Serpinc1 iRNA composition and method of use thereof

Abstract

To provide iRNA, e.g., double-stranded ribonucleic acid (dsRNA), compositions targeting the Serpinc1 gene, and methods for using such iRNA, e.g., dsRNA, compositions to inhibit expression of Serpinc1 and methods of treating subjects having a bleeding disorder, such as hemophilia.SOLUTION: The present invention provides a dsRNA for inhibiting expression of Serpinc1, comprising a specific nucleotide sequence, the dsRNA optionally further comprising a ligand, as well as a pharmaceutical composition comprising dsRNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 61 / 638,952, filed on 26 April 2012; U.S. Provisional Patent Application No. 61 / 669,249, filed on 9 July 2012; U.S. Provisional Patent Application No. 61 / 734,573, filed on 7 December 2012; and U.S. Patent Publication No. 13 / 837,129, filed on 15 March 2013. The entire contents of each of the aforementioned applications are incorporated herein by reference. [Background technology]

[0002] Serpinc1 is a member of the serine proteinase inhibitor (serpine) superfamily. Serpinc1 is a plasma protease inhibitor that inhibits thrombin, as well as other activated serine proteases of the coagulation system such as factors X, IX, XI, XII, and VII, and thus modulates 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 that catalyze the formation of the thrombin:antithrombin (TAT) complex.

[0003] Both hereditary and acquired bleeding disorders are conditions in which blood clotting is insufficient. Hemophilia, for example, is a group of hereditary bleeding disorders that impair the body's ability to control blood clotting or coagulation. Hemophilia A is a recessive X-linked genetic disorder with a deficiency of functional coagulation factor VIII and accounts for 80% of hemophilia cases. Hemophilia B is a recessive X-linked genetic disorder with a deficiency of functional coagulation factor IX. It accounts for approximately 20% of hemophilia cases. Hemophilia C is an autosomal genetic disorder with a deficiency of functional coagulation factor XI. Hemophilia C is not entirely recessive, as heterozygous individuals also exhibit increased bleeding.

[0004] Currently, there is no cure for hemophilia, but it can be controlled by regular infusions of the deficient clotting factor, such as factor VIII in hemophilia A. However, some hemophilia patients develop antibodies (inhibitors) against the administered surrogate factors, making the condition refractory to surrogate factors. Consequently, bleeding in such patients cannot be adequately controlled.

[0005] For example, the development of high-titer inhibitors against factor VIII and other coagulation factors is the most serious complication of hemophilia therapy, making the treatment of bleeding extremely difficult. Currently, the only strategies to stop bleeding in such patients are the use of "bypass agents" such as factor VIII inhibitor bypass activity (FEIBA), and activated recombinant factor VII (rFVIIa), plasmapheresis, continuous factor replacement, and immunotherapy, none of which are completely effective. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Therefore, in this technological field, there is a need for alternative therapeutic agents for patients with bleeding disorders such as hemophilia. [Means for solving the problem]

[0007] The present invention provides an iRNA composition that results in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the Serpinc1 gene. The Serpinc1 gene may be present in cells, such as cells 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 Serpinc1 gene expression and / or for treating subjects with disorders, such as bleeding disorders like hemophilia, who would benefit from the inhibition or reduction of Serpinc1 gene expression.

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

[0009] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting Serpinc1 expression. The dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense sequences listed in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

[0010] In one embodiment, the sense and antisense chains are 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-5 The dsRNA comprises a sequence selected from the group consisting of 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 2'-O-methyl-modified nucleotides, nucleotides comprising a 5'-phosphorothioate group, and terminal nucleotides 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, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base-containing nucleotides.

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

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

[0013] At least one dsRNA strand may contain a 3' overhang of at least one nucleotide or at least two 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 via a divalent or trivalent branched linker is one or more N-acetylgalactosamine (GalNAc) derivatives. In certain embodiments, the ligand is [ka] That is the case.

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

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

[0018] In one embodiment, the complementary region of the dsRNA consists of one of the antisense sequences listed in 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 present invention provides cells containing the dsRNA of the present invention.

[0021] In yet another embodiment, the present invention provides a vector encoding at least one dsRNA strand, wherein the dsRNA comprises a complementary region with at least a portion of the mRNA encoding Serpinc1, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage.

[0022] The complementary region may be at least 15 nucleotides long or 19 to 21 nucleotides long.

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

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

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

[0026] In another embodiment, the present invention provides a method for inhibiting Serpinc1 expression in cells. The method comprises the steps of contacting cells with the dsRNA or vector of the present invention and keeping the resulting cells for a sufficient time to obtain degradation of the mRNA transcript of the Serpinc1 gene, thereby inhibiting Serpinc1 gene expression in cells.

[0027] The cells may be present within a human subject, such as a human subject suffering from a bleeding disorder like hemophilia.

[0028] In one embodiment of the method of the present invention, Serpinc1 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 embodiment, the present invention provides a method for treating a subject with a disorder that benefits from reduced Serpinc1 expression, such as a bleeding disorder like hemophilia. The method comprises the step of administering a therapeutically effective amount of the dsRNA or vector of the present invention to the subject, thereby treating the subject.

[0030] In one embodiment, the present invention provides a method for preventing at least one symptom, such as bleeding, in subjects having a disorder, such as hemophilia, that would benefit from reduced Serpinc1 expression. The method includes the step of administering a therapeutically effective amount of an iRNA or vector, such as the dsRNA of the present invention, to a subject, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced Serpinc1 expression.

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

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

[0033] In one embodiment, the dsRNA is coupled 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, dsRNA is, for example, approximately 0.05 mg / kg to approximately 5 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.2 mg / kg to approximately 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 .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 It is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg, such as approximately 10 mg / kg, approximately 6.5 mg / kg to approximately 10 mg / kg, approximately 7 mg / kg to approximately 10 mg / kg, approximately 7.5 mg / kg to approximately 10 mg / kg, approximately 8 mg / kg to approximately 10 mg / kg, approximately 8.5 mg / kg to approximately 10 mg / kg, approximately 9 mg / kg to approximately 10 mg / kg, or approximately 9.5 mg / kg to approximately 10 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0035] For example, dsRNA is 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 It may be administered in doses of 0.6, 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0036] In another embodiment, dsRNA is present in concentrations of approximately 0.5 to 50 mg / kg, 0.75 to 50 mg / kg, 1 to 50 mg / kg, 1.5 to 50 mg / kb, 2 to 50 mg / kg, 2.5 to 50 mg / kg, 3 to 50 mg / kg, 3.5 to 50 mg / kg, 4 to 50 mg / kg, 4.5 to 50 mg / kg, 5 to 50 mg / kg, 7.5 to 50 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, 20 to 50 mg / kg, 20 to 50 mg / kg, 25 to 50 mg / kg, and 25 to 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 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 in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention. .

[0037] For example, the target values ​​are 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, 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 Therapeutic doses of iRNA such as 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 approximately 50 mg / kg may be administered. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

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

[0039] In another embodiment, the present invention provides a method for inhibiting Serpinc1 expression in a subject. The method comprises the step of administering a therapeutically effective amount of the dsRNA or vector of the present invention to the subject, thereby inhibiting Serpinc1 expression in the subject.

[0040] In one embodiment, the dsRNA is coupled 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, dsRNA is, for example, approximately 0.05 mg / kg to approximately 5 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.2 mg / kg to approximately 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 .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 It is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg, such as approximately 10 mg / kg, approximately 6.5 mg / kg to approximately 10 mg / kg, approximately 7 mg / kg to approximately 10 mg / kg, approximately 7.5 mg / kg to approximately 10 mg / kg, approximately 8 mg / kg to approximately 10 mg / kg, approximately 8.5 mg / kg to approximately 10 mg / kg, approximately 9 mg / kg to approximately 10 mg / kg, or approximately 9.5 mg / kg to approximately 10 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0042] For example, dsRNA is 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 It may be administered in doses of 0.6, 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0043] In another embodiment, dsRNA is present in concentrations of approximately 0.5 to 50 mg / kg, 0.75 to 50 mg / kg, 1 to 50 mg / kg, 1.5 to 50 mg / kb, 2 to 50 mg / kg, 2.5 to 50 mg / kg, 3 to 50 mg / kg, 3.5 to 50 mg / kg, 4 to 50 mg / kg, 4.5 to 50 mg / kg, 5 to 50 mg / kg, 7.5 to 50 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, 20 to 50 mg / kg, 20 to 50 mg / kg, 25 to 50 mg / kg, and 25 to 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 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 in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention. .

[0044] For example, the target values ​​are 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, 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 Therapeutic doses of iRNA such as 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 approximately 50 mg / kg may be administered. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

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

[0046] In yet another embodiment, 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 a method for inhibiting Serpinc1 expression in cells by contacting cells with a double-stranded RNAi agent in an amount effective to inhibit Serpinc1 gene expression in cells. The kit comprises the RNAi agent, instructions for use, and means for selectively administering the RNAi agent to a target. [Brief explanation of the drawing]

[0047] [Figure 1] This is a schematic diagram of the blood coagulation cascade. [Figure 2A] This graph shows the inhibition of Serpinc1 expression in Hep3B cells following a single administration of the indicated iRNA. [Figure 2B] This graph shows the inhibition of Serpinc1 expression in Hep3B cells following a single administration of the indicated iRNA. [Figure 3A] This graph shows the inhibition of Serpinc1 mRNA (A) and protein (B) expression in CD-1 mice following a single administration of the AD-50509 or AD-1955 LNP formulations. [Figure 3B] This graph shows the inhibition of Serpinc1 mRNA (A) and protein (B) expression in CD-1 mice following a single administration of the AD-50509 or AD-1955 LNP formulations. [Figure 4A] This graph shows the duration of inhibition of Serpinc1 mRNA (A) and protein (B) expression in CD-1 mice following a single administration of 1 mg / kg of AD-50509 or AD-1955 LNP preparations. [Figure 4B] This graph shows the duration of inhibition of Serpinc1 mRNA (A) and protein (B) expression in CD-1 mice following a single administration of 1 mg / kg of AD-50509 or AD-1955 LNP preparations. [Figure 4C] This graph shows the inhibition of Serpinc1 activity and Serpinc1 protein expression in CD1 mice following a single administration of 1 mg / kg of AD-50509 or AD-1955 LNP preparations. [Figure 5] This graph shows the percentage knockdown of Serpinc1 mRNA and protein levels following a single administration of 10 mg / kg of GalNAc-coupled iRNA. [Figure 6]This graph shows the 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, as well as repeated doses of 5 × 5 mg / kg. [Figure 7A] This graph shows the effect of repeated administration of GalNAc-coupled AD-54944 on the duration of inhibition of Serpinc1 protein expression in C57BL / 6 mice. [Figure 7B] This graph shows the effect of repeated administration of GalNAc-coupled AD-54944 on the duration of inhibition of Serpinc1 protein expression in C57BL / 6 mice. [Figure 8] This graph shows the effect of the proposed divided dosing plan on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-coupled AD-54944. [Figure 9] This graph shows the effect of the proposed divided dosing plan on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-coupled AD-54944. [Figure 10] This graph shows the percentage knockdown of Serpinc1 protein levels following a single administration of 10 mg / kg (A) or 3 mg / kg (B) of the indicated GalNAc-coupled iRNA. [Figure 11] This graph shows the percentage knockdown of Serpinc1 protein levels following a single administration of 10 mg / kg or 3 mg / kg of the indicated GalNAc-coupled iRNA. [Figure 12] This graph shows the percentage of Serpinc1 activity knockdown following a single administration of 10 mg / kg or 3 mg / kg of the indicated GalNAc-coupled iRNA. [Figure 13] This graph shows the dose-response response to a single dose of AD-57213. [Figure 14]This graph shows the duration of Serpinc1 silencing following a single dose of AD-57213 at 1 mg / kg, 3 mg / kg, or 10 mg / kg in patients with hemophilia A mau. [Figure 15] This graph shows the inhibition of Serpinc1 mRNA expression in C57BL / 6 mice following single doses of AD-57213 at 30 mg / kg, 10 mg / kg, 3 mg / kg, 1 mg / kg, and 0.3 mg / kg. [Figure 16] This graph shows the duration of Serpinc1 silencing following single doses of AD-57213(A), AD-57205(B), and AD-57214(C). [Figure 17] This graph shows the effect of the proposed divided dosing plan on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-coupled AD-57213. [Figure 18] This graph shows the effect of the proposed divided dosing plan on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-coupled AD-57213. [Figure 19] This graph shows the effect of the proposed divided dosing plan on the duration of silencing of Serpinc1 protein expression in C57BL / 6 mice administered GalNAc-coupled AD-57213. [Figure 20] This graph shows the single-dose screening effect of the compounds indicated on the duration of Serpinc1 protein expression in non-human primates. [Figure 21] This graph shows the single-dose screening effect of GalNAc-coupled AD-57213 on the duration of Serpinc1 protein expression in non-human primates. [Figure 22] This graph shows the single-dose screening effect of the compounds indicated on the duration of Serpinc1 protein expression in non-human primates. [Figure 23]This graph shows the effect of a single dose of compound AD-57213 on serum antithrombin (Serpinc1) levels in non-human primates. [Figure 24] This graph shows the effects 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 (Serpinc1) levels and the change factor of peak plasma thrombin levels in non-human primates. The change factor of peak thrombin is plotted on the secondary y-axis (gray), and the relative antithrombin levels are plotted on the primary y-axis (black). [Figure 25] This graph shows the effect of AD-57213 as a multiplier of change in peak thrombin increase as a function of relative antithrombin (Serpinc1) silencing. [Figure 26] This graph shows the effects 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. The data points represent the group mean, and the error bars represent the standard deviation (N=3). (qw = weekly; q2w = bi-weekly). [Figure 27A] This graph shows the cumulative effect of Serpinc1 silencing in non-human primates. [Figure 27B] This graph shows the cumulative effect of Serpinc1 silencing in non-human primates. [Figure 28A] This graph shows the effect of Serpinc1 silencing on platelet accumulation following microvascular laser injury. The graph shows the median value from all traumatic injuries. [Figure 28B] This graph shows the effect of Serpinc1 silencing on fibrin area following microvascular laser injury. The graph shows the median value from all damaging injuries. [Figure 29] This graph shows the duration of Serpinc1 silencing following administration of the compound AD-57213, which is formulated into lipid nucleic acid particles. [Figure 30A]The nucleotide sequence of human (Homo sapiens) serpin peptidase inhibitor, branching group C (antithrombin), member 1 (SERPINC1) (sequence number 1) is shown. [Figure 30B] This shows the nucleotide sequence of member 1 (SERPINC2) (SEQ ID NO: 1) of the serpine peptidase inhibitor, branching group C (antithrombin), from the rhesus macaque (Macaca mulatta). [Figure 30C] This shows the nucleotide sequence of member 1 (Serpinc1) (SEQ ID NO: 3) of the serine (or cysteine) peptidase inhibitor, branching group C (antithrombin), from the mouse (Mus musculus). [Figure 30D] This shows the nucleotide sequence of member 1 (Serpinc1) (SEQ ID NO: 4) of branch group C (antithrombin) of the brown rat (Rattus norvegicus) serpin peptidase inhibitor. [Figure 30E] The inverse complement (sequence number 5) of sequence number 1 is shown. [Figure 30F] The inverse complement (sequence number 6) of sequence number 2 is shown. [Figure 30G] The inverse complement (sequence number 7) of sequence number 3 is shown. [Figure 30H] The inverse complement (sequence number 8) of sequence number 4 is shown. [Figure 30I] The amino acid sequences of exemplary hydrophobic MTS-containing peptide RFGF (SEQ ID NO: 9), exemplary RFGF analogs (SEQ ID NO: 10), HIV Tat protein (SEQ ID NO: 11), Drosophila Antennapedia protein (SEQ ID NO: 12), and exemplary peptide-based cleavable linking groups are shown. [Figure 31A] This graph illustrates that a decrease in antithrombin levels increases thrombin production in factor IX-depleted human plasma in vitro. [Figure 31B] This graph illustrates that a decrease in antithrombin levels increases thrombin production in factor IX-depleted human plasma in vitro. [Modes for carrying out the invention]

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

[0049] Examples of iRNAs in this invention include 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 1 Examples include RNA strands (antisense strands) having a length of approximately 30 nucleotides or less, 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, where the region is substantially complementary to at least a portion of the Serpinc1 gene mRNA transcript. The use of these iRNAs enables targeted degradation of Serpinc1 gene mRNA in mammals. In particular, very low doses of Serpinc1 iRNA can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of Serpinc1 gene expression. The inventors have demonstrated that iRNAs targeting Serpinc1 can significantly inhibit Serpinc1 gene expression via RNAi in vitro and in vivo. Therefore, methods and compositions containing these iRNAs are useful for treating subjects who would benefit from reduced levels and / or activity of the Serpinc1 protein, such as subjects with bleeding disorders like hemophilia.

