. Octamer frequencies were normalized for heptamer frequencies using the median value from the octamer frequency range. The "mirSeedScore" was then calculated by summing ((3 × normalized octamer count) + (2 × heptamer2 count) + (1 × heptamer1 count)).

[0372] Both siRNA strands were assigned to specificity categories according to the calculated scores. A score greater than 3 was considered highly specific, a score equal to 3 was considered specific, and a score between 2.2 and 2.8 was considered moderately specific. Duplexes were sorted by antisense strand specificity, and duplexes in which the antisense oligo lacked GC at position 1, G at both positions 13 and 14, and had three or more Us or As in the seed region were selected.

[0373] siRNA sequence selection A total of 66 sense and 66 antisense-derived human / rhesus monkey, 6 sense and 6 antisense-derived human / rhesus monkey / mouse, 12 human / rhesus monkey / mouse / rat, and 21 sense and 21 antisense-derived mouse / rat siRNA oligos were synthesized and duplexed. A detailed list of the Sepinc1 sense and antisense strand sequences is shown in Tables 3 and 4.

[0374] siRNA synthesis I. General Small- and Medium-Scale RNA Synthesis Procedures RNA oligonucleotides were synthesized on a 0.2–500 μmol scale using commercially available 5'-O-(4,4'-dimethoxytrityl)-2'-Ot-butyldimethylsilyl-3'-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite monomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine, and 2-N-isobutyrylguanosine, and the corresponding 2'-O-methyl and 2'-fluorophosphoramidites, according to standard solid-phase oligonucleotide synthesis protocols. Amidite solutions were prepared at concentrations of 0.1–0.15 M, and 5-ethylthio-1H-tetrazole (0.25–0.6 M in acetonitrile) was used as the activating agent. For the oxidation step, phosphorothioate backbone modifications were introduced during synthesis using 0.2 M phenylacetyl disulfide (PADS) in lutidine:acetonitrile (1:1) (v:v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine. After synthesis was complete, the sequences were cleaved from the solid support and deprotected using methylamine followed by triethylamine·3HF to remove any 2'-Ot-butyldimethylsilyl protecting groups present.

[0375] For the synthesis of fully 2'-modified sequences (2'-fluoro and / or 2'-O-methyl, or a combination thereof) on a 5- to 500-μmol scale, oligonucleotides were deprotected using 3:1 (v / v) ethanol and concentrated (28-32%) aqueous ammonia at either 35°C for 16 hours or 55°C for 5.5 hours. Prior to ammonia deprotection, oligonucleotides were treated with 0.5 M piperidine in acetonitrile for 20 minutes on solid support. Crude oligonucleotides were analyzed by LC-MS and anion-exchange HPLC (IEX-HPLC). Oligonucleotide purification was performed by IEX HPLC using 20 mM phosphate, 10%-15% ACN, pH = 8.5 (Buffer A), and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH = 8.5 (Buffer B). Fractions were analyzed for purity by analytical HPLC. Product-containing fractions of appropriate purity were pooled and concentrated on a rotary evaporator prior to desalting. Samples were desalted by size-exclusion chromatography and lyophilized. The corresponding siRNA duplexes were prepared by annealing equimolar amounts of the sense and antisense strands in 1x PBS buffer.

