SERPINC1 iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

The invention relates to iRNA, e.g., double-stranded ribonucleic acid (dsRNA), compositions targeting the Serpinc1 gene, and methods of using such iRNA, e.g., dsRNA, compositions to inhibit expression of Serpinc1 and methods of treating subjects having a bleeding disorder, such as a hemophilia.

Description

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 14/806,084, filed Jul. 22, 2015, which is a continuation of U.S. patent application Ser. No. 13/837,129, filed Mar. 15, 2013, which claims priority to U.S. Provisional Application No. 61/638,952, filed on Apr. 26, 2012, to U.S. Provisional Application No. 61/669,249, filed on Jul. 9, 2012, and to U.S. Provisional Application No. 61/734,573, filed on Dec. 7, 2012. The entire contents of each of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 3, 2016, is named Seq_Listing_121301_00206.txt and is 458,227 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Serpinc1 is a member of the serine proteinase inhibitor (serpin) 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, regulates the blood coagulation cascade (see, e.g., FIG. 1). The anticoagulant activity of Serpinc1 is enhanced by the presence of heparin and other related glycosaminoglycans which catalyze the formation of a thrombin:antithrombin (TAT) complexes.

[0004] Bleeding disorders, either inherited or acquired, are conditions in which there is inadequate blood clotting. For example, hemophilia is a group of hereditary genetic bleeding disorders that impair the body's ability to control blood clotting or coagulation. Hemophilia A is a recessive X-linked genetic disorder involving a lack of functional clotting Factor VIII and represents 80% of hemophilia cases. Hemophilia B is a recessive X-linked genetic disorder involving a lack of functional clotting Factor IX. It comprises approximately 20% of haemophilia cases. Hemophilia C is an autosomal genetic disorder involving a lack of functional clotting Factor XI. Hemophilia C is not completely recessive, as heterozygous individuals also show increased bleeding.

[0005] Although, at present there is no cure for hemophilia, it can be controlled with regular infusions of the deficient clotting factor, e.g., factor VIII in hemophilia A. However, some hemophiliacs develop antibodies (inhibitors) against the replacement factors given to them and, thus, become refractory to replacement coagulation factor. Accordingly, bleeds in such subjects cannot be properly controlled.

[0006] The development of high-titer inhibitors to, for example, factor VIII and other coagulation factors, is the most serious complication of hemophilia therapy and makes treatment of bleeds very challenging. Currently, the only strategies to stop bleeds in such subjects are the use of "bypassing agents" such as factor eight inhibitor bypass activity (FEIBA) and activated recombinant factor VII (rFVIIa), plasmapheresis, continuous factor replacement, and immune tolerance therapy, none of which are completely effective. Accordingly, there is a need in the art for alternative treatments for subjects having a bleeding disorder, such as hemophilia.

SUMMARY OF THE INVENTION

[0007] The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a Serpinc1 gene. The Serpinc1 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using and uses of the iRNA compositions of the invention for inhibiting the expression of a Serpinc1 gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia.

[0008] Accordingly, in one aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of Serpinc1. The dsRNAs comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.

[0009] In another aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of Serpinc1. The dsRNAs comprise a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

[0010] In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of AD-50487.1, AD-50477.1, AD-50483.1, AD-50475.1, AD-50495.1, AD-50476.1, AD-50499.1, AD-50478.1, AD-50489.1, AD-50501.1, AD-50507.1, AD-50484.1, AD-50515.1, AD-50540.1, AD-50528.1, AD-50549.1, AD-50539.1, AD-50534.1, AD-50527.1, AD-50514.1, AD-50509.1, AD-50529.1, AD-54944, AD-56813, AD-57205, AD-57214, and AD-57213 of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21. In certain embodiments of the invention, the dsRNAs comprise at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

[0011] The region of complementarity of the dsRNAs may be at least 17 nucleotides in length, between 19 and 21 nucleotides in length, or 19 nucleotides in length.

[0012] In one embodiment, each strand of a dsRNA is no more than 30 nucleotides in length.

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

[0014] In certain embodiments, a 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 is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker. In particular embodiments, the ligand is

##STR00001##

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

##STR00002##

[0017] In another embodiment, the RNAi agent is conjugated to the ligand as shown in the following schematic, wherein X is O or S.

##STR00003##

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

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

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

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

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

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

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

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

[0026] In another aspect, the present invention provides methods of inhibiting Serpinc1 expression in a cell. The methods include contacting the cell with the dsRNA or a vector of the invention, and maintaining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of a Serpinc1 gene, thereby inhibiting expression of the Serpinc1 gene in the cell.

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

[0028] In one embodiment of the methods of the 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 aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in Serpinc1 expression, e.g., a bleeding disorder, such as a hemophilia. The methods include administering to the subject a therapeutically effective amount of the dsRNA or vector of the invention, thereby treating the subject.

[0030] In one aspect, the invention provides methods of preventing at least one symptom, e.g., bleeding, in a subject having a disorder that would benefit from reduction in Serpinc1 expression, e.g., hemophilia. The methods include administering to the subject a therapeutically effective amount of thei RNA, e.g., dsRNA, or vector of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in Serpinc1 expression.