[0050] The following detailed description discloses methods for preparing and utilizing compositions containing iRNAs that inhibit Serpinc1 gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from the inhibition and / or reduction of expression of this gene.

[0051] I. Definition To make the present invention easier to understand, certain terms are defined first. In addition, it should be noted that whenever parameter values ​​or ranges of values ​​are enumerated, intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention.

[0052] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical purposes of the article. For example, "an element" means one factor or two or more factors, such as multiple factors.

[0053] The term "including" is used herein to mean "including, but not limited to," and is used without distinction from that term.

[0054] In this specification, the term "or" is used to mean, and is used without distinction from, the term "and / or" unless the context clearly indicates otherwise.

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

[0056] As used herein, the term “Serpinc1” also refers to specific polypeptides expressed in cells due to spontaneous DNA sequence diversity of the Serpinc1 gene, such as single nucleotide polymorphisms (SNPs) in the Serpinc1 gene. Numerous SNPs within the Serpinc1 gene have been identified, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp). Non-exclusive examples of SNPs within the Serpinc1 gene are found in NCBIdbSNP acceptance numbers rs677;rs5877;rs5878;rs5879;rs941988;rs941989;rs1799876;rs19637711;rs2008946; and rs2227586.

[0057] In the use of this specification, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Serpinc1 gene, including mRNA, which is the RNA processing product of the primary transcript. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for iRNA-directed cleavage, either in or near the portion of the nucleotide sequence of the mRNA molecule formed during transcription of the Serpinc1 gene.

[0058] The target sequence may be approximately 9 to 36 nucleotides long, for example, approximately 15 to 30 nucleotides long. For example, the target sequences 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 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, etc., which may be approximately 15-30 nucleotide lengths. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0059] In the use of this specification, the term “sequence-containing chain” refers to an oligonucleotide comprising a nucleotide chain described by the sequence referred to, using standard nucleotide nomenclature.

[0060] "G," "C," "A," "T," and "U" typically represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, the terms "ribonucleotide" or "nucleotide" are also understood to refer to modified nucleotides or substituted portions, 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 portions without substantially altering the base-pairing properties of oligonucleotides containing such substituted portions. As an example not intended to be limiting, a nucleotide containing inosine as a base can base-pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the dsRNAs discussed in this invention with, for example, nucleotides containing inosine. In another embodiment, adenine and cytosine may be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods discussed in this invention.

[0061] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used synonymously herein and refer to agents containing RNA as defined herein that mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNAs induce sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate, for example, inhibit Serpinc1 expression in cells, such as those in mammalian subjects.

[0062] In one embodiment, the RNAi agent of the present invention is a single-stranded RNA that interacts with a target RNA sequence, such as a Serpinc1 target mRNA sequence, to induce cleavage of the target RNA. Although theoretical constraints are not desired, it is thought that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into a short interfering RNA of 19-23 base pairs with a characteristic 2-base 3' overhang (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 double-strand, allowing a complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Therefore, in one embodiment, the present invention relates to single-stranded RNA (siRNA) generated in cells to promote RISC complex formation and result in the silencing of a target gene, namely the Serpinc1 gene. Thus, 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 a target mRNA. Single-stranded siRNAs typically consist of 15 to 30 nucleotides and are chemically modified. Designs and tests of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, each of which is incorporated herein by reference in its entirety. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or chemically modified in the manner described in Lima et al., (2012) Cell 150:883-894.

[0064] In another embodiment, the drug is a single-stranded antisense RNA molecule that inhibits the target by an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to the sequence in the target mRNA. The single-stranded antisense RNA molecule can inhibit translation in a stoichiometric manner by physically interfering with the translation mechanism by forming base pairs with the mRNA. 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 with the target (hydridizing) and cleaving the target through an RNaseH cleavage event. The single-stranded antisense RNA molecule may be about 15 to 30 nucleotides long and have a sequence complementary to the target sequence. For example, a single-stranded antisense RNA molecule may contain at least approximately 15, 16, 17, 18, 19, or 20 consecutive nucleotide sequences from any one of the antisense sequences in 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 herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a double-stranded ribonucleic acid molecule complex comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having “sense” and “antisense” orientations with respect to the target RNA, i.e., the Serpinc1 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0066] The double-stranded region may be of any length that enables the specific degradation of the desired target RNA via the RISC pathway, and may be in the range of approximately 9 to 36 base pairs in length, for example, approximately 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, approximately 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, These include base pair lengths of 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. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0067] The two strands forming the double-stranded structure may be different parts of a larger RNA molecule, or they may be different RNA molecules. If the two strands are parts of one larger molecule, and thus the 3' end of one strand forming the double-stranded structure is joined to the 5' end of the other strand by an uninterrupted nucleotide strand, the joined RNA strands are referred to as a "hairpin loop." A hairpin loop may consist of at least one unpaired nucleotide; in some embodiments, a hairpin loop may consist of 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, and at least 23 or more unpaired nucleotides.

[0068] If two substantially complementary strands of dsRNA are composed of another RNA molecule, these molecules may, but do not necessarily, be covalently linked. If the two strands are covalently linked between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand by means other than an uninterrupted nucleotide chain, the linking structure is called a "linker." RNA strands may have the same or different nucleotide numbers. The maximum base pair count is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain 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 dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends over the 5' end of the other strand, or vice versa. A dsRNA may consist of an overhang of at least one nucleotide; alternatively, the overhang may consist of at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang may consist of or comprise nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0070] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, which overhang at the 3' and / or 5' ends. In another embodiment, one or more nucleotides in the overhang are substituted with thiophosphate nucleosides.

[0071] As used herein with respect to dsRNA, the terms “blunted” or “blunt-terminated” mean that a given end of the dsRNA has no unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs. One or both ends of a dsRNA can be blunted. If both ends of a dsRNA are blunted, the dsRNA is said to be blunt-terminated. For clarity, a “blunt-terminated” dsRNA is a dsRNA where both ends are blunted, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule is double-stranded along its entire length.

[0072] The terms “antisense strand” or “guide strand” refer to an iRNA strand, such as dsRNA, that contains a region substantially complementary to a target sequence, such as Serpinc1 mRNA. As used herein, the term “regional complementarity” refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as the Serpinc1 nucleotide sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, there may be a mismatch in the internal or terminal regions of the molecule. Generally, the most tolerable mismatches are in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the iRNA.

[0073] The terms “sense strand” or “passenger strand” as used herein refer to an iRNA strand that includes a region substantially complementary to the antisense strand region as defined herein.

[0074] In the use of this specification, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under specific conditions to form a double-stranded structure, as will be understood by those skilled in the art when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 12–16 hours at 50°C or 70°C, 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 reasonable conditions that may be encountered in living organisms, may also be applicable. Those skilled in the art can determine the optimal set of conditions for the complementarity test of the two sequences, depending on the end use of the hybridized nucleotides.

[0075] For example, complementary sequences within iRNAs, such as those within dsRNAs, as described herein, include base pairings of an oligonucleotide or polynucleotide comprising a first nucleotide sequence spanning the entire length of one or both nucleotide sequences with an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, generally 5, 4, 3, or 2 or fewer, while retaining the ability to hybridize in a double-stranded hybridization of up to 30 base pairs under conditions most appropriate for their end use, such as inhibition of gene expression via the RISC pathway. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, is still referred to as “perfectly complementary” for the purposes described herein.

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

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

[0078] As used herein, a polynucleotide "substantially complementary to at least a portion" of messenger RNA (mRNA) means a polynucleotide substantially complementary to the continuous portion of the mRNA in question (e.g., the mRNA encoding Serpinc1). For example, a polynucleotide is complementary to at least a portion of Serpinc1 mRNA if its sequence is substantially complementary to the uninterrupted portion of the mRNA encoding Serpinc1.

[0079] Generally, the majority of nucleotides in each chain are ribonucleotides, but as described in detail herein, one or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, “iRNA” can refer to chemically modified ribonucleotides. Such modifications include all types of modifications disclosed herein or known in the art. For the purposes of this specification and the claims, any such modification is encompassed by “iRNA” in its use in an iRNA molecule.

[0080] The term “inhibit” as used herein is synonymous with “reduce,” “silence,” “downcontrol,” “suppress,” and other similar terms, and includes all levels of inhibition.

[0081] As used herein, the phrase "inhibit Serpinc1 expression" includes the 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 any variant or mutant of the Serpinc1 gene that encodes 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, including inhibition of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.

[0083] Serpinc1 gene expression may be assessed based on the levels of any variables associated with Serpinc1 gene expression, such as Serpinc1 mRNA levels, Serpinc1 protein levels, or thrombin:antithrombin complex levels as a measure of thrombin production capacity (portential), bleeding time, prothrombin time (PT), platelet count, and / or activated partial thromboplastin time (aPTT). Inhibition may be assessed by a decrease in one or more absolute or relative levels of these variables compared to control levels. Control levels may be any type of control level available in the art, such as pre-administration baseline levels, or levels measured from similar subjects, cells, or samples that have been untreated or treated with a control (e.g., a buffer-only control or a non-activating drug control).

[0084] In one embodiment, at least partial repression of Serpinc1 gene expression is assessed by a decrease in the amount of Serpinc1 mRNA isolated from or detectable in the first cell or cell group in which the Serpinc1 gene is transcribed, compared to a second cell or cell group (control cell) that is substantially identical to a first cell or cell group but not appropriately treated, in which Serpinc1 gene expression is inhibited. The degree of inhibition is,

number

[0085] In the usage herein, the phrase "contacting cells with an RNAi agent such as dsRNA" includes the step of contacting cells by any possible means. Contacting cells with an RNAi agent includes the step of contacting cells with iRNA in a test tube or contacting cells with iRNA in vivo. Contact may be carried out directly or indirectly. Thus, for example, the person performing the method may physically contact the RNAi agent with cells, or alternatively, place the RNAi agent in a situation where subsequent contact with cells is possible or induced.

[0086] Cell contact in vitro may be performed, for example, by incubating cells with an RNAi agent. Cell contact in vivo may be performed, for example, by injecting the RNAi agent into or near the tissue in which the cells are located, or by injecting the RNAi agent into another region, such as the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue in which the cells to be contacted are located. For example, the RNAi agent may contain and / or be coupled with a ligand such as GalNAc3 that induces 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 may be contacted with an RNAi agent in vitro and then subsequently transplanted into a target.

[0087] In one embodiment, the step of bringing cells into contact with iRNA includes the step of “introducing” or “delivering iRNA to cells,” thereby facilitating or resulting in its uptake or absorption into the cells. Absorption or uptake of iRNA may occur through unassisted diffusive or activated cellular processes, or by aids or devices. Introduction of iRNA into cells may be in vitro and / or in vivo. For example, for in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be carried out by β-glucan delivery systems, such as those described in U.S. Patent No. 5,032,401 and U.S. Patent No. 5,607,677, and U.S. Patent Application Publication 2005 / 0281781, whose entire contents are incorporated herein by reference. Extracellular introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below herein and / or are known in the art.

[0088] The term “lipid nanoparticle” or “LNP” refers to a vesicle comprising a lipid layer that encapsulates a nucleic acid molecule, such as iRNA, or a pharmacologically active molecule, such as a plasmid from which iRNA is transcribed. LNPs are described, for example, in U.S. Patent No. 6,858,225, U.S. Patent No. 6,815,432, U.S. Patent No. 8,158,601, and U.S. Patent No. 8,058,069, which are incorporated herein by reference in their entirety.

[0089] The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP is a lipid vesicle lining a reducing aqueous interior, comprising a nucleic acid such as iRNA or a plasmid from which 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, whose entire contents are incorporated herein by reference. Examples of “SNALP” formulations are given below.

[0090] In the use of this specification, “Subject” means a mammal, including primates (such as humans, non-human primates such as monkeys and chimpanzees), non-primates (such as cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or an animal such as a bird (such as a duck or goose). In one embodiment, the subject is a human being treated or evaluated for a disease, disorder or condition that would benefit from reduced Serpinc1 expression; a human being at risk of a disease, disorder or condition that would benefit from reduced Serpinc1 expression; a human being having a disease, disorder or condition that would benefit from reduced Serpinc1 expression; and / or a human being treated for a disease, disorder or condition that would benefit from reduced Serpinc1 expression as described herein. In the context of this specification, the terms “to treat” or “to cure” refer to a beneficial or desired outcome, including, but not limited to, the alleviation or improvement of one or more symptoms, whether detectable or undetectable; a reduction in the degree of bleeding; stabilization (i.e., no worsening) of a bleeding condition; improvement or temporary relief of bleeding. “Treatment” may also mean an extension of survival compared to the predicted survival in the absence of treatment.

[0091] "To reduce" means a statistically significant reduction in such a level in the context of a disease marker or symptom. The reduction may be, for example, at least 10%, at least 20%, at least 30%, or at least 40% or more, and preferably to a level that is generally recognized as being within the normal range for individuals without such disease.

[0092] In the context of this specification, “prevention” or “prevention” as used with respect to a disease, disorder, or condition that benefits from reduced expression of the Sertpinc1 gene means a reduced likelihood of the subject developing symptoms associated with such 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 bleeding of less severe severity compared to a population with the same risk factors but who do not receive the treatment described herein. Effective prevention is considered to be the absence of the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically recognized scale of the disease or disorder), or a delay in the onset of delayed symptoms (e.g., by several days, weeks, months, or years).

[0093] As used herein, the term “hemorrhagic disorder” means a disease or disorder resulting in unfavorable blood clotting and / or excessive bleeding. A hemorrhagic disorder may be a hereditary disorder such as hemophilia or von Willebrand disease, or an acquired disorder such as disseminated intravascular coagulation, pregnancy-related 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 medication such as aspirin, heparin, or warfarin, diabetes mellitus, 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, a hereditary hemorrhagic disorder is, for example, hemophilia such as hemophilia A, B, or C. In one embodiment, the subject has a hereditary bleeding disorder, such as hemophilia, and has developed inhibitors, such as alloantibody inhibitors, in response to alternative coagulation therapy, and is referred to herein as the "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] In the use herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to induce treatment of a disease (for example, by reducing, improving, or maintaining the symptoms of a pre-existing disease or one or more disease symptoms) when administered to a subject with bleeding disorders and bleeding. “Therapeutic dose” may vary depending on the RNAi agent, method of drug administration, disease and its severity, and the medical history, age, weight, family history, genetic predisposition, type of preceding or concomitant treatment, and any other individual characteristics of the subject being treated.

[0095] In the use herein, “prophylactic effective dose” is intended to contain an amount of iRNA sufficient to prevent or improve the disease, or one or more disease symptoms, when administered to a subject with a bleeding disorder but not bleeding, such as a subject with a bleeding disorder who is scheduled for surgery. Disease improvement includes a slowing of the disease course or a reduction in the severity of the disease that subsequently develops. The “prophylactic effective dose” 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 predisposition, type of prior or concomitant treatment, and any other individual characteristics.

[0096] The “therapeutic effective dose” or “preventive effective dose” may also refer to the amount of RNAi agent that produces several desired local or systemic effects with a reasonable benefit / risk ratio applicable to any therapeutic agent. The iRNA used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such therapies.