[0376] Small-scale (0.2–1 μmol) synthesis was performed on a MerMade 192 synthesizer in a 96-well configuration. For fully 2'-modified sequences (2'-fluoro and / or 2'-O-methyl, or a combination thereof), oligonucleotides were deprotected using methylamine for 30–60 min at room temperature, followed by a 30-min incubation at 60°C, or using 3:1 (v / v) ethanol and concentrated (28–32%) aqueous ammonia for 30–60 min at room temperature, followed by a 1.5-h incubation at 40°C. Crude oligonucleotides were then precipitated in acetonitrile:acetone (9:1) solution, isolated by centrifugation, and the supernatant was decanted. The crude oligonucleotide pellet was resuspended in 20 mM NaOAc buffer and analyzed by LC-MS and anion-exchange HPLC. Crude oligonucleotide sequences were desalted in a 96-deep-well plate on a 5 mL HiTrap Sephadex G25 column (GE Healthcare). Approximately 1.5 mL of sample corresponding to an individual sequence was collected in each well. These purified, desalted oligonucleotides were analyzed by LC-MS and anion exchange chromatography. Duplexes were prepared by annealing equimolar amounts of sense and antisense sequences on a Tecan robot. The concentration of the duplex was adjusted to 10 μM in 1× PBS buffer.

[0377] II. Synthesis of GalNAc-conjugated oligonucleotides for in vivo analysis Oligonucleotides conjugated at the 3' end with GalNAc ligands were synthesized on a 0.2-500 μmol scale using a solid support preloaded with a Y-shaped linker bearing a 4,4'-dimethoxytrityl (DMT)-protected primary hydroxyl group and a GalNAc ligand attached via a tether.

[0378] For GalNAc conjugate synthesis on the 5- to 500-μmol scale, the RNA synthesis protocol above was followed with the following modifications. For polystyrene-based synthetic supports, 5% dichloroacetic acid in toluene was used for DMT cleavage during synthesis. Cleavage and deprotection from the support were performed as described above. Phosphorothioate-rich sequences (typically greater than five phosphorothioates) were synthesized without removing the final 5'-DMT group ("DMT-on") and, after cleavage and deprotection as described above, were purified by reverse-phase HPLC using 50 mM ammonium acetate in water (Buffer A) and 50 mM ammonium acetate in 80% acetonitirile (Buffer B). Fractions were analyzed for purity by analytical HPLC and / or LC-MS. Product-containing fractions of appropriate purity were pooled and concentrated on a rotary evaporator. DMT groups were removed to completion using 20% ​​to 25% acetic acid in water. The samples were desalted by size-exclusion chromatography and lyophilized. The corresponding siRNA duplexes were prepared by annealing equimolar amounts of the sense and antisense strands in 1× PBS buffer.

[0379] For small-scale synthesis of GalNAc conjugates (0.2–1 μmol) containing sequences with multiple phosphorothioate linkages, the protocol described above for the synthesis of RNA or entirely 2'-F / 2'-OMe-containing sequences on the MerMade platform was adapted. Synthesis was performed on a pre-packed column containing a GalNAc-functionalized controlled-pore glass support.

[0380] Example 2: In vitro screening Cell culture and transfection Hep3B cells (ATCC, Manassas, VA) were grown to near confluence in Eagle's minimum essential medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in a 5% CO2 atmosphere and then released from the plate by trypsinization. For mouse cross-reactive duplexes, mouse primary hepatocytes (PMH) were freshly isolated within 1 hour prior to transfection and cultured in primary hepatocyte medium. Transfection of both Hep3B and PMH was performed in individual wells of a 96-well plate by adding 14.8 μl of Opti-MEM and 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA; catalog number 13778-150) per well to 5 μl of each siRNA duplex. The mixture was then incubated at room temperature for 15 minutes. Approximately 2 × 10 4 80 μl of complete growth medium without antibiotics containing Hep3B cells was added to the siRNA mixture. Cells were cultured for 24 hours prior to RNA purification. Single-dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose-response experiments were performed using 8 x 5-fold serial dilutions ranging from 10 nM to 128 pM (see Figures 2A and 2B).

[0381] Free uptake transfection In each well of a 96-well plate, 4 × 10 cells resuspended in 95 μl of In Vitro Gro CP medium (In Vitro Technologies-Celsis, Baltimore, MD) were cultured. 4 Freshly thawed cryopreserved cynomolgus monkey hepatocytes were combined with 5 μl of each GalNac-conjugated siRNA in PBS. The mixtures were incubated at 37°C in a 5% CO atmosphere for approximately 24 hours. siRNAs were tested for effective free uptake assays at final concentrations of 100 nM, 10 nM, and 0.1 nM.