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

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

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

[0034] In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/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 to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0035] For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

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

[0037] For example, subjects can be administered a therapeutic amount of iRNA, such as about 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, 21.5, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

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

[0039] In another aspect, the present invention provides methods of inhibiting the expression of Serpinc1 in a subject. The methods include administering to the subject a therapeutically effective amount of the dsRNA or a vector of the invention, thereby inhibiting the expression of Serpinc1 in the subject.

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

[0041] In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/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 to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0042] For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

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

[0044] For example, subjects can be administered a therapeutic amount of iRNA, such as about 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, 21.5, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 is a schematic of the blood coagulation cascade.

[0048] FIG. 2A is a graph showing the inhibition of Serpinc1 expression in Hep3B cells following a single dose of the indicated iRNAs.

[0049] FIG. 2B is a graph showing the inhibition of Serpinc1 expression in Hep3B cells following a single dose of the indicated iRNAs.

[0050] FIG. 3A is a graph showing the inhibition of Serpinc1 mRNA expression in CD-1 mice following a single dose, as indicated, of an LNP formulation of AD-50509 or AD-1955.

[0051] FIG. 3B is a graph showing the inhibition of Serpinc1 protein expression in CD-1 mice following a single dose, as indicated, of an LNP formulation of AD-50509 or AD-1955.

[0052] FIG. 4A is a graph showing the duration of inhibition of Serpinc1 mRNA expression in CD-1 mice following a single 1 mg/kg dose of an LNP formulation of AD-50509 or AD-1955.

[0053] FIG. 4B is a graph showing the duration of inhibition of Serpinc1 mRNA protein expression in CD-1 mice following a single 1 mg/kg dose of an LNP formulation of AD-50509 or AD-1955.

[0054] FIG. 4C is a graph showing the inhibition of Serpinc1 activity and Serpinc1 protein expression in CD1 mice following a single 1 mg/kg dose of an LNP formulation of AD-50509 or AD-1955.

[0055] FIG. 5 is a graph showing the percent knock-down of Serpinc1 mRNA and protein levels following a single 10 mg/kg dose of the indicated iRNA conjugated to GalNAc.

[0056] FIG. 6 is a graph showing the inhibition of Serpinc1 protein expression in C57BL/6 mice following a single 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg, and a repeat dose of 5.times.5 mg/kg of AD-54944 conjugated to GalNAc.

[0057] FIG. 7A is a graph showing the effect of repeat-dosing on the duration of inhibition of Serpinc1 protein expression in C57BL/6 mice of GalNAc conjugated AD-54944.

[0058] FIG. 7B is a graph showing the effect of repeat-dosing on the duration of inhibition of Serpinc1 protein expression in C57BL/6 mice of GalNAc conjugated AD-54944.

[0059] FIG. 8 is a graph showing the effects of the indicated split-dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL/6 mice administered GalNAc conjugated AD-54944.

[0060] FIG. 9 is a graph showing the effects of the indicated split-dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL/6 mice administered GalNAc conjugated AD-54944.

[0061] FIG. 10A is a graph showing the percent knock-down of Serpinc1 protein levels following a single 10 mg/kg dose of the indicated iRNA conjugated to GalNAc.

[0062] FIG. 10B is a graph showing the percent knock-down of Serpinc1 protein levels following a single 3 mg/kg dose of the indicated iRNA conjugated to GalNAc.

[0063] FIG. 11 is a graph showing the percent knock-down of Serpinc1 protein levels following a single 10 mg/kg or 3 mg/kg dose of the indicated iRNA conjugated to GalNAc.

[0064] FIG. 12 is a graph showing the percent knock-down of Serpinc1 activity following a single 10 mg/kg or 3 mg/kg dose of the indicated iRNA conjugated to GalNAc.

[0065] FIG. 13 is a graph showing a dose effect response to a single dose of AD-57213.

[0066] FIG. 14 is a graph showing the duration of silencing of Serpinc1 with AD-57213 following a single dose of 1 mg/kg, 3 mg/kg or 10 mg/kg in Hemophilia A mice.

[0067] FIG. 15 is a graph showing the inhibition of Serpinc1 mRNA expression in C57BL/6 mice following a single 30 mg/kg, 10 mg/kg, 3 mg/kg, 1 mg/kg, and 0.3 mg/kg dose of AD-57213.

[0068] FIG. 16A is a graph showing the duration of silencing of Serpinc1 with AD-57213 following a single dose as indicated.

[0069] FIG. 16B is a graph showing the duration of silencing of Serpinc1 with AD-57205 following a single dose as indicated.

[0070] FIG. 16C is a graph showing the duration of silencing of Serpinc1 with AD-57214 following a single dose as indicated.

[0071] FIG. 17 is a graph showing the effects of the indicated split-dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL/6 mice administered GalNAc conjugated AD-57213.

[0072] FIG. 18 is a graph showing the effects of the indicated split-dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL/6 mice administered GalNAc conjugated AD-57213.

[0073] FIG. 19 is a graph showing the effects of the indicated split-dosing regimens on the duration of silencing of Serpinc1 protein expression in C57BL/6 mice administered GalNAc conjugated AD-57213.

[0074] FIG. 20 is a graph showing the effects of the single dose screen of the indicated compounds on the duration of Serpinc1 protein expression in non-human primates.