[0097] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with the tissues of human and animal subjects, provided that there is no excessive toxicity, irritation, allergic response, or other problems or complications that balance out in a reasonable benefit-to-risk ratio.

[0098] In the use herein, the term “pharmacoagulably acceptable carrier” means a pharmacoagulably acceptable material, composition, or vehicle, such as a liquid or solid extender, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate, or steric acid), or a solvent encapsulating material involved in transporting or delivering a compound of interest from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Some examples of materials that may serve as pharmacoagulably 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 (1) Smoothing agents such as sodium lauryl sulfate and talc; (2) Excipients such as cocoa butter and suppository wax; (3) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (4) Glycols such as propylene glycol; (5) Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (6) Esters such as ethyl oleate and ethyl laurate; (7) Agar; (8) Magnesium hydroxide Examples include buffers such as nesium and aluminum hydroxide; (15) alginic acid; (16) pyrogenic substance-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyacid anhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic and suitable substances used in pharmaceutical formulations.

[0099] As used herein, the term “sample” includes similar collections of bodily fluids, cells, or tissues isolated from a subject, as well as bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples include samples from tissues, organs, or localized areas. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may originate from the liver (e.g., the entire liver or a specific part of the liver, or a specific type of liver cell, such as hepatocytes). In some embodiments, “sample derived from subject” refers to blood or plasma collected from the subject.

[0100] II. The iRNA of the present invention This specification describes iRNAs that inhibit Serpinc1 gene expression. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits Serpinc1 gene expression in cells, such as cells in a target organism, such as a human with a bleeding disorder, such as a hereditary bleeding disorder. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during Serpinc1 gene expression. The complementary region is approximately 30 nucleotides or less in length (for example, approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less). Upon contact with cells expressing the Serpinc1 gene, the iRNA inhibits the expression of the Serpinc1 gene (e.g., human, primate, non-primate, or avian Serpinc1 gene) by at least approximately 10% using assays such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry.

[0101] A dsRNA contains two complementary RNA strands, which hybridize under the conditions in which the dsRNA is used to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a complementary region, which is substantially complementary to the target sequence and generally fully complementary. The target sequence may originate from the mRNA sequence formed during the expression of the Serpinc1 gene. The other strand (the sense strand) contains a region complementary to the antisense strand, such that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere in this specification and as known in the art, the complementary sequence of a dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to that on a separate oligonucleotide.

[0102] Generally, double-stranded structures are, 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 These range from 15 to 30 base pairs in length, such as 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. Intermediate ranges and lengths beyond the listed ranges and lengths are also intended to be part of the present invention.

[0103] Similarly, complementary regions of target sequences are, 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, 19-2 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 nucleotide lengths, etc. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0104] In some embodiments, the dsRNA is about 15–20 nucleotides long, or about 25–30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. It is well known in the art that dsRNA longer than about 21–23 nucleotides may serve as a substrate for Dicer. As those skilled in the art will recognize, the target region of the RNA to be cleaved is in most cases part of a larger RNA molecule, which is often an mRNA molecule. Where applicable, the “part” of the mRNA target is a continuous sequence of mRNA targets long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0105] Those skilled in the art can, for example, use approximately 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-3 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 double-stranded regions of approximately 9 to 36 base pairs, such as ~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, are the main functional parts of dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs, within the range that it is processed into a functional double-strand of 15 to 30 base pairs that targets a desired RNA for cleavage, is a dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a native miRNA. In another embodiment, iRNA agents useful for targeting Serpinc1 expression are not generated in the target cell by cleaving larger dsRNAs.

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

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

[0108] The iRNA compounds of the present invention may be prepared using a two-step method. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the constituent strands are annealed. The individual strands of the siRNA compound can be prepared using solution phase, solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotide chains containing non-natural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase, solid-phase organic synthesis, or both.

[0109] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences: a sense sequence and an antisense sequence. The 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 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, and one of the sequences is substantially complementary to the mRNA sequence that occurs during Serpinc1 gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand from any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and the second oligonucleotide described as the antisense strand corresponding to the sense strand from any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21. In one embodiment, substantially complementary sequences of dsRNA are contained on separate oligonucleotides. In another embodiment, substantially complementary sequences of dsRNA are contained on a single oligonucleotide.

[0110] While 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 present invention, such as the dsRNA of the present invention, may include any one of the sequences described in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, which are unmodified, unconjugated, and / or modified and / or conjugated in a manner different from those described.

[0111] Those skilled in the art are well aware that dsRNAs having double-stranded structures of approximately 20–23 base pairs, such as 21 base pairs, are supported as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877–6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures can also be equally effective (Chu and Rana (2007) RNA 14:1714–1719; Kim et al. (2005) Nat Biotech 23:222–226). In the embodiments described above, 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 comprise at least one strand of a minimum length of 21 nucleotides. It can be reasonably predicted that shorter double-stranded sequences having one of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, with only a few nucleotides missing from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having at least 15, 16, 17, 18, 19, or 20 or more consecutive nucleotide sequences derived from one of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and having the ability to inhibit Serpinc1 gene expression, differing from dsRNAs comprising a 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 Serpinc1 transcript that are highly sensitive to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target within one of these sequences. In the use herein, an iRNA is said to target within that specific site of the RNA transcript if it promotes cleavage of the transcript somewhere within a particular site. Such iRNAs generally consist of about 15 consecutive nucleotides from one of the sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, which are linked to an additional nucleotide sequence from a region adjacent to a selected sequence in the Serpinc1 gene.

[0113] Target sequences are generally about 15–30 nucleotides long, but there is a wide range of variation in the suitability of specific sequences within this range to induce cleavage of any given target RNA. The various software packages and guidelines presented herein provide guidance for identifying the optimal target sequence for any given gene target, but an empirical approach can also be taken to identify sequences within a size range that could act as the target sequence by actually or figuratively (including, for example, by computer simulation) placing a “window” or “mask” of a given size (21 nucleotides as an unrestricted example) on the target RNA sequence. By sequentially 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 to identify optimally functioning sequences (using assays described herein or known in the art), can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, while a sequence identified in any one of Tables 2, 3, 4, 8, 11, 12, 14, 15, 20, and 21 represents an effective target sequence, it is possible to further optimize inhibition efficiency by identifying sequences with equivalent or better inhibitory properties by sequentially "walking a window" one nucleotide upstream or downstream of a given sequence.

[0114] Furthermore, it is explored that further optimization of any sequence identified in any one of Tables 2, 3, 4, 8, 11, 12, 14, 15, 20, and 21 may be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking a window of size longer or shorter than the target RNA from that position. Again, combining this approach to creating new target candidates with testing the efficacy of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein may lead to further improvements in inhibition efficiency. Moreover, such optimized sequences may be modulated by further optimizing the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, targeting of specific sites or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc.) by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or considered herein.

[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 the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA drug chain complementary to the SERPINC1 gene region, the RNA chain generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it may be determined whether an iRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the SERPINC1 gene. Examining the effectiveness of mismatched iRNAs in inhibiting SERPINC1 gene expression is important, especially when a particular complementary region of the Serpinc1 gene is known to have polymorphic sequence variations within the population.

[0116] III. Modified iRNA of the Invention In one embodiment, the RNA of the iRNA of the present invention, such as dsRNA, is undenatured and free from chemical modifications and / or binding, for example, those known in the art and described herein. In another embodiment, the RNA of the iRNA of the invention, such as dsRNA, is chemically modified to enhance stability or other beneficial properties. The nucleic acids addressed in the present invention may be synthesized and / or modified by methods established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Examples of modifications include terminal modifications such as 5'-end modifications (phosphorylation, conjugation, inversion) or 3'-end modifications (conjugation, DNA nucleotide, inversion, etc.); base modifications such as substitution, base removal (debasing nucleotide), or conjugated bases, whether at a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanding partner repertoire; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and main chain modifications, including modifications or substitutions of phosphate diester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified main chain or RNA without natural internucleoside bonds. RNA having a modified main chain is particularly limited to those that do not have a phosphorus atom in the main chain. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside main chains are also considered oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside main chain.

[0117] Examples of modified RNA backbone include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylenephosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages and their 2'-5' linked analogues, as well as boranophosphates with reversed polarity where adjacent nucleoside unit pairs are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

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

[0119] Modified RNA backchains that do not contain a phosphorus atom have backchains formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar portion of nucleosides); siloxane backchains; sulfide, sulfoxide, and sulfone backchains; formacetyl and thioformacetyl backchains; methyleneformacetyl and thioformacetyl backchains; alkene-containing backchains; sulfamate backchains; methyleneimino and methylenehydrazino backchains; sulfonate and sulfonamide backchains; those having amide backchains; and others having mixed N, O, S, and CH2 components.

[0120] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides are U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, all of which are incorporated herein by reference in their entirety. Examples of U.S. Patent Nos. include, but are not limited to, U.S. Patent 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, a suitable RNA mimetic is considered for use in iRNA, in which both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with a new group. The base units are maintained for hybridization with a suitable nucleic acid target compound. Such an oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bind directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, each of which is incorporated herein by reference in its entirety. Furthermore, PNA compounds suitable for use in iRNA according to the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0122] Some embodiments of the present invention include RNA having a phosphorothioate backbone, and oligonucleosides having a heteroatom backbone which is --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [natural phosphate diester backbone is represented as --O--P--O--CH2--], and an amide backbone as described in U.S. Patent No. 5,602,240. In some embodiments, the RNA discussed herein has the morpholino backbone structure described in the aforementioned U.S. Patent No. 5,034,506.

[0123] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs such as the dsRNAs discussed herein can contain at the 2’ position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C 10 alkyl, or C2-C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) 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 contains at the 2’ position one of: C1-C 10 lower alkyl, substituted lower alkyl, aralkyl, aralkyl, O-aralkyl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaararyl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of the iRNA, or a group that improves the pharmacodynamic properties of the iRNA, and other substituents having similar properties. In some embodiments, the modification is 2’-methoxyethoxy (also known as 2’-O-CH2CH2OCH3, 2’-O-(2-methoxyethyl) or 2’-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Other exemplary modifications are 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 below in the examples herein.

[0124] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA, specifically at the 3' position of the sugar on the 3' terminal nucleotide, or in the 2'-5' linked dsRNA, and at the 5' position of the 5' terminal nucleotide. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of the above-mentioned modified sugar structures include, specifically, U.S. Patent No. 4,981,957; U.S. Patent No. 5,118,800; U.S. Patent No. 5,319,080; U.S. Patent No. 5,359,044; U.S. Patent No. 5,393,878; U.S. Patent No. 5,446,137; U.S. Patent No. 5,466,786; U.S. Patent No. 5,514,785; U.S. Patent No. 5,519,134; U.S. Patent No. 5, U.S. Patent Nos. 567,811; U.S. Patent Nos. 5,576,427; U.S. Patent Nos. 5,591,722; U.S. Patent Nos. 5,597,909; U.S. Patent Nos. 5,610,300; U.S. Patent Nos. 5,627,053; U.S. Patent Nos. 5,639,873; U.S. Patent Nos. 5,646,265; U.S. Patent Nos. 5,658,873; U.S. Patent Nos. 5,670,633; and U.S. Patent Nos. 5,700,920, among others. The entire contents of each of the above are incorporated herein by reference.

[0125] iRNAs may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). In the use of this specification, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases 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-thio Examples 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 other synthetic and natural nucleic acid bases such as 3-deazaguanine and 3-deazaadenine.Furthermore, examples of nucleic acid bases 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, STand Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), making it an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0126] Representative U.S. patents teaching the preparation of the specific modified nucleic acid bases and other modified nucleic acid bases described above are, as their entire contents are incorporated herein by reference, U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469. U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent Nos. 5,614,617; U.S. Patent Nos. 5,681,941; U.S. Patent Nos. 5,750,692; U.S. Patent Nos. 6,015,886; U.S. Patent Nos. 6,147,200; U.S. Patent Nos. 6,166,197; U.S. Patent Nos. 6,222,025; U.S. Patent Nos. 6,235,887; U.S. Patent Nos. 6,380,368; U.S. Patent Nos. 6,528,640; U.S. Patent Nos. 6,639,062; U.S. Patent Nos. 6,617,438; U.S. Patent Nos. 7,045,610; U.S. Patent Nos. 7,427,672; and U.S. Patent Nos. 7,495,088.

[0127] The RNA of iRNA can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, in which the ribose moiety includes additional crosslinks connecting the 2' and 4' carbons. This structure effectively "locks" the ribose within the 3'-end 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 U.S. patents teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0129] Potential stabilization modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3''-phosphate, and the reversed base dT (idT). These modifications are disclosed in International Publication No. 2011 / 005861.

[0130] IV. Ligand-coupled iRNAs Another modification of the iRNA of the present invention involves chemically linking one or more ligands, moieties, or complexes to the RNA, which enhance the activity, cell distribution, or intracellular uptake of the iRNA. Such parts include lipid portions such as the cholesterol portion (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770) and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); and thioethers such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO Aliphatic chains such as J,1991,10:1111-1118;Kabanov et al.,FEBS Lett.,1990,259:327-330;Svinarchuk et al.,Biochimie,1993,75:49-54); phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 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 acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Examples include, but are not limited to, octadecylamine or the hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0131] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity to selected targets, such as molecules, cells or cell types, compartments such as intracellular or organelle compartments, tissues or organs or regions of the body, compared to chemical species in which such ligand is absent. The preferred ligand does not participate in double-strand pairing in the double-stranded nucleic acid.

[0132] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid 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 polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or α-helical peptides.

[0133] The ligand may also include targeting groups such as antibodies that bind to specific cell types, such as kidney cells, or cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins. The targeting groups may be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetylglucosamine (gulucoseamine), polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimetic compounds.

[0134] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 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) lithoglycerol Examples include lic acid, O3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole complexes, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, or AP.

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

[0136] Ligands can be substances such as drugs that can increase the uptake of iRNA agents into cells by disrupting, for example, the cellular microtubules, microfibrils, and / or intermediate filaments, or by disrupting the cellular cytoskeleton. Drugs may include, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0137] In some embodiments, the ligands attached to iRNAs described herein refer to pharmacokinetic modifiers (PK modifiers). Examples of PK modifiers include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, and vitamins. Exemplary PK modifiers include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate bonds are also known to bind to serum proteins, and therefore, for example, short-chain oligonucleotides such as approximately 5-base, 10-base, 15-base, or 20-base oligonucleotides containing multiple phosphorothioate bonds in the main chain are also suitable as ligands (e.g., as PK modulating ligands) in 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] The ligand-conjugated oligonucleotides of the present invention may be synthesized using oligonucleotides having pendant-reactive functional groups, such as those derived from the addition of a binding molecule onto the oligonucleotide (described below). These reactive oligonucleotides may be reacted directly with commercially available ligands, synthesized ligands having any of the various protecting groups, or ligands having a binding site to which attachment is possible.

[0139] The oligonucleotides used in the complexes of the present invention may, conveniently and conventionally, be produced through well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including Applied Biosystems (Foster City, Calif.). Alternatively, any other means known in the art for such synthesis may be used. It is also known that other oligonucleotides, such as phosphorothioates and alkylated derivatives, can be prepared using similar techniques.

[0140] In the ligand-conjugated oligonucleotides and sequence-specific binding nucleosides containing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, nucleotides or nucleoside complex precursors already containing binding sites, ligand-nucleotide or nucleoside complex precursors already containing ligand molecules, or basic units containing non-nucleoside ligands.

[0141] When using a nucleotide complex precursor that already has a binding site, the synthesis of a sequence-specific bound nucleoside is typically completed, and then the ligand molecule reacts with the binding site to produce a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or bound nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside complexes, in addition to standard and non-standard phosphoramidites that are commercially available and conventionally used in oligonucleotide synthesis.

[0142] A. Lipid complexes In one embodiment, the ligand or complex is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the complex to target tissues, such as non-renal target tissues of the body. Target tissues, for example, could be the liver, including hepatic parenchymal cells. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the degradation resistance of the complex, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate the binding of serum proteins, such as HSA.