[0382] Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen; part number 610-12) The cells were collected and lysed in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the procedure). 10 μl of magnetic beads and 80 μl of the lysis / binding buffer mixture were added to a round-bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were recaptured and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, captured, and the supernatant was removed. The beads were then washed with 150 μl of elution buffer, captured, and the supernatant was removed. Finally, the beads were dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 70 minutes at 5°C. The beads were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and placed in another 96-well plate.

[0383] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA; catalog number 4368813) Per reaction, a master mix of 2 μl of 10× buffer, 0.8 μl of 25× dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of H O was added to 10 μl of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and a 4°C hold.

[0384] Real-time PCR Two microliters of cDNA was added per well of a 384-well plate to a master mix containing 0.5 μl of human GAPDH TaqMan probe (Applied Biosystems; catalog no. 4326317E), 0.5 μl of human SERPINC1 TaqMan probe (Applied Biosystems; catalog no. Hs00892758_m1) for human cells or 0.5 μl of mouse GAPDH TaqMan probe (Applied Biosystems; catalog no. 4308313) for mouse cells, 0.5 μl of mouse SERPINC1 TaqMan probe (Applied Biosystems; catalog no. Mm00446573_m1), and 5 μl of Lightcycler 480 Probe Master Mix (Roche; catalog no. 04887301001). Real-time PCR was performed in an ABI7900HT real-time PCR system (Applied Biosystems) using the ΔΔCt (RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate unless otherwise noted in the summary table.

[0385] To calculate relative fold changes in Serpinc1 mRNA levels, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. A four-parameter fitting model using XLFit was used to calculate the IC. 50 was calculated and normalized to cells transfected with AD-1955 over the same dose range or to its own lowest dose. Table 5 shows the results of a single-dose screen in Hep3B and PMH cells transfected with the indicated iRNAs. Table 6 shows the dose-response results of transfection of Hep3B and PMH cells with the indicated iRNAs.

[0386] The sense and antisense sequences of AD-1955 are as follows: Sense cuuAcGcuGAGuAcuucGAdTsdT- (SEQ ID NO: 13) Antisense UCGAAGuACUcAGCGuAAGdTsdT- (SEQ ID NO: 14).

[0387] [Table 2-1]

[0388] [Table 2-2]

[0389] [Table 2-3]

[0390] [Table 3-1]

[0391] [Table 3-2]

[0392] [Table 4-1]

[0393] [Table 4-2]

[0394] [Table 4-3]

[0395] [Table 5-1]

[0396] [Table 5-2]

[0397] [Table 6]

[0398] Some of the siRNAs were also synthesized with 2'-OMe modifications, and these siRNA duplexes in Lipofectamine formulations were used to transfect Hep3B cells. The results of the single-dose screening of the modified duplexes are shown in Table 7.

[0399] [Table 7-1]

[0400] [Table 7-2]

[0401] Examples 3-4: Lead optimization and in vivo testing Table 8 provides a detailed list of double-stranded siRNA sequences targeting Serpinc1 that were formulated as lipid nanoparticles (LNPs) (i.e., with MC3) or conjugated to GalNAc for lead optimization and in vivo delivery.