[0075] FIG. 21 is a graph showing the effects of the single dose screen of AD-57213 conjugated to GalNAc on the duration of Serpinc1 protein expression in non-human primates.

[0076] FIG. 22 is a graph showing the effects of the single dose screen of the indicated compounds on the duration of Serpinc1 protein expression in non-human primates.

[0077] FIG. 23 is a graph showing the effects of the single dose of compound AD-57213 on serum antithrombin (Serpinc1) levels in non-human primates.

[0078] FIG. 24A is a graph showing the effects of the single dose of compound AD-57213 at 1 mg/kg on the relationship between serum antithrombin (Serpinc1) levels and fold change in peak plasma thrombin levels in non-human primates. Fold change in peak thrombin is depicted on the secondary y-axis (grey) and relative antithrombin level is depicted on the primary y-axis (black).

[0079] FIG. 24B is a graph showing the effects of the single dose of compound AD-57213 at 3 mg/kg on the relationship between serum antithrombin (Serpinc1) levels and fold change in peak plasma thrombin levels in non-human primates. Fold change in peak thrombin is depicted on the secondary y-axis (grey) and relative antithrombin level is depicted on the primary y-axis (black).

[0080] FIG. 24C is a graph showing the effects of the single dose of compound AD-57213 at 10 mg/kg on the relationship between serum antithrombin (Serpinc1) levels and fold change in peak plasma thrombin levels in non-human primates. Fold change in peak thrombin is depicted on the secondary y-axis (grey) and relative antithrombin level is depicted on the primary y-axis (black).

[0081] FIG. 24D is a graph showing the effects of the single dose of compound AD-57213 at 30 mg/kg on the relationship between serum antithrombin (Serpinc1) levels and fold change in peak plasma thrombin levels in non-human primates. Fold change in peak thrombin is depicted on the secondary y-axis (grey) and relative antithrombin level is depicted on the primary y-axis (black).

[0082] FIG. 25 is a graph showing the effects of AD-57213 as a fold change increase in peak thrombin as a function of relative antithrombin (Serpinc1) silencing.

[0083] FIG. 26A is a graph showing the cumulative effects of Serpinc1 silencing in non-human primates.

[0084] FIG. 26B is a graph showing the cumulative effects of Serpinc1 silencing in non-human primates.

[0085] FIG. 27A is a graph showing the effects of a multi-dose administration (1.5 mg/kg qw, 3 mg/kg q2w) of a Serpinc1 siRNA on serum antithrombin levels in non-human primates. Data points represent group mean, error bars represent standard deviation (N=3). (qw=weekly; q2w=every other week).

[0086] FIG. 27B is a graph showing the effects of a multi-dose administration (0.5 mg/kg qw, 1 mg/kg q2w) of a Serpinc1 siRNA on serum antithrombin levels in non-human primates. Data points represent group mean, error bars represent standard deviation (N=3). (qw=weekly; q2w=every other week).

[0087] FIG. 28A is a graph showing the effect of Serpinc1 silencing on platelet accumulation following microvessel laser injury. The graph shows the median values from all inflicted injuries.

[0088] FIG. 28B is a graph showing the effect of Serpinc1 silencing on fibrin area following microvessel laser injury. The graph shows the median values from all inflicted injuries.

[0089] FIG. 29 is a graph showing the duration of silencing of Serpinc1 following administration of compound AD-57213 formulated in a lipid nucleic acid particle.

[0090] FIG. 30A shows the nucleotide sequence of Homo sapiens serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) (SEQ ID NO:1); FIG. 30B shows the nucleotide sequence of Macaca mulatta serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) (SEQ ID NO:2); FIG. 30C shows the nucleotide sequence of Mus musculus serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1 (Serpinc1) (SEQ ID NO:3); FIG. 30D shows the nucleotide sequence of Rattus norvegicus serpin peptidase inhibitor, clade C (antithrombin), member 1 (Serpinc1) (SEQ ID NO:4); FIG. 30E shows the reverse complement of SEQ ID NO:1 (SEQ ID NO:5); FIG. 30F shows the reverse complement of SEQ ID NO:2 (SEQ ID NO:6); FIG. 30G shows the reverse complement of SEQ ID NO:3 (SEQ ID NO:7); FIG. 30H shows the reverse complement of SEQ ID NO:4 (SEQ ID NO:8); and FIG. 30I shows the amino acid sequence of an exemplary hydrophobic MTS-containing peptide, RFGF (SEQ ID NO: 9); the amino acid sequence of an exemplary RFGF analogue (SEQ ID NO: 10); the amino acid sequence of the HIV Tat protein (SEQ ID NO: 11); the amino acid sequence of the Drosophila Antennapedia protein (SEQ ID NO: 12); and the amino acid sequence of an exemplary Peptide-based Cleavable Linking Group.

[0091] FIG. 31A is a graph depicting that antithrombin reduction increases thrombin generation in Factor IX-depleted human plasma in vitro.

[0092] FIG. 31B is a graph depicting that antithrombin reduction increases thrombin generation in Factor IX-depleted human plasma in vitro.

DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a Serpinc1 gene. The Serpinc1 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a Serpinc1 gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia. The present invention further provides methods for preventing at least one symptom, e.g., bleeding, in a subject having a disorder that would benefit from inhibiting or reducing the expression of a Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia.

[0094] The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a Serpinc1 gene. The use of these iRNAs enables the targeted degradation of mRNAs of a Serpinc1 gene in mammals. Very low dosages of Serpinc1 iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of a Serpinc1 gene. The present inventors have demonstrated that iRNAs targeting Serpinc1 can mediate RNAi in vitro and in vivo, resulting in significant inhibition of expression of a Serpinc1 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of a Serpinc1 protein, such as a subject having a bleeding disorder, e.g., hemophilia.

[0095] The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a Serpinc1 gene, as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.

I. DEFINITIONS

[0096] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0097] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0098] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0099] The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

[0100] As used herein, "Serpinc1" refers to a particular polypeptide expressed in a cell. Serpinc1 is also known as serpin peptidase inhibitor, clade C (antithrombin), member 1; antithrombin III; AT3; antithrombin; and heparin cofactor 1. The sequence of a human Serpinc1 mRNA transcript can be found at, for example, GenBank Accession No. GI:254588059 (NM_000488; SEQ ID NO:1). The sequence of rhesus Serpinc1 mRNA can be found at, for example, GenBank Accession No. GI:157167169 (NM_001104583; SEQ ID NO:2). The sequence of mouse Serpinc1 mRNA can be found at, for example, GenBank Accession No. GI:237874216 (NM_080844; SEQ ID NO:3). The sequence of rat Serpinc1 mRNA can be found at, for example, GenBank Accession No. GI:58865629 (NM_001012027; SEQ ID NO:4).

[0101] The term "Serpinc1," as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the Serpinc1 gene, such as a single nucleotide polymorphism in the Serpinc1 gene. Numerous SNPs within the Serpinc1 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the Serpinc1 gene may be found at, NCBI dbSNP Accession Nos. rs677; rs5877; rs5878; rs5879; rs941988; rs941989; rs1799876; rs19637711; rs2008946; and rs2227586.

[0102] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a Serpinc1 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a Serpinc1 gene.

[0103] The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0104] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0105] "G," "C," "A," "T" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

[0106] The terms "iRNA", "RNAi agent," "iRNA agent,", "RNA interference agent" as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of Serpinc1 in a cell, e.g., a cell within a subject, such as a mammalian subject.

[0107] In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., a Serpinc1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down 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 the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a Serpinc1 gene. Accordingly, the term "siRNA" is also used herein to refer to an RNAi as described above.

[0108] In another embodiment, the RNAi agent may be a single-stranded siRNA that is introduced into a cell or organism to inhibit a target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150; 883-894.

[0109] In another aspect, the agent is a single-stranded antisense RNA molecule that inhibits a target via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense RNA molecule inhibits a target mRNA by hydridizing to the target and cleaving the target through an RNaseH cleavage event. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

[0110] In another embodiment, an "iRNA" for use in the compositions, uses, and methods of the invention is a double-stranded RNA and is referred to herein as a "double stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA". The term "dsRNA", refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations with respect to a target RNA, i.e., a Serpinc1 gene. In some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

[0111] The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0112] The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop." A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

[0113] Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a "linker." The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

[0114] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA.

[0115] In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3'-end and/or the 5'-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3'-end and/or the 5'-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

[0116] The terms "blunt" or "blunt ended" as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a "blunt ended" dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

[0117] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a Serpinc1 mRNA. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a Serpinc1 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5'- and/or 3'-terminus of the iRNA.

[0118] The term "sense strand," or "passenger strand" as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

[0119] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0120] Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as "fully complementary" for the purposes described herein.

[0121] "Complementary" sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0122] The terms "complementary," "fully complementary" and "substantially complementary" herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

[0123] As used herein, a polynucleotide that is "substantially complementary to at least part of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding Serpinc1). For example, a polynucleotide is complementary to at least a part of a Serpinc1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Serpinc1.

[0124] In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, an "iRNA" may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA molecule, are encompassed by "iRNA" for the purposes of this specification and claims.

[0125] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating," "suppressing" and other similar terms, and includes any level of inhibition.

[0126] The phrase "inhibiting expression of a Serpinc1," as used herein, includes inhibition of expression of any Serpinc1 gene (such as, e.g., a mouse Serpinc1 gene, a rat Serpinc1 gene, a monkey Serpinc1 gene, or a human Serpinc1 gene) as well as variants or mutants of a Serpinc1 gene that encodes a Serpinc1 protein.