[0143] Lipid-based ligands can be used for inhibition, for example, by controlling the binding of the complex to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the complex to the kidneys.

[0144] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the complex is distributed to non-renal tissues. However, the affinity is preferably not so strong as to prevent the HSA ligand binding from being reversed.

[0145] In another preferred embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the complex is preferably distributed to the kidney. Other portions that target renal cells may also be used instead of, or in addition to, the lipid-based ligand.

[0146] In another embodiment, ligands are portions of vitamins, for example, that are taken up by target cells such as proliferating cells. These are particularly useful in treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant forms, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins such as folic acid, B12, riboflavin, biotin, and pyridoxal, or other vitamins or nutrients that are taken up by target cells such as liver cells. HSA and low-density lipoprotein (LDL) are also examples.

[0147] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the cell permeabilizer is amphiphilic. Exemplary cell permeabilizers are peptides such as tat or antennopedia. If the cell permeabilizer is a peptide, it may be modified, including peptidyl mimetic, inverted isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having lipophilic and oleophobic phases.

[0148] The ligand may be a peptide or a peptide mimetic. Peptidimides (also referred to herein as oligopeptide mimes) are molecules that can fold into a defined three-dimensional structure similar to natural peptides. The addition of peptides and peptide mimes to iRNA agents may affect the pharmacokinetic distribution of the iRNA, such as by enhancing cell recognition and absorption. The peptide or peptide mimetic moiety may be approximately 5 to 50 amino acid lengths, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths.

[0149] Peptides or peptide mimetic drugs may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a bound peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane transition sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) may also be target moieties. The peptide moiety may be a “delivery” peptide capable of transporting a number of polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. Sequences from, for example, HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been shown to function as delivery peptides. Peptides or peptide mimetic drugs 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, examples of peptides or peptide mimetic drugs anchored to dsRNA activators via incorporated monomer units include arginine-glycine-aspartate (RGD) peptides or RGD mimetic drugs. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase stability or induce conformational properties. Any of the structural modifications described below may be used.

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

[0151] "Cell-permeable peptides" can penetrate cells such as microbial cells, including bacterial or fungal cells, or mammalian cells, including human cells. Microbial cell-permeable peptides may be, for example, α-helical linear peptides (e.g., LL-37 or ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphimotic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the 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 the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, “carbohydrate” means a carbohydrate itself, which consists of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound which has as part a carbohydrate portion consisting of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical 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. Examples of specific monosaccharides include sugars with 5 or more C molecules (e.g., C5, C6, C7, or C8); disaccharides include sugars having 2 or 3 monosaccharide units (e.g., C5, C6, C7, or C8).

[0153] In one embodiment, the carbohydrate complex used in the composition and method of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

[0154] In another embodiment, the carbohydrate complex used in the compositions and methods of the present invention is [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0155] Another representative carbohydrate complex used in the embodiments described herein is: [ka] (In the formula, Examples include, but are not limited to, a (where either X or Y is an oligonucleotide and the other is hydrogen) array.

[0156] In some embodiments, the carbohydrate complex further comprises one or more of the above-mentioned additional ligands, such as but not limited to PK regulators and / or cell-permeable peptides.

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

[0158] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, such as by covalently bonding two parts of the compound together. Linkers are typically directly bonded, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryl, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, Alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl hererocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkyl heteroaryl, alkenyl heteroaryl, alkynylhereroaryl, etc., comprising an atomic chain, one or more of which are O, S, S(O), SO2, N(R) 8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (wherein R 8 The linker can be interrupted or terminated by hydrogen, acyl, aliphatic, or substituted aliphatic atoms. In one embodiment, the linker is approximately 1 to 24 atoms, 2 to 24 atoms, 3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 16 atoms, or 8 to 16 atoms.

[0159] The cleavable linking group is sufficiently stable outside the cell but is cleaved upon entry into the target cell, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster in the target cell or under a first standard condition (which may be selected to mimic or be equivalent to intracellular conditions) than in the target blood or under a second standard condition (which may be selected to mimic or be equivalent to conditions found in blood or serum).

[0160] Cleavable linkers are susceptible to the influence of 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 oxidative or reductases or reducing agents such as mercaptans present in cells that can degrade redox-cleavable linkers by reduction, and redox-selected or non-substrate-specific redox agents selected for specific substrates; esterases; agents that can create acidic environments, such as endosomes or those that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0161] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While human serum has a pH of 7.4, the mean intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a favorable pH, thereby releasing cationic lipids from ligands within the cell or to desired compartments of the cell.

[0162] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, a ligand targeting the liver may link to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore linkers are cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0163] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

[0164] Generally, the suitability of candidate cleavable linkers can be evaluated by testing the ability of a degrading agent (condition) to cleave the candidate linker. It is also desirable to test the candidate cleavable linker's resistance to cleavage in the blood or in contact with other non-target tissues. Therefore, the relative susceptibility to cleavage between a first and second condition can be determined, with the first condition selected to demonstrate cleavage in target cells and the second condition selected to demonstrate cleavage in other tissues or in biological fluids such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and then confirm it with further evaluation in whole animals. 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 faster 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 linking groups In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). Methods described herein can be relied upon to determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate may 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 may also be evaluated under conditions selected to mimic blood or serum conditions. One candidate compound is cleaved by up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at least 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 cleavage rate of candidate compounds can be determined using a standard enzyme kinetics assay under conditions selected to mimic an extracellular medium, compared to conditions selected to mimic an extracellular medium.

[0166] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group can be cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves the 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-, and -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-, and -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 linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of approximately 6.5 or less (e.g., approximately 6.0, 5.75, 5.5, 5.25, or less) or by an active agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable group may have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when the carbon is attached to the oxygen of the ester (alkoxy group), it may be an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can 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 linking group. The ester-based cleavable linking group is cleaved in the cell by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkylylene groups. The ester-based cleavable linking group has 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 linking group. The peptide-based cleavable linking group is cleaved in cells by enzymes such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond, which is formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable linking group does not contain an amide group (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond that is formed between amino acids to produce peptides and proteins. The peptide-based cleavable linking group is generally limited to peptide bonds (i.e., amide bonds) that are formed between amino acids to produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0170] In one embodiment, the iRNA of the present invention is coupled to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrates coupled to the linker of the composition and method of the present invention include: [ka] [ka] (In the formula, Examples include, but are not limited to, a (where either X or Y is an oligonucleotide and the other is hydrogen) array.

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

[0172] In one embodiment, the dsRNA of the present 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 repeating 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 Each occurrence is independently of the others: absence, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each occurrence is independently of the others: absent, alkylene, substituted alkylene, and one or more methylene groups: O, S, S(O), SO2, N(R) N ), C(R')=C(R''), C≡C or C(O) may be interrupted or terminated by one or more of these; R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5CEach occurrence is independently: absence, 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 heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, each occurrence is independently a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a It is conjugated with a divalent or trivalent branched linker, selected from a group of structures represented by either H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are Formula (XXXV), [ka] (In the formula, L 5A , L 5B and L 5C It is particularly useful when used in conjunction with RNAi agents to inhibit the expression of target genes (such as monosaccharides represented by GalNAc derivatives).

[0173] Suitable divalent and trivalent branched linker groups conjugated to GalNAc derivatives include, but are not limited to, the structures listed above as formulas II, VII, XI, X, and XIII.

[0174] Representative U.S. patents teaching the preparation of RNA complexes are, as are incorporated herein by reference in their entirety: U.S. Patent No. 4,828,979; U.S. Patent No. 4,948,882; U.S. Patent No. 5,218,105; U.S. Patent No. 5,525,465; U.S. Patent No. 5,541,313; U.S. Patent No. 5,545,730; U.S. Patent No. 5,552,538; U.S. Patent No. 5,578,717; U.S. Patent No. 5,580,731; and U.S. Patent No. 5,591,584. Detailed Statement; U.S. Patent No. 5,109,124; U.S. Patent No. 5,118,802; U.S. Patent No. 5,138,045; U.S. Patent No. 5,414,077; U.S. Patent No. 5,486,603; U.S. Patent No. 5,512,439; U.S. Patent No. 5,578,718; U.S. Patent No. 5,608,046; U.S. Patent No. 4,587,044; U.S. Patent No. 4,605,735; U.S. Patent No. 4,667,025; U.S. Patent No. 4,762,779; U.S. Patent No. 4,789,737 Details Document; U.S. Patent No. 4,824,941; U.S. Patent No. 4,835,263; U.S. Patent No. 4,876,335; U.S. Patent No. 4,904,582; U.S. Patent No. 4,958,013; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,245,022; U.S. Patent No. 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;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 are examples, but are not limited to these.

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

[0176] In the context of this invention, a "chimeric" iRNA compound or "chimeras" is an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased intracellular uptake, and / or increased binding affinity to a target nucleic acid. The additional region of the iRNA may act as an enzyme substrate capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded molecule. Therefore, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region, comparable results are often obtained with shorter iRNAs. Cleavage of RNA targets can conventionally be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0177] In some cases, the RNA of iRNA can be modified with non-ligand groups. Several non-ligand molecules are conjugated to iRNA to enhance its activity, cell distribution, or intracellular uptake, and procedures for performing such conjugations are available in the academic literature.These non-ligand portions include lipid portions 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. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids 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 & It contains Nucleotides (1995, 14:969), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents teaching the preparation of such RNA complexes are listed above. A typical conjugation protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with a molecule that conjugates the amino group using an appropriate coupling or activating reagent. The conjugation reaction can be carried out in the solution phase while the RNA is still bound to a solid support, or following RNA cleavage. Purification of the RNA complex by HPLC typically yields a pure complex.

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

[0179] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, whose entire contents are incorporated herein by reference). For in vivo delivery, factors to be considered for delivering the iRNA molecule include, for example, the biological stability of the delivery molecule, prevention of nonspecific effects, and accumulation of the delivery molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or transplantation into tissue or local administration of the formulation. Local administration to the treatment site maximizes the local concentration of the active substance, limits exposure of systemic tissues to the active substance which could otherwise be harmed or degraded by the active substance, and allows for administration of lower total doses of the iRNA molecule. Several studies have shown successful gene product knockdown when iRNA is administered locally. For example, intravitreal injection of VEGF dsRNA in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ., et al (2003) Mol.Vis.9:210-216) both demonstrated the prevention of 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 extended the survival time of mice with tumors (Kim, WJ., et al (2006) Mol.Ther.14:343-350; Li, S., et al (2007) Mol.Ther.15:515-523).RNA interference can be administered 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 lungs by intranasal administration (Howard, KA., et al. (2006) Mol.Ther.14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med.11:50-55) Successful local delivery has been demonstrated. To treat diseases, or to administer iRNA systemically, RNA can be modified or, alternatively, delivered using drug delivery systems; both methods act to prevent rapid degradation of dsRNA by endogenous and exonucleases. Modification of RNA or pharmaceutical carriers can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical bonding of lipophilic groups such as cholesterol to enhance intracellular uptake and prevent degradation. For example, when an iRNA that counteracts ApoB, which is conjugated to the lipophilic cholesterol portion, was systemically injected into mice, apoB mRNA knockdown was induced in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). The conjugation of iRNA to aptamers has been shown to suppress tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNA may 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 the iRNA molecule (which is negatively charged) and also enhance interactions with the negatively charged cell membrane, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may be induced to bind to iRNA or form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), cited above; Verma, UN., et al (2003), cited above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441:111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME., et al. Examples include al (2008) Pharm. Res., published online on August 16; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D., 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 forms a complex with cyclodextrin. Methods of administering iRNA and cyclodextrin and pharmaceutical compositions are described in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0180] A. Vector encoding the iRNA of the present invention Serpinc1 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., International Publication No. 00 / 22113; Conrad, International Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a few hours to several weeks) or persistent (from several weeks to several months or more), 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 may be embedded or non-embedded vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (see, for example, Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

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

[0182] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be generated 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 suppliers. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors may include systemic administration, such as intravenous or intramuscular administration; administration to target cells explanted from a patient and subsequent reintroduction into the patient; or any other means that allows introduction into desired target cells.

[0183] iRNA expression plasmids can be translocated into target cells as complexes with cationic lipid carriers (e.g., oligofectamines) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid translocation for iRNA-mediated knockdown, targeting different regions of the target RNA over a period of more than one week, is also explored in this invention. Successful introduction of the vector into host cells can be monitored using various known methods. For example, transient translocation can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable translocation into cells in vitro can be ensured by using markers that provide the translocated cells with resistance to specific environmental factors (e.g., antibiotics and drugs), 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) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (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, including vaccinia virus vectors, or avipox, including canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may optionally include a viral sequence for translocation. Alternatively, the construct may be incorporated into an episomal replication vector, such as EPV and EBV vectors. Constructs for the recombinant expression of iRNAs generally require regulatory factors, such as promoters and enhancers, to ensure iRNA expression in target cells. Other aspects of vectors and constructs that are considered are described in more detail below.

[0185] A vector useful for delivering iRNA contains sufficient regulatory factors (promoters, enhancers, etc.) to express the iRNA in the desired target cells or tissues. These regulatory factors can be selected to provide either constitutive or regulatory / inducible expression.

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

[0187] Viral vectors containing nucleic acid sequences encoding iRNA 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 the correct packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors to facilitate delivery of the nucleic acid to the patient. For more details on retroviral vectors, see Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to generate stem cells that exhibit higher resistance 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 considered 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 the delivery of the iRNAs of the invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, airway epithelium. Adenoviruses infect the airway epithelium naturally and cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, the 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 an adenovirus vector for transferring genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy are 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 Publication No. 94 / 12649 pamphlet; and Wang, et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing the iRNAs addressed in the present invention, methods of constructing recombinant AV vectors, and methods of 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 iRNA of the present invention (Walsh et al., Proc.Soc.Exp.Biol.Med.204:289-300(1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNA may be expressed as two distinct complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNAs addressed in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described herein by reference in their entirety: Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Publication No. 94 / 13788; and International Publication No. 93 / 24641.

[0190] Other viral vectors suitable for delivering the iRNA of the present invention include, for example, vaccinia viruses such as modified virus Ankara (MVA) or attenuated vaccinia such as NYVAC, and poxviruses such as avipox such as fowlpox or canarypox.

[0191] The affinity of the viral vector can be modified, if desired, by pseudotyping the vector with an envelope protein or other surface antigen from another virus, or by substituting the capsid protein from a different virus. 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 engineering the vector to express different capsid protein serotypes; see, for example, Rabinowitz J E et al. (2002), J Virol 76:791-801, which is incorporated herein by reference in its entirety.

[0192] The pharmaceutical of the vector can contain the vector in an acceptable diluent or can contain a sustained release matrix in which the gene delivery vehicle is encapsulated. As an alternative, if a complete gene delivery vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical can contain one or more cells that produce the gene delivery system.

[0193] V. Pharmaceutical Composition of the Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, the present invention provides a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier. The iRNA-containing pharmaceutical composition is useful for treating diseases or disorders related to the expression or activity of the Serpinc1 gene, 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, for example, by intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma, for example, by intracerebral infusion, such as continuous pump infusion. The pharmaceutical composition of the present invention may be administered in a dose sufficient to inhibit Serpinc1 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 body weight of the recipient per day, generally ranging from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered in single doses of approximately 0.01 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg.

[0194] For example, dsRNA is approximately 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, It may be administered in doses 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0195] In another embodiment, dsRNA is approximately 0.1 to 50 mg / kg, approximately 0.25 to 50 mg / kg, approximately 0.5 to 50 mg / kg, approximately 0.75 to 50 mg / kg, approximately 1 to 50 mg / mg, approximately 1.5 to 50 mg / kb, approximately 2 to 50 mg / kg, approximately 2.5 to 50 mg / kg, approximately 3 to 50 mg / kg, approximately 3.5 to 50 mg / kg, approximately 4 to 50 mg / kg, approximately 4.5 to 50 mg / kg, approximately 5 to 50 mg / kg, approximately 7.5 to 50 mg / kg, approximately 10 to 50 mg / kg, approximately 15 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 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 It is administered in doses of g / kg, approximately 0.25–20 mg / kg, approximately 0.5–20 mg / kg, approximately 0.75–20 mg / kg, approximately 1–20 mg / kg, approximately 1.5–20 mg / kg, approximately 2–20 mg / kg, approximately 2.5–20 mg / kg, approximately 3–20 mg / kg, approximately 3.5–20 mg / kg, approximately 4–20 mg / kg, approximately 4.5–20 mg / kg, approximately 5–20 mg / kg, approximately 7.5–20 mg / kg, approximately 10–20 mg / kg, or approximately 15–20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of this invention.