[0402] [Table 8-1]

[0403] [Table 8-2]

[0404] [Table 8-3]

[0405] [Table 8-4]

[0406] [Table 8-5]

[0407] [Table 8-6]

[0408] [Table 8-7]

[0409] [Table 8-8]

[0410] [Table 8-9]

[0411] [Table 8-10]

[0412] [Table 8-11]

[0413] [Table 8-12]

[0414] Example 3: LNP-mediated siRNA delivery The above-mentioned in vitro single dose and IC 50Based on the results, modified AD-50509 was selected for lipid nanoparticle (LNP) formulation. To determine the effective dose of LNP-mediated delivery of AD-50509, a single dose of LNP formulation of AD-50509 siRNA (AF-011) was intravenously injected into CD1 mice at 0.003, 0.01, 0.03, 0.1, 0.3, or 1.0 mg / kg. Animals were sacrificed 48 hours later, and Serpincl mRNA levels relative to GAPDH and Serpincl protein levels were measured as described herein. As shown in Figures 3A and 3B, an ED of approximately 0.1 mg / kg was observed. 50 At 85%, a maximum Serpinc1 mRNA silencing by AF-011-AD-50509 was achieved (Figure 3A), with an ED of approximately 0.05 mg / kg. 50 , a maximum Serpinc1 protein silencing of 90% was achieved ( Fig. 3B ).

[0415] The silencing duration of AD-50509 siRNA (Af-011-50509) LNP formulation was determined in CD1 mice following a single 1 mg / kg intravenous injection of siRNA. Animals were sacrificed 1, 2, 3, 7, 14, 21, or 28 days after administration, and relative Serpincl mRNA and Serpincl protein levels were measured. Figure 4A demonstrates that Af-011-formulated AD-50509 achieve...

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting Serpinc1 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides.

2. A double-stranded ribonucleic acid (dsRNA) for inhibiting Serpincl expression, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

3. The sense and antisense strands are selected from the group consisting of AD-50487.1, AD-50477.1, AD-50483.1, AD-50475.1, AD-50495.1, AD-50476.1, AD-50499.1, AD-50478.1, AD-50489.1, AD-50501.1, AD-50507.1, AD-50484.1, AD-50515.1, AD-50540.1, AD-505 28.1, AD-50549.1, AD-50539.1, AD-50534.1, AD-50527.1, AD-50514.1, AD-50509.1, AD-50529.1, and AD-54944, and any of the sequences listed in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

4. 3. The dsRNA of claim 1 or 2, wherein the dsRNA comprises at least one modified nucleotide.

5. 5. The dsRNA of claim 4, wherein at least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

6. The dsRNA of claim 4, wherein the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base-containing nucleotides.

7. 3. The dsRNA of claim 2, wherein the complementary region is at least 17 nucleotides in length.

8. 3. The dsRNA of claim 2, wherein the complementary region is 19 to 21 nucleotides in length.

9. 9. The dsRNA of claim 8, wherein the complementary region is 19 nucleotides in length.

10. 3. The dsRNA of claim 1 or 2, wherein each strand is 30 nucleotides or less in length.

11. 3. The dsRNA of claim 1 or 2, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

12. 3. The dsRNA of claim 1 or 2, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

13. The dsRNA of claim 1 or 2, further comprising a ligand.

14. 14. The dsRNA of claim 13, wherein the ligand is attached to the 3' end of the sense strand of the dsRNA.

15. 14. The dsRNA of claim 13, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

16. The ligand is 【Chemistry 1】 The dsRNA of claim 15, wherein

17. Schematic diagram below, 【Chemistry 2】 (In the formula, 16. The dsRNA of claim 15, wherein X is O or S.

18. 18. The dsRNA of claim 17, wherein X is O.

19. 3. The dsRNA of claim 2, wherein the region of complementarity consists of one of the antisense sequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

20. 3. The dsRNA of claim 1 or 2, comprising a sense strand consisting of a sense strand sequence selected from any one of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and an antisense strand consisting of an antisense sequence selected from any one of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

21. A cell containing the dsRNA of claim 1 or 2.

22. A vector encoding at least one dsRNA strand, wherein the dsRNA comprises a region of complementarity to at least a portion of an mRNA encoding Serpincl, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage.

23. 23. The vector of claim 22, wherein the region of complementarity is at least 15 nucleotides in length.

24. 23. The vector of claim 22, wherein the complementary region is 19 to 21 nucleotides in length.