[0127] "Inhibiting expression of a Serpinc1 gene" includes any level of inhibition of a Serpinc1 gene, e.g., at least partial suppression of the expression of a Serpinc1 gene, such as an inhibition by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0128] The expression of a Serpinc1 gene may be assessed based on the level of any variable associated with Serpinc1 gene expression, e.g., Serpinc1 mRNA level, Serpinc1 protein level, or, for example, thrombin:antithrombin complex levels as a measure of thrombin generation potential, bleeding time, prothrombin time (PT), platelet count, and/or activated partial thromboplastin time (aPTT). Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0129] In one embodiment, at least partial suppression of the expression of a Serpinc1 gene, is assessed by a reduction of the amount of Serpinc1 mRNA which can be isolated from or detected in a first cell or group of cells in which a Serpinc1 gene is transcribed and which has or have been treated such that the expression of a Serpinc1 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of

( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in control cells ) 100 % ##EQU00001##

[0130] The phrase "contacting a cell with an RNAi agent," such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0131] Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

[0132] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into the cell" by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

[0133] The term "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

[0134] The term "SNALP" refers to a stable nucleic acid-lipid particle. A SNALP is a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817, the entire contents of which are hereby incorporated herein by reference. Examples of "SNALP" formulations are described below.

[0135] As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in Serpinc1 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in Serpinc1 expression; a human having a disease, disorder or condition that would benefit from reduction in Serpinc1 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in Serpinc1 expression as described herein. As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms, diminishing the extent of bleeding, stabilized (i.e., not worsening) state of bleeding, amelioration or palliation of the bleeding, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0136] By "lower" in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.

[0137] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of a Sertpinc1 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with a such a disease, disorder, or condition, e.g., a symptom such as a bleed. The likelihood of developing a bleed is reduced, for example, when an individual having one or more risk factors for a bleed either fails to develop a bleed or develops a bleed with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

[0138] As used herein, the term "bleeding disorder" is a disease or disorder that results in poor blood clotting and/or excessive bleeding. A bleeding disorder may be an inherited disorder, such as a hemophilia or von Willebrand's disease, or an acquired disorder, associated with, for example, disseminated intravascular coagulation, pregnancy-associated eclampsia, vitamin K deficiency, an autoimmune disorder, inflammatory bowel disease, ulcerative colitis, a dermatologic disorder (e.g., psoriasis, pemphigus), a respiratory disease (e.g., asthma, chronic obstructive pulmonary disease), an allergic drug reaction, e.g., the result of medications, such as aspirin, heparin, and warfarin, diabetes, acute hepatitis B infection, acute hepatitis C infection, a malignancy or solid tumor (e.g., prostate, lung, colon, pancreas, stomach, bile duct, head and neck, cervix, breast, melanoma, kidney, and/or a hematologic malignancy). In one embodiment, an inherited bleeding disorder is a hemophilia, e.g., hemophilia A, B, or C. In one embodiment, a subject having an inherited bleeding disorder, e.g., a hemophilia, has developed inhibitors, e.g., alloantibody inhibitors, to replacement coagulation therapies and is referred to herein as an "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.

[0139] "Therapeutically effective amount," as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a bleeding disorder and bleeding, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0140] "Prophylactically effective amount," as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a bleeding disorder but not bleeding, e.g., a subject having a bleeding disorder and scheduled for surgery, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The "prophylactically effective amount" may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0141] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

[0142] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0143] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laureate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0144] The term "sample," as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject.

II. IRNAS OF THE INVENTION

[0145] Described herein are iRNAs which inhibit the expression of a Serpinc1 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a Serpinc1 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a bleeding disorder, e.g., an inherited bleeding disorder. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a Serpinc1 gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the Serpinc1 gene, the iRNA inhibits the expression of the Serpinc1 gene (e.g., a human, a primate, a non-primate, or a bird Sertpinc1 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

[0146] A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a Serpinc1 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0147] Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0148] Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0149] In some embodiments, the dsRNA is between about 15 and about 20 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a "part" of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

[0150] One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 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, 14-33, 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, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target Serpinc1 expression is not generated in the target cell by cleavage of a larger dsRNA.

[0151] A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA.

[0152] A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0153] iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

[0154] In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a Serpinc1gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0155] It will be understood that, although some of the sequences in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 are described as modified and/or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 that is un-modified, un-conjugated, and/or modified and/or conjugated differently than described therein.

[0156] The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and differing in their ability to inhibit the expression of a Serpinc1 gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

[0157] In addition, the RNAs provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 identify a site(s) in a Serpinc1 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a Serpinc1 gene.

[0158] While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a "window" or "mask" of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence "window" progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

[0159] Further, it is contemplated that for any sequence identified, e.g., in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

[0160] An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5'- or 3'-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of a Serpinc1 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a Serpinc1 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a Serpinc1 gene is important, especially if the particular region of complementarity in a Serpinc1 gene is known to have polymorphic sequence variation within the population.

III. MODIFIED IRNAS OF THE INVENTION

[0161] In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

[0162] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0163] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0164] Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.

[0165] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0166] In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0167] Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH.sub.2--NH--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2--[known as a methylene (methylimino) or MMI backbone], --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and --N(CH.sub.3)--CH.sub.2--CH.sub.2--[wherein the native phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0168] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2)..sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.

[0169] Other modifications include 2'-methoxy (2'-OCH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F) Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0170] An iRNA can also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. 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, J. L, ed. John Wiley & Sons, 1990, these 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, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0171] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0172] The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0173] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.

[0174] Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

IV. IRNAS CONJUGATED TO LIGANDS

[0175] Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (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), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0176] In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

[0177] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a 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 polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0178] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucoseamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0179] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0180] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-.kappa.B.

[0181] The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoseverin.