[0196] For example, dsRNA is 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, It may be administered in doses of 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0197] In another embodiment, dsRNA is present in concentrations of approximately 0.5 to 50 mg / kg, 0.75 to 50 mg / kg, 1 to 50 mg / kg, 1.5 to 50 mg / kb, 2 to 50 mg / kg, 2.5 to 50 mg / kg, 3 to 50 mg / kg, 3.5 to 50 mg / kg, 4 to 50 mg / kg, 4.5 to 50 mg / kg, 5 to 50 mg / kg, 7.5 to 50 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, 20 to 50 mg / kg, 20 to 50 mg / kg, 25 to 50 mg / kg, and 25 to 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 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 in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. In one embodiment, dsRNA is administered at a dose of approximately 10 mg / kg to approximately 30 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0198] For example, the target values ​​are 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, 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 Therapeutic doses of iRNA such as 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 approximately 50 mg / kg may be administered. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0199] The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two or three or more partial doses at appropriate intervals throughout the day, or even by delivery via continuous infusion or controlled-release formulation. In this case, the amount of iRNA contained in each partial dose must be correspondingly smaller in order to achieve the total daily dose. The dosing unit may also be formulated for delivery over several days using conventional sustained-release formulations that provide sustained release of iRNA over several days, for example. Sustained-release formulations are well known in the art and are particularly useful for the delivery of the active ingredient to a specific site, which may be used with the active ingredient of the present invention. In this embodiment, the dosing unit contains a number of corresponding daily doses.

[0200] In another embodiment, a single dose of the pharmaceutical composition may be administered over a longer period, with subsequent doses given at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less. 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 pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective dose of the composition may consist of a single treatment or a series of treatments. The effective doses and in vivo half-lives of individual iRNAs incorporated in this invention may be estimated using conventional procedures or based on in vivo studies using appropriate animal models, as described elsewhere in this specification.

[0202] Advances in mouse genetics have led to the creation of several mouse models for the study of various human diseases, such as bleeding disorders that benefit from reduced Serpinc1 expression. Such models can be used for in vivo testing of iRNAs and for determining therapeutically effective doses. For example, suitable mouse models such as hemophilia A mouse models and hemophilia B mouse models are known in the art, such as mice with coagulation factor gene knockouts, as described in Bolliger, et al. (2010) Thromb Haemost 103:1233-1238, Bi L, et al. (1995) Nat Genet 10:119-21, Lin et al. (1997) Blood 90:3962-6, Kundu et al. (1998) Blood 92:168-74, Wang et al. (1997) Proc Natl Acad Sci USA 94:11563-6, and Jin, et al. (2004) Blood 104:1733.

[0203] The pharmaceutical compositions of the present invention may be administered in several ways, depending on whether topical or systemic treatment is desired and on the treatment area. Administration may be topical (e.g., by a transdermal patch), transpulmonary by inhalation or blowing of powder or fume, including by a nebulizer; intratracheal, intranasal, transepidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal administration via an implantable device, for example; or intracranial administration, such as into the brain parenchyma, subarachnoid space or ventricles. iRNA may be delivered in a manner that targets specific tissues, such as the liver (e.g., hepatic parenchymal cells). Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, etc., may be necessary or desirable. Covered condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the iRNA addressed in this invention is in a miscible material with a topically delivered substance such as a lipid, liposome, fatty acid, fatty acid ester, steroid, chelating agent, and surfactant. 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 addressed in this invention can be encapsulated within liposomes, or can form complexes with them, particularly with cationic liposomes. Alternatively, the iRNA can form complexes 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-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C. 1~20 Examples include, but are not limited to, alkyl esters (e.g., isopropylmyristate 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 preparation comprising a membrane-like molecular assembly 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. In this specification, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one or more bilayers. Liposomes include monolayer or multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition but may optionally. Liposomes are useful for the migration and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to a biological membrane, when liposomes are applied to tissue, the liposomal bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and cell progresses, the internal aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to target RNA and mediate RNAi. In some cases, liposomes are also specifically targeted, for example, to induce iRNAs into specific cell types.

[0205] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed without the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid complex. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. Next, the RNAi agent preparation is added to the micelles containing the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain the RNAi agent liposome preparation.

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

[0207] A method for generating a stable polynucleotide delivery vehicle by incorporating a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle is further described, for example, in International Publication No. 96 / 37194, which is incorporated herein by reference in its entirety. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent 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. This may also include one or more embodiments of the exemplary methods described in al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing appropriately sized lipid aggregates for use as delivery vehicles include sonication and combinations of freeze-thaw 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, micro-solution preparation may be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable to the packing of RNAi preparations into liposomes.

[0208] Liposomes are broadly classified into two 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 taken into the endosome. Due to the acidic pH inside the endosome, the liposomes 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 form complexes with nucleic acids; rather, they encapsulate them. Because nucleic acids and lipids both have similar charges, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used in culture to deliver nucleic acids encoding thymidine kinase genes to cell monolayers. Expression of exogenous genes was detected in 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 derived 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 fusion liposomes are mainly formed from dioleoyl sphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Yet another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0211] Examples of other methods of introducing liposomes into cells in vitro and in vivo include U.S. Patent No. 5,283,185; U.S. Patent No. 5,171,678; International Publication No. 94 / 00569 pamphlet; International Publication No. 93 / 24640 pamphlet; International Publication No. 91 / 16024 pamphlet; 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 liposome systems, particularly those comprising a nonionic surfactant and cholesterol, have been studied and their utility in drug delivery to the skin has been determined. Cyclosporin A was delivered intradermally into mouse skin using a nonionic liposome formulation comprising Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposome systems are effective in facilitating the deposition of cyclosporin A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0213] Liposomes also include "sterically stabilized" liposomes, which term as used herein refers to liposomes comprising one or more specialized lipids which, when incorporated into liposomes, result in an improved circulation lifetime compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1(B) These consist of one or more glycolipids, or are derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While we do not wish to be constrained by any particular theory, in the art, in sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivativeized lipids, the improvement in the circulating half-life of these sterically stabilized liposomes is thought to be due to reduced uptake by reticuloendothelial system (RES) cells (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. NYAcad. Sci., 1987, 507, 64) described monosialoganglioside G M1 The ability of galactocerebroside sulfate and phosphatidylinositol to improve the half-life of liposomes in the blood has been reported. These findings were described in detail by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Both U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924, both granted to Allen et al., describe (1) sphingomyelin and (2) ganglioside G M1 Liposomes comprising or galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0215] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot efficiently fuse with the plasma membrane, but they can be taken up by macrophages in vivo and used to deliver RNAi agents to macrophages.

[0216] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; they can encapsulate a wide range of water- and lipid-soluble drugs; and they can protect RNAi agents encapsulated within their internal compartments from metabolism and degradation (Rosoff, “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Key considerations in the preparation of liposomal formulations include the lipid surface charge, vesicle size, and aqueous capacity of the liposomes.

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

[0218] The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA complexing vesicles. Lipofectin® (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to cultured tissue cells, and 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 complex is also positive. The positively charged complex thus prepared spontaneously adheres to negatively charged cell surfaces, fuses with the plasma membrane, and efficiently delivers functional nucleic acids, for example, into 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 portion is linked by an ester rather than an ether bond.

[0219] Other reported cationic lipid compounds include those conjugated to a variety of moieties, such as carboxyspermine conjugated to one of two lipid types, and compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, for example, U.S. Patent No. 5,171,678).

[0220] Another cationic lipid complex involves lipid derivatization by cholesterol ("DC-Chol") combined 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 translocation 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 translocation 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 oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0221] Liposome formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver RNAi agents intracutaneously. In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and to enhance their penetration into skin tissues, such as within the skin. For example, liposomes can be applied topically. Topical delivery of therapeutic drugs 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, R. Jand 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. Rand Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. 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, and their efficacy in drug delivery to the skin has been evaluated. 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, including RNAi agents, are useful for treating skin diseases.

[0223] Liposomes containing iRNA can be made highly deformable. Such deformability can allow the liposome to penetrate pores smaller than the average radius of the liposome. Transferosomes, for example, are a type of deformable liposome. Transferosomes can be created by adding surface edge activators, usually surfactants, to a standard liposome composition. Transferosomes containing RNAi agents can be delivered subcutaneously, for example by infection, to deliver the RNAi agent to keratinocytes in the skin. To cross intact mammalian skin, the lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transcutaneous 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 often self-load.

[0224] Other formulations to which the present invention can be applied are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on 2 January 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed on 2 January 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed on 26 March 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed on 22 April 2008; and U.S. Provisional Patent Application No. 61 / 051,528, filed on 8 May 2008. PCT Application PCT / US2007 / 080331, filed on 3 October 2007, also describes formulations to which the present invention can be applied.

[0225] Transfersomes are yet another type of liposome, highly deformable lipid aggregates, that are attractive candidates for drug delivery vehicles. Because they are so highly deformable, transfersomes can be described as lipid droplets that can easily penetrate through pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to skin pore shapes), self-repair, and often reach and self-load their targets without fragmentation. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. 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 serum albumin-containing solution.

[0226] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and grading the properties of the many different types of surfactants, both natural and synthetic, is the use of the hydrophile / lipophile balance (HLB). The properties of the hydrophilic group (also known as the "head") provide 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] When a 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 across a wide pH range. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Examples of 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, also belong to this class. Polyoxyethylene surfactants are the most commonly found components of the nonionic surfactant class.

[0228] Surfactants are classified as anionic when their molecules retain a negative charge when dissolved or dispersed in water. Examples of anionic surfactants include carboxylates such as soap, acyl lactylate, acylamides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0229] Surfactants are classified as cationic if their molecules retain a positive charge when dissolved or dispersed in water. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used components in this class.

[0230] A surfactant is classified as amphoteric if its molecule has the ability to have either a positive or negative charge. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phospholipids.

[0231] The use of surfactants in pharmaceuticals, formulations, and emulsions is outlined (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0232] The iRNA used in the method of the present invention may also be provided as a micelle formulation. A “micelle” is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. The opposite configuration exists if the environment is hydrophobic.

[0233] Mixed micelle formulations suitable for transdermal delivery include siRNA compositions and alkali metals C8-C8. 22 The mixture may be prepared by mixing an aqueous solution of an alkyl sulfate and a micelle-forming compound. Examples of micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocolanyglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogues, polydocanol alkyl ether and its analogues, 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 will form regardless of how the components are mixed substantially, but vigorous mixing is preferable to provide smaller micelles.

[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, the micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by the addition of 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 preparation and protect it from bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the mixed micelle composition has been formed.

[0236] To deliver a micelle formulation as a spray, the formulation can be placed in a fumigant metering and dispensing device, and the spray can be loaded into the device. Under pressurization, the spray is in liquid form within the metering and dispensing device. The ratio of components is adjusted so that the aqueous phase and the spray phase are one, i.e., a single phase. If there are two phases, the metering and dispensing device needs to be shaken, for example, before dispersing a portion of the contents through a metering valve. The dispensing dose of the pharmaceutical is sprayed in a fine mist from the metering valve.

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

[0238] The specific concentration of essential components can be determined by relatively simple experimental methods. For oral absorption, it is often desirable to increase the dose to, for example, at least two or three times the dose administered by injection or via the gastrointestinal tract.

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

[0240] In the use of this specification, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLPs. In the use of this specification, 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 complexes). SNALPs and SPLPs exhibit a long circulatory lifetime following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic applications. Examples of SPLPs include "pSPLPs" containing encapsulation condenser-nucleic acid complexes as described in International Publication No. 00 / 03683. The particles of the present invention are substantially non-toxic and 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. In addition, when present in the nucleic acid-lipid particles of the present invention, the nucleic acids are resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent No. 5,976,567; U.S. Patent No. 5,981,501; U.S. Patent No. 6,534,484; U.S. Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and International Publication No. 96 / 40964.

[0241] In one embodiment, the ratio (mass / mass ratio) of lipids to drugs (e.g., lipid to dsRNA ratio) is in the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1. Ranges between the ranges cited above are also considered to be part of the present 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-dioleyloxy)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-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylamine. Minopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleythio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyoxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyoyl-3-trimethylaminopropane chloride (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-propanediol (propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) The analogs thereof may be (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)piperazine-1-yl)ethylazandiyl)didodecane-2-ol (Tech G1), or mixtures thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol% or 40 mol% of the total lipids present in the particles.

[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 consist of 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (molar percentage), with a particle size of 63.0 ± 20 nm and an siRNA / lipid ratio of 0.027.

[0245] Ionic / noncationic lipids include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerin (DOPG), dipalmitoyl phosphatidylglycerin (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimide). These may include, but are not limited to, anionic or neutral lipids, such as 1-Cyl-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. If cholesterol is present, noncationic lipids may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0246] Conjugated lipids that inhibit particle aggregation may include, without limitation, polyethylene glycol (PEG)-lipids, such as PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, and PEG-ceramide (Cer), or mixtures thereof. PEG-DAA complexes may include, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C8). The amount of conjugated lipids that prevent particle aggregation may be 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

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

[0248] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be prepared using the lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, whose contents are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be combined in a molar ratio, for example, 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (for example, in sodium acetate at pH 5) so that the final ethanol concentration is about 35–45% and the final sodium acetate concentration is about 100–300 mM. Lipid-dsRNA nanoparticles typically form spontaneously during mixing. 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 a 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 replaced with phosphate-buffered saline (PBS) at approximately pH 7, such as approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka]

[0249] The LNP01 formulation is described, for example, in International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0250] Additional exemplary lipid dsRNA preparations are listed 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 International Publication No. 2009 / 127060, filed on 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 on 29 January 2009; U.S. Provisional Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Application No. 1 filed on 10 June 2009; U.S. Provisional Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Application No. 61 / 239,686 filed on 3 September 2009; and International Application PCT / US2010 / 022614 filed on 29 January 2010.

[0256] Formulations comprising MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010, which is incorporated herein by reference in its entirety.

[0257] Formulations containing ALNY-100 are described, for example, in the international application PCT / US 09 / 63933, filed on November 10, 2009, which is incorporated herein by reference.

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

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

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

[0261] An alkenyl is an alkyl group, as defined above, that contains at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative linear and branched alkenyls include ethyleneyl, propyrenyl, 1-butenyl, 2-butenyl, isobutyrenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.

[0262] "Alkynyl" refers to any alkyl or alkenyl as defined above, which further contains at least one triple bond between adjacent carbon atoms. Representative linear and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0263] "Acyl" refers to any alkyl, alkenyl, or alkynyl group in which a carbon atom is substituted with an oxo group at the bonding site, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0264] A "heterocycle" means a saturated, unsaturated, or aromatic 5- to 7-membered monocycle or 7- to 10-membered dicycle, heterocycle containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, including a dicycle in which any of the lower heterocycles is fused to a benzene ring, wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocycles can be attached via any heteroatom or carbon atom. Examples of heterocycles include heteroaryls as defined below. Examples of heterocycles include morpholinyl, pyrrolidinonyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxylanil, oxetanyl, tetrahydrofuranil, tetrahydropyranil, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranil, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyranil.

[0265] The terms "alkyl which may or may not be substituted", "alkenyl which may or may not be substituted", "alkynyl which may or may not be substituted", "acyl which may or may not be substituted", and "heterocyclic ring which may or may not be substituted", when substituted, mean that at least one hydrogen atom is substituted with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, the substituents include oxo, halogen, heterocyclic ring, -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 are mentioned, n is 0, 1 or 2, and R x and R y are the same or different and independently are hydrogen, alkyl or heterocyclic ring, and each of the alkyl and heterocyclic ring substituents is one or more of oxo, halogen, -OH, -CN, alkyl, -OR x 、heterocyclic ring, -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 and may be further substituted by.