25. A cell comprising the vector of claim 22.

26. A pharmaceutical composition for inhibiting Serpinc1 gene expression, comprising the dsRNA of claim 1 or 2, or the vector of claim 22.

27. (a) contacting a cell with the dsRNA of claim 1 or 2, or the vector of claim 22; (b) maintaining the cells produced in step (a) for a time sufficient to allow degradation of mRNA transcripts of the Serpincl gene, thereby inhibiting Serpincl gene expression in said cells; A method for inhibiting Serpinc1 expression in a cell, comprising:

28. 28. The method of claim 27, wherein the cell is in a subject.

29. 29. The method of claim 28, wherein the subject is a human.

30. 30. The method of claim 29, wherein the human subject suffers from a bleeding disorder.

31. 31. The method of claim 30, wherein the bleeding disorder is hemophilia.

32. The method of any one of claims 27 to 31, wherein the Serpincl expression is inhibited by at least about 30%.

33. A method for treating a subject having a disorder that would benefit from reduced expression of Serpincl, comprising administering to the subject a therapeutically effective amount of the dsRNA of claim 1 or 2 or the vector of claim 22, thereby treating the subject.

34. A method for preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpincl expression, comprising the step of administering to the subject a therapeutically effective amount of the dsRNA described in claim 1 or 2 or the vector described in claim 22, thereby preventing at least one symptom in the subject having the disorder that would benefit from reduced Serpincl expression.

35. 35. The method of claim 33 or 34, wherein the disorder is a bleeding disorder.

36. 36. The method of claim 35, wherein the bleeding disorder is hemophilia.

37. The method of claim 33 or 34, wherein administration of the dsRNA to the subject causes increased blood clotting and / or decreased Serpincl protein accumulation.

38. 35. The method of claim 33 or 34, wherein the dsRNA is conjugated to a ligand.

39. 39. The method of claim 38, wherein the ligand is attached to the 3' end of the sense strand of the dsRNA.

40. 40. The method of claim 39, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

41. 35. The method of claim 34, wherein the dsRNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

42. 42. The method of claim 41, wherein the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg.

43. 42. The method of claim 41, wherein the dsRNA is administered at a dose selected from the group consisting of 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg.

44. 44. The method of claim 42 or 43, wherein the dsRNA is administered to the subject once a week.

45. 44. The method of claim 42 or 43, wherein the dsRNA is administered to the subject twice a month.

46. 35. The method of claim 33 or 34, further comprising measuring thrombin levels in the subject.

47. 36. The method of claim 34 or 35, wherein the dsRNA is administered subcutaneously to the subject at a cumulative weekly dose of about 0.5 mg / kg to about 5 mg / kg, and the dsRNA is AD-57213.

48. A method for inhibiting Serpincl expression in a subject, comprising the step of administering to the subject a therapeutically effective amount of the dsRNA described in claim 1 or 2 or the vector described in claim 22, thereby inhibiting Serpincl expression in the subject.

49. 49. The method of claim 48, wherein the dsRNA is conjugated to a ligand.

50. 50. The method of claim 49, wherein the ligand is attached to the 3' end of the sense strand of the dsRNA.

51. 51. The method of claim 50, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

52. 49. The method of claim 48, wherein the dsRNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

53. 53. The method of claim 52, wherein the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg.

54. 53. The method of claim 52, wherein the dsRNA is administered at a dose selected from the group consisting of 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg.

55. 55. The method of claim 54, wherein the dsRNA is administered to the subject once a week.

56. 55. The method of claim 54, wherein the dsRNA is administered to the subject twice a month.

57. 49. The method of claim 48, wherein the dsRNA is administered subcutaneously to the subject at a cumulative weekly dose of about 0.5 mg / kg to about 5 mg / kg, and the dsRNA is AD-57213.

58. 49. The method of claim 48, further comprising measuring thrombin levels in the subject.

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