[0182] In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0183] Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

[0184] The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

[0185] In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0186] When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0187] A. Lipid Conjugates

[0188] In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[0189] A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

[0190] In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

[0191] In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

[0192] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

[0193] B. Cell Permeation Agents

[0194] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[0195] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0196] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0197] An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0198] A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a .alpha.-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0199] C. Carbohydrate Conjugates

[0200] In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0201] In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

##STR00004##

[0202] In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

##STR00005## ##STR00006## ##STR00007## ##STR00008##

[0203] Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

##STR00009##

when one of X or Y is an oligonucleotide, the other is a hydrogen.

[0204] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

[0205] D. Linkers

[0206] In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

[0207] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO.sub.2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0208] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0209] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0210] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

[0211] A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[0212] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

[0213] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations 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 about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0214] i. Redox Cleavable Linking Groups

[0215] In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (--S--S--). To determine if a candidate cleavable linking group is a suitable "reductively cleavable linking group," or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0216] ii. Phosphate-Based Cleavable Linking Groups

[0217] In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are --O--P(O)(ORk)-O--, --O--P(S)(ORk)-O--, --O--P(S)(SRk)-O--, --S--P(O)(ORk)-O--, --O--P(O)(ORk)-S--, --S--P(O)(ORk)-S--, --O--P(S)(ORk)-S--, --S--P(S)(ORk)-O--, --O--P(O)(Rk)-O--, --O--P(S)(Rk)-O--, --S--P(O)(Rk)-O--, --S--P(S)(Rk)-O--, --S--P(O)(Rk)-S--, --O--P(S)(Rk)-S--. Preferred embodiments are --O--P(O)(OH)--O--, --O--P(S)(OH)--O--, --O--P(S)(SH)--O--, --S--P(O)(OH)--O--, --O--P(O)(OH)--S--, --S--P(O)(OH)--S--, --O--P(S)(OH)--S--, --S--P(S)(OH)--O--, --O--P(O)(H)--O--, --O--P(S)(H)--O--, --S--P(O)(H)--O, --S--P(S)(H)--O--, --S--P(O)(H)--S--, --O--P(S)(H)--S--. A preferred embodiment is --O--P(O)(OH)--O--. These candidates can be evaluated using methods analogous to those described above.

[0218] iii. Acid Cleavable Linking Groups

[0219] In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula --C.dbd.NN--, C(O)O, or --OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

[0220] iv. Ester-Based Linking Groups

[0221] In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula --C(O)O--, or --OC(O)--. These candidates can be evaluated using methods analogous to those described above.

[0222] v. Peptide-Based Cleaving Groups

[0223] In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (--C(O)NH--). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula --NHCHRAC(O)NHCHRBC(O), where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

[0224] In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

##STR00010## ##STR00011##

when one of X or Y is an oligonucleotide, the other is a hydrogen.

[0225] In certain embodiments of the compositions and methods of the invention, a ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

[0226] In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):

##STR00012##

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P.sup.2A, P.sup.2B, P.sup.3A, P.sup.3B, P.sup.4A, P.sup.4B, P.sup.5A, P.sup.5B, P.sup.5C, T.sup.2A, T.sup.2B, T.sup.3A, T.sup.3B, T.sup.4A, T.sup.4B, T.sup.4A, T.sup.5B, T.sup.5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2, CH.sub.2NH or CH.sub.2O; Q.sup.2A, Q.sup.2B, Q.sup.3A, Q.sup.3B, Q.sup.4A, Q.sup.4B, Q.sup.5A, Q.sup.5B, Q.sup.5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO.sub.2, N(R.sup.N), C(R').dbd.C(R''), C.ident.C or C(O); R.sup.2A, R.sup.2B, R.sup.3A, R.sup.3B, R.sup.4A, R.sup.4B, R.sup.5A, R.sup.5B, R.sup.5C are each independently for each occurrence absent, NH, O, S, CH.sub.2, C(O)O, C(O)NH, NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO, CH.dbd.N--O,

##STR00013##

or heterocyclyl;

[0227] L.sup.2A, L.sup.2B, L.sup.3A, L.sup.3B, L.sup.4A, L.sup.4B, L.sup.5A, L.sup.5B, and L.sup.5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R.sup.a is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

##STR00014##

[0228] wherein L.sup.5A, L.sup.5B and L.sup.5C represent a monosaccharide, such as GalNAc derivative.

[0229] Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

[0230] Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 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; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

[0231] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0232] "Chimeric" iRNA compounds or "chimeras," in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up 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 wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0233] In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. DELIVERY OF AN IRNA OF THE INVENTION

[0234] The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a bleeding disorder) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

[0235] In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., 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). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

[0236] A. Vector Encoded iRNAs of the Invention

[0237] iRNA targeting the Serpinc1 gene 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 PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0238] The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

[0239] iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

[0240] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO.TM.). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0241] Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

[0242] Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

[0243] Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

[0244] Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

[0245] Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

[0246] Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

[0247] Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

[0248] The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

[0249] The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

V. PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

[0250] The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of a Serpinc1 gene, e.g. a bleeding disorder. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a Serpinc1 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.