[0266] "Halogen" refers to fluoro, chloro, bromo, and iodine.

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

[0268] Synthesis of Equation A In some embodiments, the nucleic acid lipid particles of the present invention are of formula A, [ka] The preparation is carried out using a cationic lipid of formula A (wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl molecules, each of which may be substituted or unsubstituted, and R3 and R4 are independently lower alkyl molecules, or R3 and R4 together may form an optionally substituted heterocycle). In some embodiments, the cationic lipid is XTC(2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be prepared by reaction scheme 1 or 2 below, where all substituents are as defined above unless otherwise specified.

[0269] [ka] Lipid A, in which R1 and R2 are independently alkyl, alkenyl, or alkynyl compounds, each optionally substituted, and R3 and R4 are independently lower alkyl compounds, or R3 and R4 together may form an optionally substituted heterocycle, 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. The reaction of 1 and 2 yields ketal 3. Ketal 3 is treated with amine 4 to obtain the lipid of formula A. The lipid of formula A can be converted to the corresponding ammonium salt by an organic salt of formula 5 (wherein X is an anion counterion selected from halogens, hydroxides, phosphates, sulfates, etc.).

[0270] [ka] Alternatively, the starting materials for ketone 1 may 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. The reaction of 6 and 7 yields ketone 1. The conversion of ketone 1 to the corresponding lipid of formula A is as described in Scheme 1.

[0271] MC3 synthesis The preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid) was as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyrate (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 diluted aqueous sodium bicarbonate. The organic fraction was dried on 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. The fractions containing the purified product were combined, and the solvent was removed to obtain a colorless oil (0.54 g).

[0272] Synthesis of ALNY-100 Ketal 519 [ALNY-100] was synthesized using the following scheme 3. [ka]

[0273] 515 synthesis To a suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml of anhydrous THF stirred in 1 L of RBF, a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF was slowly added at 0°C under a nitrogen atmosphere. After the addition was complete, the reaction mixture was heated to room temperature, then heated and refluxed for 4 hours. 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 careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 hours and filtered. The residue was thoroughly washed with THF. The filtrate and washings were mixed and diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 minutes. The volatilities were evaporated under vacuum to obtain 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] 516 synthesis To a stirred solution of compound 515 in 100 mL of dry DCM in 250 mL double-necked RBF, NEt3 (37.2 mL, 0.2669 mol) was added and the mixture was cooled to 0°C under a nitrogen atmosphere. After the slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was allowed to stand until it warmed to room temperature. After the reaction was complete (2-3 hours by TLC), the mixture was successively washed with 1N HCl solution (1 × 100 mL) and saturated NaHCO3 solution (1 × 50 mL). The organic layer was then dried on anhydrous Na2SO4, and the solvent was evaporated to obtain the crude product, which was purified by silica gel column chromatography to obtain 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] Combination of 517A and 517B At room temperature, cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of acetone and water (10:1) in a 500 mL bottle of RBF. N-methylmorpholine N-oxide (7.6 g, 0.06492 mol) was added, followed by 4.2 mL of 7.6% OsO4 (0.275 g, 0.00108 mol) solution in tert-butanol. After the reaction was complete (approximately 3 hours), the mixture was quenched by adding solid Na2SO3, and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with DCM (300 mL), washed with water (2 × 100 mL), and then with saturated NaHCO3 (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed under vacuum. A diastereomer mixture was obtained by silica gel column chromatography purification of the crude product, and then separated by preliminary HPLC. Yield: -6g crude product

[0276] 517A - Peak-1 (white solid), 5.13 g (96%). 1H-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+NH4+]-283.5 present, HPLC - 97.86%. Stereochemistry was confirmed by X-ray.

[0277] 518 synthesis Compound 518 was obtained as a colorless oil (1.2 g, 41%) using a procedure similar to that described for the synthesis of compound 505. ¹H-NMR (CDCl3, 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] Basic 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 hours and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous solution Na2SO4 and then filtered through Celite to concentrate into oil. Column chromatography yielded 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 of C44H80NO2(M+H)+, calculated value 654.6, measured value 654.6.

[0279] Formulations prepared by either the standard method or a non-extrusion method can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be whitish, translucent solutions free of aggregates or precipitates. The particle size and particle size distribution of lipid nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). The 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 formulations and inclusion fractions was estimated using a dye exclusion assay. Samples of formulated dsRNA can be incubated with RNA-binding dyes such as Ribogreen (Molecular Probes) in or out of the presence of a formulation-disrupting surfactant, for example, 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the surfactant-containing sample compared to a standard curve. The inclusion fraction is determined by subtracting the "free" dsRNA content (measured by the signal in the absence of surfactant) from the total dsRNA content. The percentage of inclusion dsRNA is typically >85%. In SNALP formulations, particle sizes are at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Appropriate ranges are typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0280] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, the oral formulation is administered in combination with one or more osmotic surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. 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-dihydrofusidate, 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-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers are used, such as fatty acid / salt combined with bile acid / salt. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA addressed in this invention may be delivered orally in granular form, including spray-dried particles, or may be complexed to form micro or nanoparticles.Examples of DsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivativeized 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-hexyl Examples include acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, methyl polyacrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparations are described in detail in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Patent No. 6,747,014, respectively, which are incorporated herein by reference.

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

[0282] Examples of the pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but are not limited to, pre-made liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver disorders such as liver cancer, formulations targeting the liver are particularly preferred.

[0283] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage forms, can be prepared according to the prior art well known in the pharmaceutical industry. Such art involves the step of combining the active ingredient with a pharmaceutical carrier or excipient. Generally, formulations are prepared by uniformly and closely combining the active ingredient with a liquid carrier or an ultrafine particle solid carrier or both, and then shaping the product if necessary.

[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 may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions may also contain stabilizers.

[0285] C. Additional formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (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 that are closely mixed and dispersed from one another. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed in a bulk oily phase as microdroplets, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed in a bulk aqueous phase as microdroplets, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to a dispersed phase and an active agent, which may exist as a solution in either an aqueous or oily phase, or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multi-phase emulsions comprising more than two phases, such as oil-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer specific advantages that simple two-component emulsions do not. Among these, a multi-phase emulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, an oil droplet system encapsulated in small water spheres and stabilized within a continuous oily phase provides an o / w / o emulsion.

[0286] Emulsions are characterized by having little to no thermodynamic stability. Often, the dispersed or discontinuous phases of an emulsion are well dispersed externally or within the continuous phase and maintained in this form through emulsifiers or means of increasing the formulation viscosity. In the case of emulsion-type ointment bases and creams, any of the emulsion phases may be semi-solid or solid. Another means of stabilizing an emulsion involves the use of emulsifiers, which may be incorporated into any of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorbent bases, and finely dispersed solids (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).

[0287] Synthetic surfactants, also known as surfactants, have a wide range of applications in emulsion formulations and are outlined in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, 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 consist of hydrophilic and hydrophobic moieties. The ratio of hydrophilic to hydrophobic groups is called the hydrophilic / lipophilic balance (HLB) of a surfactant and is a useful means of classifying and selecting surfactants in the preparation of pharmaceutical formulations. Surfactants can be classified into different classes based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, 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 that can absorb water and form w / o emulsions, such as anhydrous lanolin and hydrophilic petrolatum, still maintain their semi-solid viscosity. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, particularly in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-expanding clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloids of aluminum silicate and 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. These include 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] Examples of hydrophilic colloids include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse in water or swell in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the outer phase.

[0291] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can readily support microbial growth; therefore, preservatives are frequently incorporated into these formulations. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene; 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 cutaneous, oral, and parenteral routes, and methods for manufacturing them, are outlined 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 widely used due to their ease of preparation and efficiency in terms of absorption and bioavailability (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, 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, fat-soluble vitamins, and high-fat nutritional supplements are among the materials commonly administered orally as o / w emulsions.

[0293] ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid composition is prepared as a microemulsion. A microemulsion can be defined as a single optically isotropic and thermodynamically stable solution of water, oil, and an amphiphilic substance (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, a microemulsion is a system prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, which is generally an alcohol of intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surfactant 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 a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, 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, as well as 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] Phenomenological approaches using phase diagrams have been extensively studied, providing those skilled in the art with comprehensive knowledge regarding the formulation of microemulsions (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, 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 spontaneously formed, thermodynamically stable droplet formulations.

[0295] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with co-surfactants. Typically, co-surfactants, which are short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant coating, resulting in an irregular coating due to gaps between surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may, but is not limited to, water, aqueous solutions of pharmaceuticals, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, 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, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, protection of drugs from enzymatic hydrolysis, expected enhanced drug absorption due to changes in membrane fluidity and permeability induced by surfactants, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 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 compounding heat-unstable drugs, peptides, or iRNAs. Microemulsions have proven effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve local intracellular uptake of iRNAs and nucleic acids.

[0297] The microemulsion of the present invention may also contain additional components 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. Penetration enhancers used in the microemulsion of the present invention can be classified into one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0298] iii. Particulates The RNAi agent of the present invention may be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but they 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 provides efficient delivery of nucleic acids, particularly iRNAs, to animal skin using various penetration enhancers. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes if the membrane being traversed is treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

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

[0301] Surfactants (or "surface-activating agents") are chemical substances 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, thereby improving iRNA absorption through mucous membranes. In addition to bile salts and fatty acids, examples of these penetration enhancers include 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 perfluoro compound 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-monoleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and its C 1~20 Examples include alkyl esters (e.g., methyl, isopropyl, and t-butyl) and their mono- and di-glycerides (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 osmotic enhancers. Therefore, 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), glucoseic acid (sodium glucose), 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, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 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 connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with them, thereby improving iRNA absorption through mucous membranes. With regard to their use as penetration enhancers in the present invention, since most DNA nucleases require divalent metal ions for catalytic activity and are inhibited by chelating agents, chelating substances have the additional advantage of also acting as deoxyribonuclease inhibitors (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating substances 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 (enamine). (See, for example, 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] In the usage herein, non-chelating, non-surfactant osmotic enhancers can be defined as compounds that demonstrate insignificant activity as chelating agents or surfactants, but still enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of osmotic enhancers in this class include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0306] Substances that enhance iRNA uptake at the cellular level can also be added to the pharmaceuticals and other compositions of the present invention. For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188, granted to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (International Publication No. 97 / 30731, granted to Lollo et al.) are also known to enhance dsRNA uptake within cells.Examples of commercially available trait transfer reagents include, for example, Lipofectamine(trademark) (Invitrogen; Carlsbad, CA), Lipofectamine 2000(trademark) (Invitrogen; Carlsbad, CA), 293fectin(trademark) (Invitrogen; Carlsbad, CA), Cellfectin(trademark) (Invitrogen; Carlsbad, CA), DMRIE-C(trademark) (Invitrogen; Carlsbad, CA), FreeStyle(trademark)MAX(Invitrogen; Carlsbad, CA), and Lipofectamine(trademark) 2000. 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 include 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 (trademark) (B-Bridge International, Mountain View, CA, USA).

[0307] Glycols such as ethylene glycol and propylene glycol; pyrroles such as 2-pyrrole; azone; and other active ingredients including terpenes such as limonene and menthone can be used to enhance the penetration of administered nucleic acids.

[0308] v. Carrier Certain compositions of the present invention also incorporate a carrier compound during formulation. In the use herein, “carrier compound” or “carrier” may refer to a nucleic acid or analogue that is inactive (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids, for example, by degrading biologically active nucleic acids or facilitating their removal from circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter, may result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory storage sites, possibly due to competition between the carrier compound and nucleic acid for the normal receptor. For example, the recovery of partial phosphorothioate dsRNAs in liver tissue may be reduced when administered concurrently with polyinosinate, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyanostilbene-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 carrier compounds, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected with the planned mode of administration in mind so as to provide the desired bulk, viscosity, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); bulking agents (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, silicon dioxide colloids, stearic acid, metal stearate salts, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

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

[0311] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil bases. The solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not cause adverse nucleic acid reactions and are suitable for oral administration may be used.

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

[0313] vii. Other ingredients The compositions of the present invention may further contain other auxiliary components found in conventional pharmaceutical compositions, at levels of use established in the art. Therefore, for example, the compositions may contain additional suitable pharmacologically active materials such as antipruritics, tannins, topical anesthetics, or anti-inflammatory agents, or additional materials useful for physically compounding the various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, 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 acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0314] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0315] In some embodiments, the pharmaceutical compositions covered by the present invention include (a) one or more iRNA compounds, and (b) one or more agents that function by 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, antivirals, 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 diseases include terbivudine, entecavir, telaprevir, and protease inhibitors such as those disclosed in U.S. Patent Application Publication 2005 / 0148548, U.S. Patent Application Publication 2004 / 0167116, and U.S. Patent Application Publication 2003 / 0144217 granted to Tung et al., and U.S. Patent Application Publication 2004 / 0127488 granted to Hale et al.

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

[0317] Data obtained from cell culture assays and animal experiments can be used to formulate dosage ranges for human use. Doses of the compositions discussed in this invention are generally within the range of circulating concentrations, including the ED50, which is minimally or completely toxic. Doses may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods discussed in this invention, the therapeutically effective dose can first be estimated from a cell culture assay. Doses may be formulated in animal models to achieve the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence, including the IC50 (i.e., the test compound concentration that achieves maximum half-dose inhibition of symptoms) determined in cell culture (e.g., achieving a reduction in polypeptide concentration). 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] The iRNAs discussed herein may be administered in combination with other known agents effective in treating pathological processes mediated by Serpinc1 expression, in addition to the administrations considered above. In any case, the physician administering the treatment may adjust the amount and timing of iRNA administration based on the results observed using standard means of efficacy known in the art or described herein.

[0319] VI. Method of Invention The present invention also provides a method for reducing and / or inhibiting Serpinc1 expression in cells using the iRNA of the present invention and / or a composition containing the iRNA of the present invention. In other embodiments, the present invention provides the iRNA of the present invention and / or a composition containing the iRNA of the present invention for use in reducing and / or inhibiting Serpinc1 expression in cells. In yet another embodiment, the use of the iRNA of the present invention and / or a composition containing the iRNA of the present invention is provided for manufacturing a drug that reduces and / or inhibits Serpinc1 expression in cells.

[0320] The method and use include the step of contacting cells with an iRNA, such as the dsRNA of the present invention, and keeping the cells for a sufficient time to obtain degradation of the mRNA transcript of the Serpinc1 gene, thereby inhibiting Serpinc1 gene expression in the cells.

[0321] A decrease in gene expression can be assessed by any method known in the art. For example, a decrease in Serpinc1 expression may be determined by measuring the mRNA expression level of Serpinc1 using methods commonly used by those skilled in the art, such as Northern blotting or qRT-PCR; by measuring the Serpinc1 protein level using methods commonly used by 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 related to the cellular blood coagulation mechanism (or blood coagulation itself in vivo).

[0322] In the methods and uses of the present invention, cells may be brought into contact in a test tube or in vivo, i.e., cells may be present within the subject.

[0323] Cells suitable for treatment using the method of the present invention may be any cells that express the Serpinc1 gene. Cells suitable for use in the method and use of the present invention may be mammalian cells such as primate cells (human cells or non-human primate cells such as monkey cells or chimpanzee cells, for example), non-primate cells (bovine 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, for example), avian cells (duck cells or goose cells, for example), or whale cells. In one embodiment, the cells are human cells such as human liver cells.

[0324] Serpinc1 expression is present in cells at least approximately 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, 5 2, 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, or approximately 100% inhibition.