[0251] For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0252] In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, 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/kg, about 1.5 to about 45 mg/kg, 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, 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/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 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/kg, about 1.5 to about 30 mg/kg, 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, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0253] For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0254] In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, 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 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, 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, 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/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 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 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, 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, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the dsRNA is administered at a dose of about 10 mg/kg to about 30 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0255] For example, subjects can be administered a therapeutic amount of iRNA, such as about 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, 21.5, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0256] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0257] In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bi-monthly.

[0258] The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

[0259] Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as a bleeding disorder that would benefit from reduction in the expression of Serpinc1. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, Hemophilia A mouse models and Hemohphilia B mouse models, e.g., mice containing a knock-out of a clotting factor gene, such as those described in Bolliger, et al. (2010) Thromb Haemost 103:1233-1238, Bi L, et al. (1995) Nat Genet 10: 119-21, Lin et al. (1997) Blood 90: 3962-6, Kundu et al. (1998) Blood 92: 168-74, Wang et al. (1997) Proc Natl Acad Sci USA 94: 11563-6, and Jin, et al. (2004) Blood 104:1733.

[0260] The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver). Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C.sub.1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

[0261] A. iRNA Formulations Comprising Membranous Molecular Assemblies

[0262] An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have 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 an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

[0263] A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

[0264] If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

[0265] Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et 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. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging RNAi agent preparations into liposomes.

[0266] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0267] Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0268] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0269] Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, 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.

[0270] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome.TM. I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome.TM. II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

[0271] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G.sub.M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0272] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

[0273] In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

[0274] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0275] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0276] A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin.TM. Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

[0277] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam.TM., Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0278] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Chol") which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

[0279] Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, 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. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0280] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.

[0281] Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

[0282] Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

[0283] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0284] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0285] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0286] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0287] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0288] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0289] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0290] The iRNA for use in the methods of the invention can also be provided as micellar formulations. "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0291] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C.sub.8 to C.sub.22 alkyl sulphate, and a micelle forming compounds. Exemplary 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, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

[0292] In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0293] Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

[0294] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

[0295] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0296] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

[0297] B. Lipid Particles

[0298] iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in a lipid formulation, e.g., a LNP, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle.

[0299] As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean 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, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

[0300] In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

[0301] The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N--(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N--(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleoyl-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol(DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (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) or analogs thereof, (3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydr- o-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami- no)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

[0302] In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

[0303] In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0.+-.20 nm and a 0.027 siRNA/Lipid Ratio.

[0304] The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

[0305] The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci.sub.2), a PEG-dimyristyloxypropyl (Ci.sub.4), a PEG-dipalmityloxypropyl (Ci.sub.6), or a PEG-distearyloxypropyl (C).sub.8. The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

[0306] In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0307] In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such 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 upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

##STR00015##

[0308] LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

[0309] Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE-US-00001 TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP- 1,2-Dilinolenyloxy-N,N-dimethylaminopropane DLinDMA/DPPC/Cholesterol/PEG-cDMA 1 (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH- 50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA 10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1'-(2-(4-(2-((2-(bis(2- Tech Gl/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1 yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid: siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid: siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid: siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid: siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid: siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid: siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid: siRNA: 10:1

DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

[0310] XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

[0311] MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.

[0312] ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

[0313] C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Ionizable/Cationic Lipids

[0314] Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.

[0315] "Alkyl" means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0316] "Alkenyl" means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0317] "Alkynyl" means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0318] "Acyl" means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, --C(.dbd.O)alkyl, --C(.dbd.O)alkenyl, and --C(.dbd.O)alkynyl are acyl groups.

[0319] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0320] The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl", and "optionally substituted heterocycle" means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (.dbd.O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, --CN, --ORx, --NRxRy, --NRxC(.dbd.O)Ry, --NRxSO2Ry, --C(.dbd.O)Rx, --C(.dbd.O)ORx, --C(.dbd.O)NRxRy, --SOnRx and --SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, --OH, --CN, alkyl, --ORx, heterocycle, --NRxRy, --NRxC(.dbd.O)Ry, --NRxSO2Ry, --C(.dbd.O)Rx, --C(.dbd.O)ORx, --C(.dbd.O)NRxRy, --SOnRx and --SOnNRxRy.

[0321] "Halogen" means fluoro, chloro, bromo and iodo.

[0322] In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an "alcohol protecting group" is used. An "alcohol protecting group" is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

[0323] In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:

##STR00016##

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

##STR00017##

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

##STR00018##

[0324] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

[0325] Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

[0326] Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

##STR00019##

Synthesis of 515

[0327] To a stirred suspension of LiA1H4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0.degree. C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0.degree. C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): .delta.=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

[0328] To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0.degree. C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1.times.100 mL) and saturated NaHCO3 solution (1.times.50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): .delta.=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%).

Synthesis of 517A and 517B

[0329] The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (.about.3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2.times.100 mL) followed by saturated NaHCO3 (1.times.50 mL) solution, water (1.times.30 mL) and finally with brine (1.times.50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: -6 g crude

[0330] 517A--Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): .delta.=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 confirmed by X-ray.

Synthesis of 518

[0331] Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): 6=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%.

General Procedure for the Synthesis of Compound 519

[0332] A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1M, 2 eq). After complete addition, the mixture was heated at 40.degree. C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR .delta.=130.2, 130.1 (.times.2), 127.9 (.times.3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (.times.2), 29.7, 29.6 (.times.2), 29.5 (.times.3), 29.3 (.times.2), 27.2 (.times.3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+Calc. 654.6. Found 654.6.