[0325] The in vivo methods and uses of the present invention may include the step of administering a composition containing iRNA to a target, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the Serpinc1 gene of the mammal being treated. When an organism such as a mammal such as a human is being treated, 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 subarachnoid), intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), nasal, rectal, and 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 accumulation injection. Accumulation injection may release iRNA consistently over a long period. Therefore, accumulation injection may reduce the frequency of administration required to obtain desired effects, such as desired Serpinc1 inhibition or therapeutic or prophylactic effects. Accumulation injection may also provide more consistent serum concentrations. Accumulation injections may be subcutaneous or intramuscular injections. In preferred embodiments, the accumulation injection is subcutaneous.

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

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

[0329] In one embodiment, the present invention also provides a method for inhibiting Serpinc1 gene expression in mammals, such as humans. The present invention also provides a composition comprising an iRNA, such as a dsRNA that targets the Serpinc1 gene in mammalian cells, for use in inhibiting Serpinc1 gene expression in mammals. In another embodiment, the present invention provides the use of an iRNA, such as a dsRNA that targets the Serpinc1 gene in mammalian cells, for producing a drug that inhibits Serpinc1 gene expression in mammals.

[0330] The method and use include the step of administering a composition comprising an iRNA, such as a dsRNA that targets the Serpinc1 gene in mammalian cells, to a mammal, such as a human, to obtain degradation of the mRNA transcript of the Serpinc1 gene, thereby maintaining the mammal for a sufficient time to inhibit Serpinc1 gene expression in the mammal.

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

[0332] The present invention further provides a method for treating subjects having disorders that benefit from reduced Serpinc1 expression, such as hemophilia. The treatment (and use) of the present invention comprises the step of administering a therapeutically effective amount of an iRNA that targets the Serpinc1 gene, or a pharmaceutical composition comprising an iRNA that targets the Serpinc1 gene, to a subject, for example, a human, thereby treating the subject having a disorder that benefits from reduced Serpinc1 expression.

[0333] In one embodiment, the present invention provides a method for preventing at least one symptom in subjects having a disorder that benefits from reduced Serpinc1 expression. The method includes the step of administering a therapeutically effective amount of an iRNA or vector, such as the dsRNA of the present invention, to a subject, thereby preventing at least one symptom in a subject having a disorder that benefits from reduced Serpinc1 expression. For example, the present invention provides a method for preventing bleeding in subjects suffering from a disorder that benefits from reduced Serpinc1 expression, such as hemophilia.

[0334] In another embodiment, the present invention provides the use of therapeutically effective amounts of the iRNA of the present invention for treating subjects such as those who would benefit from reduced and / or inhibition of Serpinc1 expression. Examples of iRNAs include iRNAs that target the Serpinc1 gene, or pharmaceutical compositions comprising iRNAs that target the Serpinc1 gene.

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

[0336] In another aspect, the present invention provides the use of an iRNA, such as the dsRNA of the present invention, to prevent at least one symptom in subjects suffering from disorders that benefit from reduced and / or inhibition of Serpinc1 expression, such as bleeding disorders like hemophilia.

[0337] In a further embodiment, the present invention provides the use of the iRNA of the present invention in the manufacture of a drug for preventing at least one symptom in subjects suffering from disorders that benefit from reduced and / or inhibition of Serpinc1 expression, such as bleeding disorders like hemophilia. The iRNA of the present invention may be administered in “naked” or “free iRNA” form. Naked iRNA is administered in the absence of a pharmaceutical composition. Naked iRNA may be in a suitable buffer solution. The buffer solution may contain acetate, citrate, prolamin, carbonate, or phosphoric acid, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and molar osmotic concentration of the buffer solution containing the iRNA may be adjusted to suit administration to the subject.

[0338] Alternatively, the iRNA of the present invention may be administered as a pharmaceutical composition such as a dsRNA liposome formulation.

[0339] Subjects who would benefit from reduced and / or inhibition of Serpinc1 gene expression are those with bleeding disorders, such as hereditary or acquired bleeding disorders as described herein. In one embodiment, subjects with hereditary bleeding disorders have hemophilia, such as hemophilia A, B, or C. In one embodiment, subjects with hereditary bleeding disorders, such as hemophilia, are inhibitor subjects. 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 inhibition of Serpinc1 gene expression include therapeutic measures (e.g., on-demand, such as when the subject is bleeding (spontaneous bleeding or bleeding as a result of trauma) and unable to form blood clots), and preventive measures (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 iRNA or a pharmaceutically acceptable composition thereof to treat subjects who would benefit from reduced and / or inhibition of Serpinc1 expression, such as subjects with bleeding disorders, in combination with other pharmaceuticals and / or therapeutics, such as known pharmaceuticals and / or known therapies, such as those currently used to treat these disorders. For example, in certain embodiments, an iRNA targeting Serpinc1 is administered in combination with a drug useful for treating bleeding disorders, for example, as described elsewhere in this specification. Suitable additional therapeutic agents and treatments for treating subjects who would benefit from reduced Serpinc1 expression (reducton), 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 in combination with immunosuppression with or without antifibrinolytic therapy and infusion of FVIII or FIX; immune tolerance induction (ITI) with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and / or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytic agents such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophiliacs; corticosteroids; immunosuppressants; and estrogens. iRNA and additional therapeutic agents and / or treatments may be administered, for example, parenterally simultaneously and / or in the same combination, or the additional therapeutic agents may be administered as part of another composition, or at a different time, and / or by other methods known in the art or described herein.

[0341] In one embodiment, the method and use include the step of administering a composition of this specification such that the expression of the target Serpinc1 gene is reduced over a period of time such as 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, the expression of the target Serpinc1 gene is reduced over a longer period of time such as at least about 2, 3, 4, 5, 6, or 7 days, for example, about 1 week, 2 weeks, 3 weeks, or about 4 weeks or more.

[0342] Preferably, the iRNAs useful in the methods, uses, and compositions described herein specifically target the (primary or processed) RNA of the target Serpinc1 gene. Compositions, uses, and methods using iRNAs to inhibit the expression of these genes can be prepared and carried out as described herein.

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

[0344] The efficacy of treating or preventing a disease can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, bleeding incidence, pain reduction, quality of life, drug dose required to maintain therapeutic effect, disease marker levels, or any other measurable parameter levels appropriate to a given disease that are being treated or are the target of prevention. Monitoring therapeutic or preventive efficacy by measuring any one of these parameters, or any combination of parameters, is well within the capabilities of those skilled in the art. For example, the efficacy of treating bleeding disorders may be evaluated by periodically monitoring, for example, thrombin:antithrombin levels. A comparison of initial and subsequent readings provides the physician with an indicator of whether the treatment is effective. Monitoring therapeutic or preventive efficacy by measuring any one of these parameters, or any combination of parameters, is well within the capabilities of those skilled in the art. In relation to the administration of iRNA or pharmaceutical compositions targeting Serpinc1, "effective against" bleeding disorders means that administration in a clinically appropriate manner results in beneficial effects in at least a statistically significant proportion of patients, such as symptom improvement, cure, disease reduction, life extension, improved quality of life, or other effects generally recognized as favorable by physicians familiar with the treatment of bleeding disorders and related causes.

[0345] The 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 no expected worsening or onset of symptoms without treatment. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, or 50% or more, in the measurable parameters of the disease may suggest an effective treatment. The efficacy of a given iRNA agent or a formulation of that agent can also be determined using experimental animal models for a given disease known in the art. When using experimental animal models, the efficacy of the treatment is demonstrated when a statistically significant reduction in markers or symptoms is observed.

[0346] Alternatively, efficacy may be assessed by a diagnostician skilled in the art based on a clinically recognized disease severity assessment scale, such as the Child-Pugh score (sometimes also known as the Child-Turcotte-Pugh score), by a reduction in disease severity. Any favorable change resulting in a reduction in disease severity, as assessed using an appropriate scale, indicates appropriate treatment with the iRNA or iRNA formulation described herein.

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

[0348] For example, in a specific embodiment in which the composition of the present invention comprises the dsRNA described herein and lipids, the target range is approximately 0.01 mg / kg to approximately 5 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.05 mg / kg to approximately 5 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.2 mg / kg to approximately 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 Therapeutic doses of iRNA can be administered in amounts such as approximately 5.5 mg / kg to 10 mg / kg, approximately 6 mg / kg to 10 mg / kg, approximately 6.5 mg / kg to 10 mg / kg, approximately 7 mg / kg to 10 mg / kg, approximately 7.5 mg / kg to 10 mg / kg, approximately 8 mg / kg to 10 mg / kg, approximately 8.5 mg / kg to 10 mg / kg, approximately 9 mg / kg to 10 mg / kg, or approximately 9.5 mg / kg to 10 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0349] For example, dsRNA is approximately 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 It may be administered in doses of 0.2, 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0350] For example, in another embodiment in which the composition of the present invention comprises the dsRNA described herein and N-acetylgalactosamine, the target range is approximately 0.1 to approximately 50 mg / kg, approximately 0.25 to approximately 50 mg / kg, approximately 0.5 to approximately 50 mg / kg, approximately 0.75 to approximately 50 mg / kg, approximately 1 to approximately 50 mg / mg, approximately 1.5 to approximately 50 mg / kb, approximately 2 to approximately 50 mg / kg, approximately 2.5 to approximately 50 mg / kg, approximately 3 to approximately 50 mg / kg, approximately 3.5 to approximately 50 mg / kg, approximately 4 to approximately 50 mg / kg, approximately 4.5 to approximately 50 mg / kg, approximately 5 to approximately 50 mg / kg, and approximately 7.5 to approximately 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 can be administered, such as doses of approximately 0.1 to 20 mg / kg, approximately 0.25 to 20 mg / kg, approximately 0.5 to 20 mg / kg, approximately 0.75 to 20 mg / kg, approximately 1 to 20 mg / kg, approximately 1.5 to 20 mg / kg, approximately 2 to 20 mg / kg, approximately 2.5 to 20 mg / kg, approximately 3 to 20 mg / kg, approximately 3.5 to 20 mg / kg, approximately 4 to 20 mg / kg, approximately 4.5 to 20 mg / kg, approximately 5 to 20 mg / kg, approximately 7.5 to 20 mg / kg, approximately 10 to 20 mg / kg, or approximately 15 to 20 mg / kg. In one embodiment, if the composition of the present invention contains the dsRNA and N-acetylgalactosamine described herein, a therapeutic dose of approximately 10 to 30 mg / kg of dsRNA may be administered to the subject. The intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of this invention.

[0351] For example, the target values ​​are approximately 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, 2 Therapeutic doses 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 approximately 50 mg / kg may be administered. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0352] iRNA can be administered by intravenous infusion over 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or over approximately 25 minutes. Administration may be repeated regularly, weekly or bi-weekly (i.e., every two weeks), for a period of, for example, one month, two months, three months, four months or more. After the initial treatment plan, treatment may be performed at a lower frequency. For example, after weekly or bi-weekly administration for three months, monthly administration may be repeated for six months or more than one year.

[0353] In one embodiment, the present invention provides a method for treating a subject suffering from a bleeding disorder, such as hemophilia, by subcutaneously administering compound AD-57213 to the subject 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 a step of subcutaneous administration targeting a cumulative weekly dose of approximately 0.5 mg / kg. For example, in one embodiment, the method may include a step of administration targeting a cumulative weekly dose of 0.5 mg / kg, at approximately 0.5 mg / kg per week. In another embodiment, the method may include a step of administration targeting a cumulative weekly dose of 0.5 mg / kg, at 1 mg / kg every other week.

[0355] In another embodiment, the method may include a step of subcutaneous administration for a cumulative weekly dose of approximately 1.5 mg / kg. For example, in one embodiment, the method may include a step of administration for a cumulative weekly dose of 1.5 mg / kg, at approximately 1.5 mg / kg per week. In another embodiment, the method may include a step of administration for a cumulative weekly dose of 1.5 mg / kg, at 3 mg / kg every other week.

[0356] In another embodiment, the method may include a step of subcutaneous administration targeting a cumulative weekly dose of approximately 2 mg / kg. For example, in one embodiment, the method may include a step of administration targeting a cumulative weekly dose of 2 mg / kg, at approximately 2 mg / kg per week. In another embodiment, the method may include a step of administration targeting a cumulative weekly dose of 2 mg / kg, at 4 mg / kg every other week.

[0357] In another embodiment, the method may include a step of subcutaneous administration targeting a cumulative weekly dose of approximately 3 mg / kg. For example, in one embodiment, the method may include a step of administration targeting a cumulative weekly dose of 3 mg / kg, at approximately 3 mg / kg per week. In yet another embodiment, the method may include a step of administration targeting 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 for preventing at least one symptom of a bleeding disorder, such as hemophilia, in a subject by subcutaneously administering compound AD-57213 to the subject 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 a step of subcutaneous administration targeting a cumulative weekly dose of approximately 0.5 mg / kg. For example, in one embodiment, the method may include a step of administration targeting a cumulative weekly dose of 0.5 mg / kg, at approximately 0.5 mg / kg per week. In another embodiment, the method may include a step of administration targeting a cumulative weekly dose of 0.5 mg / kg, at 1 mg / kg every other week.

[0360] In another embodiment, the method may include a step of subcutaneous administration for a cumulative weekly dose of approximately 1.5 mg / kg. For example, in one embodiment, the method may include a step of administration for a cumulative weekly dose of 1.5 mg / kg, at approximately 1.5 mg / kg per week. In another embodiment, the method may include a step of administration for a cumulative weekly dose of 1.5 mg / kg, at 3 mg / kg every other week.

[0361] In another embodiment, the method may include a step of subcutaneous administration targeting a cumulative weekly dose of approximately 2 mg / kg. For example, in one embodiment, the method may include a step of administration targeting a cumulative weekly dose of 2 mg / kg, at approximately 2 mg / kg per week. In another embodiment, the method may include a step of administration targeting a cumulative weekly dose of 2 mg / kg, at 4 mg / kg every other week.

[0362] In another embodiment, the method may include a step of subcutaneous administration targeting a cumulative weekly dose of approximately 3 mg / kg. For example, in one embodiment, the method may include a step of administration targeting a cumulative weekly dose of 3 mg / kg, at approximately 3 mg / kg per week. In yet another embodiment, the method may include a step of administration targeting a cumulative weekly dose of 3 mg / kg, at 6 mg / kg every other week.

[0363] iRNA administration raises Serpinc1 levels to at least approximately 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, 44, 45, 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 approximately 99% or more.

[0364] In one embodiment, a therapeutic and / or prophylactic method involves subcutaneously administering compound AD-57213 to a subject in a dose sufficient to inhibit reduce Serpinc1 levels in, for example, the patient's cells, tissues, blood, urine, or other compartments 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] Before administering the total dose of iRNA, a smaller dose, such as a 5% infusion response, may be administered to the patient to monitor for adverse effects such as allergic reactions. In another embodiment, the patient may be monitored for undesirable 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 those generally understood by those skilled in the art to which this invention pertains. Similar or equivalent methods and materials may be used in carrying out or testing the iRNAs and methods discussed herein, but suitable methods and materials are as listed below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are intended for illustrative purposes only and not as limitations. [Examples]

[0368] Example 1: iRNA synthesis Reagent supplier Unless otherwise specified herein, such reagents may be obtained from any supplier of molecular biology reagents meeting quality / purity standards for molecular biology applications.

[0369] Transfer siRNA designs were 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 designs used the following transcripts from the NCBIRefSeq 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 double-stranded compounds were designed in several separate batches, including, but not limited to, batches containing only double-stranded human and rhesus monkey transcripts; only human, rhesus monkey, and dog transcripts; only human, rhesus monkey, mouse, and rat transcripts; and only mouse and rat transcripts. All siRNA double-stranded compounds were designed to share 100% identity with the enumerated human and other species transcripts considered in each design batch (above).

[0370] siRNA design, specificity, and efficacy prediction The predicted specificity of all possible 19-mers was predicted from each sequence. Next, candidate 19-mers lacking repeats longer than 7 nucleotides were selected. These 874 candidate human / rhesus monkey, 67 human / rhesus monkey / dog, 103 human / rhesus monkey / mouse / rat, and 569 mouse / rat siRNAs were used in a comprehensive search against appropriate transcriptomes (defined as the NM_ and XM_records sets within the human, rhesus monkey, 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 alignments and generated scores based on the placement and number of mismatches between the siRNA and any possible "off-target" transcripts. Off-target scores were weighted to highlight differences within the "seed" region of the siRNAs at positions 2–9 from the 5' end of the molecule.

[0371] Each oligotranscript pair from a brute-force attack search was assigned a mismatch score by adding its individual mismatch score; mismatches at positions 2–9 were considered 2.8, mismatches at cleavage sites at positions 10–11 were considered 1.2, and mismatches in region 12–19 were considered 1.0. Off-target prediction was further performed by comparing the occurrence frequencies of heptamers and octamers derived from three different seed-derived hexamers for each oligo. Two heptamers and one octamer were created using hexamers from positions 2–7 relative to the 5' start point. "Hepamer 1" was created by adding 3'-A to the hexamer; heptamer 2 was created by adding 5'-A to the hexamer; and the octamer was created by adding A to both the 5' and 3' ends of the hexamer. The occurrence frequencies of octamers and heptamers in human, rhesus monkey, mouse, or rat 3'UTRome (defined as a transcriptome subsequence from the NCBI Refseq database, with the coding region terminus "CDS" clearly defined) were pre-calculated. The octamer occurrence frequency was normalized for the heptamer occurrence frequency using the median from the octamer frequency range. The "mirSeedScore" was then calculated by summing ((3 × normalized number of octamers) + (2 × number of heptamers) + (1 × number of heptamers)).

[0372] Based on the calculated scores, both siRNA strands were assigned to specificity categories. Scores greater than 3 were classified as highly specific, scores equal to 3 as specific, and scores between 2.2 and 2.8 as moderately specific. The double strands were sorted by the specificity of the antisense strand to select those in which the antisense oligo lacked a GC at position 1, lacked Gs at both positions 13 and 14, and had three or more U or A in the seed region.

[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 formed into double strands. A detailed list of 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 in 0.2–500 μmol scales according to a standard solid-phase oligonucleotide synthesis protocol using commercially available uridine 5'-O-(4,4'-dimethoxytrityl)-2'-Ot-butyldimethylsilyl-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite monomers, 4-N-acetylcytidine, 6-N-benzoyladenosine, and 2-N-isobutyrylguanosine, and their corresponding 2'-O-methyl and 2'-fluorophosphoramidites. 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 an activator. For the oxidation step, modifications to the phosphorothioate backbone 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 sequence was cleaved from the solid support and deprotected with methylamine followed by triethylamine·3HF to remove any present 2'-Ot-butyldimethylsilyl protecting groups.

[0375] For the synthesis of fully modified sequences (2'-fluoro and / or 2'-O-methyl or combinations thereof) in 5–500 μmol scales, 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 on a solid support with 0.5 M piperidine in acetonitrile for 20 minutes. 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. The product-containing fraction of appropriate purity was stored and concentrated on a rotary evaporator prior to desalting. The sample was desalted by size exclusion chromatography and lyophilized. Equimolar amounts of sense and antisense strands were annealed in 1×PBS buffer to prepare the corresponding siRNA double strands.

[0376] For small-scale (0.2–1 μmol) synthesis, the synthesis was carried out on a 96-well MerMade 192 synthesizer. For fully 2'-modified sequences (2'-fluoro and / or 2'-O-methyl or a combination thereof), the oligonucleotides were deprotected using methylamine at room temperature for 30–60 minutes, followed by incubation at 60°C for 30 minutes, or using 3:1 (v / v) ethanol and concentrated (28–32%) aqueous ammonia at room temperature for 30–60 minutes, followed by incubation at 40°C for 1.5 hours. The crude oligonucleotides were then precipitated in acetonitrile:acetone (9:1) solution, isolated by centrifugation, and the supernatant was removed by gradient osmosis. 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 each individual sequence was collected in each well. These purified desalted oligonucleotides were analyzed by LC-MS and anion exchange chromatography. Double-stranded oligonucleotides were prepared by annealing equimolar amounts of sense and antisense sequences on a Tecan robot. The concentration of the double-stranded oligonucleotides 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 ligand were synthesized in quantities ranging from 0.2 to 500 μmol using a solid support pre-loaded with a Y-type linker possessing a 4,4'-dimethoxytrityl (DMT) protected primary hydroxyl group for oligonucleotide synthesis and a GalNAc ligand attached via anchoring.

[0378] For GalNAc complex synthesis on a 5–500 μmol scale, the above RNA synthesis protocol 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 more than 5 phosphorothioates) were synthesized without removing the last 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. Fractions containing product of appropriate purity were stored and concentrated on a rotary evaporator. DMT groups were removed until complete using 20–25% acetic acid in water. The samples were desalted by size exclusion chromatography and lyophilized. Equimolar amounts of sense and antisense strands were annealed in 1×PBS buffer to prepare the corresponding siRNA double-stranded molecules.

[0379] For the small-scale synthesis of GalNAc complexes (0.2–1 μmol) containing sequences with multiple phosphorothioate bonds, the protocol described above for the synthesis of RNA or complete 2'-F / 2'-OMe-containing sequences on the MerMade platform was applied. 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 translocation Hep3B cells (ATCC, Manassas, VA) were cultured to near confluence in Eagle's Minimum Essential Medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C and in a 5% CO2 atmosphere, and then released from the plate by trypsin treatment. For mouse cross-reactive double-stranded siRNA, fresh primary cultured mouse hepatocytes (PMH) were isolated within 1 hour prior to translocation and cultured in primary cultured hepatocyte medium. Translocation 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 no. 13778-150) to each 5 μl siRNA double-stranded siRNA per well. The mixture was then incubated at room temperature for 15 minutes. Next, approximately 2 × 10⁶ 4 80 μl of antibiotic-free complete growth medium containing 10 Hep3B cells was added to the siRNA mixture. The cells were cultured for 24 hours prior to RNA purification. Single-dose experiments were performed at final double-strand concentrations of 10 nM and 0.1 nM, and dose-response experiments were conducted using 8 × 5-fold serial dilutions in the range of 10 nM to 128 pM (see Figures 2A and 2B).

[0381] Free uptake of traits In each well of a 96-well plate, 4 × 10¹⁶ molecules were resuspended in 95 μl of In Vitro Gro CP medium (In Vitro Technologies-Celsis, Baltimore, MD). 4 Freshly thawed, cryopreserved cynomolgus monkey liver parenchyma cells were combined with 5 μl of each GalNac-coupled siRNA in PBS. The mixture was cultured at 37°C and in a 5% CO2 atmosphere for approximately 24 hours. The 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 the DYNABEADS mRNA isolation kit (Invitrogen; part number 610-12) Cells were collected and dissolved in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing rate was consistent throughout the process). 10 μl of magnetic beads and 80 μl of the lysis / binding buffer mixture were placed in 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 washing buffer A and mixed for 1 minute. The beads were captured again and the supernatant was removed. Next, the beads were washed with 150 μl of washing buffer B, captured, and the supernatant was removed. Next, the beads were 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 at 5°C for 70 minutes. The beads were captured on a magnet for 5 minutes. 40 μl of supernatant was removed and placed in another 96-well plate.

[0383] cDNA synthesis using the ABI high-volume cDNA reverse transcription kit (Applied Biosystems, Foster City, CA; catalog number 4368813) For each reaction, 10 μl of total RNA was detached to a master mixture consisting 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 H2O. cDNA was prepared using a Bio-RadC-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C, and retention at 4°C.

[0384] Real-time PCR Each well of a 384-well plate contained a master mixture containing 0.5 μl of human GAPDH TaqMan probe (Applied Biosystems; catalog number 4326317E), 0.5 μl of human SERPINC1 TaqMan probe (Applied Biosystems; catalog number Hs00892758_m1) for human cells, or 0.5 μl of mouse GAPDH TaqMan probe (Applied Biosystems; catalog number 4308313), 0.5 μl of mouse SERPINC1 TaqMan probe (Applied Biosystems; catalog number Mm00446573_m1) for mouse cells, and 5 μl of Lightcycler 480 probe master mixture (Roche; catalog number 04887301001), to which 2 μl of cDNA was added. Real-time PCR was performed in an ABI7900HT real-time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each double-stranded cell was tested with two independent transfusions, and unless otherwise noted in the summary table, each transfusion was assayed with replicat...

Claims

1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent for prophylactic use to prevent or reduce the frequency of bleeding in patients with hemophilia A or hemophilia B who have or do not have inhibitors, Here, the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprises a complementary region to the mRNA encoding Serpinc1, and here the complementary region comprises at least 21 consecutive nucleotides that differ by one nucleotide or less from the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562), Here, the antisense chain comprises at least one phosphorothioate nucleoside bond and at least one nucleotide 3' overhang. Here, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides. Here, the nucleotide modification is selected from the group consisting of 2'-O-methyl (2'-OMe) and 2'-fluoronucleotide modifications, and Here, a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to the 3' end of the sense strand of the dsRNA agent. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein each chain has a length of 30 nucleotides or less.

3. The pharmaceutical composition according to claim 1, wherein the double-stranded region is 17-25 nucleotide pairs long.

4. The pharmaceutical composition according to claim 1, wherein the double-stranded region is 21-23 nucleotide pairs long.

5. The pharmaceutical composition according to claim 1, wherein the double-stranded region is 17-23 nucleotide pairs long.

6. The pharmaceutical composition according to claim 1, wherein the double-stranded region has a length of 23-27 nucleotide pairs.

7. The pharmaceutical composition according to claim 1, wherein the double-stranded region is 19-21 nucleotide pairs long.

8. The pharmaceutical composition according to claim 1, wherein each chain has a length of approximately 19-25 nucleotides.

9. The pharmaceutical composition according to claim 1, wherein each chain has a length of 21-23 nucleotides.

10. The pharmaceutical composition according to claim 1, wherein the sense chain is 21 nucleotides long and the antisense chain is 23 nucleotides long.

11. The pharmaceutical composition according to claim 1, wherein the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562).

12. The pharmaceutical composition according to claim 1, wherein the sense strand comprises the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 294), and the antisense strand comprises the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562).

13. The pharmaceutical composition according to claim 1, wherein the dsRNA agent comprises a sense strand having the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 294) and an antisense strand having the nucleotide sequence 5'-UUGAAGUAAAAUGGGUGUUAACCAG-3' (SEQ ID NO: 562).

14. The ligand 【Chemistry 1】 The pharmaceutical composition according to claim 1.

15. The dsRNA agent is shown in the following schematic diagram. 【Chemistry 2】 The pharmaceutical composition according to claim 1, conjugated with the ligand as shown in the formula (wherein X is O or S).

16. The pharmaceutical composition according to claim 1, wherein X is O in the formula.

17. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent for prophylactic use to prevent or reduce the frequency of bleeding in patients with hemophilia A or hemophilia B who have or do not have inhibitors, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941), and the antisense strand comprising the nucleotide sequence 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 960), A pharmaceutical composition in which a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, and U; and s is a phosphorothioate bond.

18. The pharmaceutical composition according to claim 17, further comprising a ligand.

19. The pharmaceutical composition according to claim 18, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA.

20. The pharmaceutical composition according to claim 18, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

21. The ligand The pharmaceutical composition according to claim 20.

22. The aforementioned dsRNA is shown in the following schematic diagram. 【Transformation 3】 The pharmaceutical composition according to claim 20, conjugated with the ligand as shown in the formula (wherein X is O or S).

23. The pharmaceutical composition according to claim 22, wherein X is O in the formula.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the dsRNA agent is in a free acid form.

25. The pharmaceutical composition according to any one of claims 1 to 23, wherein the dsRNA agent is in salt form.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the pharmaceutical composition comprises phosphate-buffered saline (PBS).

27. The pharmaceutical composition according to any one of claims 1 to 25, wherein the pharmaceutical composition prevents bleeding.

28. The pharmaceutical composition according to any one of claims 1-25, wherein the patient is a hemophilia A patient with an inhibitor.

29. The pharmaceutical composition according to any one of claims 1 to 25, wherein the patient is a hemophilia A patient without inhibitors.

30. The pharmaceutical composition according to any one of claims 1 to 25, wherein the patient is a hemophilia B patient with an inhibitor.

31. The pharmaceutical composition according to any one of claims 1 to 25, wherein the patient is a hemophilia B patient without inhibitors.

32. The pharmaceutical composition according to any one of claims 1 to 25, wherein administration of the pharmaceutical composition to the patient causes increased blood coagulation and / or decreased Serpinc1 protein accumulation.

33. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is administered to the patient once a month.

34. The pharmaceutical composition according to any one of claims 1 to 25, wherein the patient is a hemophilia A patient.

35. The pharmaceutical composition according to any one of claims 1 to 25, wherein the patient is a hemophilia B patient.

36. The pharmaceutical composition according to claim 34, wherein the patient is a hemophilia A patient with an inhibitor.

37. The pharmaceutical composition according to claim 35, wherein the patient is a hemophilia B patient with an inhibitor.

38. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent for prophylactic use to prevent or reduce the frequency of bleeding in patients with hemophilia A or hemophilia B who have or do not have inhibitors, wherein the dsRNA consists of a sense strand and an antisense strand. Here, the sequence of the sense strand is 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96-3' (SEQ ID NO: 941), and the sequence of the antisense strand is 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 960), A pharmaceutical composition in which a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, and U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol.

39. The pharmaceutical composition according to claim 38, wherein the dsRNA agent is in the form of a free acid.

40. The pharmaceutical composition according to claim 38, wherein the dsRNA agent is in salt form.

41. The pharmaceutical composition according to any one of claims 38-40, wherein administration of the dsRNA to the patient causes increased blood coagulation and / or decreased Serpinc1 protein accumulation.

42. The pharmaceutical composition according to any one of claims 38-40, wherein the patient is a hemophilia A patient.

43. The pharmaceutical composition according to claim 42, wherein the patient is a hemophilia A patient with an inhibitor.

44. The pharmaceutical composition according to claim 42, wherein the patient is a hemophilia A patient without inhibitors.

45. The pharmaceutical composition according to any one of claims 38-40, wherein the patient is a hemophilia B patient.

46. The pharmaceutical composition according to claim 45, wherein the patient is a hemophilia B patient with an inhibitor.

47. The pharmaceutical composition according to claim 45, wherein the patient is a hemophilia B patient without inhibitors.

48. A method for producing a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of Serpinc1, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a complementary region to the mRNA encoding Serpinc1, wherein the complementary region comprises at least 21 consecutive nucleotides that differ by one or less nucleotides from the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562), and the antisense strand comprising at least one phosphorothioate nucleoside bond and at least one nucleotide 3' overhang. Here, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides. The nucleotide modification is selected from the group consisting of 2'-O-methyl (2'-OMe) and 2'-fluoronucleotide modifications. The aforementioned method: (a) synthesizing the sense strand of the dsRNA; (b) synthesizing the antisense strand of the dsRNA; and (c) Annealing the sense strand and the antisense strand to produce the dsRNA agent, A manufacturing method comprising the following.

49. The method according to claim 48, wherein each chain is 30 nucleotides or less in length.

50. The method according to claim 48, wherein the double-stranded region is 17-25 nucleotide pairs long.

51. The method according to claim 48, wherein each chain is 19-25 nucleotides long.

52. The method according to claim 48, wherein each chain has a length of 21-23 nucleotides.

53. The method according to claim 48, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

54. The method according to claim 48, wherein the complementary region consists of the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562).

55. The method according to claim 48, wherein the sense strand comprises the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 294), and the antisense strand comprises the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562).

56. The method according to claim 55, wherein the sense strand comprises the nucleotide sequence 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 294), and the antisense strand comprises the nucleotide sequence 5'-UUGAAGUAAAAUGGUGUUAACCAG-3' (SEQ ID NO: 562).

57. The sense chain comprises 5'-GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 956), and the antisense chain comprises 5'-uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg-3' (SEQ ID NO: 975), The method according to claim 55, wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, and U; and s is a phosphorothioate bond.

58. The sense chain comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO: 941), and the antisense chain comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO: 960), The method according to claim 55, wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, and U; and s is a phosphorothioate bond.

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