[0333] Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is 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. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

[0334] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. 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, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxyethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, Pullulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0335] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0336] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

[0337] The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0338] The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

[0339] C. Additional Formulations

[0340] i. Emulsions

[0341] The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0342] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0343] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0344] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0345] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0346] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0347] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0348] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0349] ii. Microemulsions

[0350] In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0351] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0352] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sesquioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0353] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

[0354] Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0355] iii. Microparticles

[0356] an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0357] iv. Penetration Enhancers

[0358] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0359] Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0360] Surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0361] Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 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).

[0362] The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring 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), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0363] Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanillate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 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).

[0364] As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0365] Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine.TM. (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000.TM. (Invitrogen; Carlsbad, Calif.), 293Fectin.TM. (Invitrogen; Carlsbad, Calif.), Cellfectin.TM. (Invitrogen; Carlsbad, Calif.), DMRIE-C.TM. (Invitrogen; Carlsbad, Calif.), FreeStyle.TM. MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine.TM. (Invitrogen; Carlsbad, Calif.), Optifect.TM. (Invitrogen; Carlsbad, Calif.), 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.RTM. Reagent (Promega; Madison, Wis.), TransFast.TM. Transfection Reagent (Promega; Madison, Wis.), Tfx.TM.-20 Reagent (Promega; Madison, Wis.), Tfx.TM.-50 Reagent (Promega; Madison, Wis.), DreamFect.TM. (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec.TM./LipoGen.TM. (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TrojanPORTER.TM. transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect.TM. (B-Bridge International, Mountain View, Calif., USA), among others.

[0366] Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0367] v. Carriers

[0368] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidylic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

[0369] vi. Excipients

[0370] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

[0371] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0372] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0373] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0374] vii. Other Components

[0375] The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0376] Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

[0377] In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a bleeding disorder. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.

[0378] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

[0379] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0380] In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by Serpinc1 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. METHODS OF THE INVENTION

[0381] The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit Serpinc1 expression in a cell. In other aspects, the present invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting Serpinc1 expression in a cell. In yet other aspects, use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament for reducing and/or inhibiting Serpinc1 expression in a cell are provided.

[0382] The methods and uses include contacting the cell with an iRNA, e.g., a dsRNA, of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a Serpinc1 gene, thereby inhibiting expression of the Serpinc1 gene in the cell.

[0383] Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of Serpinc1 may be determined by determining the mRNA expression level of Serpinc1 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of Serpinc1 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, and/or by determining a biological activity of Serpinc1, such as affecting one or more molecules associated with the cellular blood clotting mechanism (or in an in vivo setting, blood clotting itself).

[0384] In the methods and uses of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

[0385] A cell suitable for treatment using the methods of the invention may be any cell that expresses a Serpinc1 gene. A cell suitable for use in the methods and uses of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

[0386] Serpinc1 expression may be inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.

[0387] The in vivo methods and uses of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Serpinc1 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.

[0388] In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of Serpinc1, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

[0389] In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

[0390] The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

[0391] In one aspect, the present invention also provides methods for inhibiting the expression of a Serpinc1 gene in a mammal, e.g., a human. The present invention also provides a composition comprising an iRNA, e.g., a dsRNA, that targets a Serpinc1 gene in a cell of a mammal for use in inhibiting expression of the Serpinc1 gene in the mammal. In another aspect, the present invention provides use of an iRNA, e.g., a dsRNA, that targets a Serpinc1 gene in a cell of a mammal in the manufacture of a medicament for inhibiting expression of the Serpinc1 gene in the mammal.

[0392] The methods and uses include administering to the mammal, e.g., a human, a composition comprising an iRNA, e.g., a dsRNA, that targets a Serpinc1 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the Serpinc1 gene, thereby inhibiting expression of the Serpinc1 gene in the mammal.

[0393] Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in Serpinc1 gene and/or protein expression. In another embodiment, a blood sample serves as the tissue material for monitoring the reduction in Serpinc1 gene and/or protein expression. In other embodiments, inhibition of the expression of a Serpinc1 gene is monitored indirectly by, for example, determining the expression and/or activity of a gene in a Serpinc1 pathway (see, e.g., FIG. 1). For example, the activity of factor Xa may be monitored to determine the inhibition of expression of a Serpinc1 gene. Antithrombin levels, clot formation, and/or endogenous thrombin potential, in a sample, e.g., a blood or liver sample, may also be measured. Suitable assays are further described in the Examples section below.

[0394] The present invention further provides methods of treating a subject having a disorder that would benefit from reduction in Serpinc1 expression, e.g., hemophilia. The treatment methods (and uses) of the invention include administering to the subject, e.g., a human, a therapeutically effective amount of an iRNA targeting a Serpinc1 gene or a pharmaceutical composition comprising an iRNA targeting a Serpinc1 gene, thereby treating the subject having a disorder that would benefit from reduction in Serpinc1 expression.

[0395] In one aspect, the invention provides methods of preventing at least one symptom in a subject having a disorder that would benefit from reduction in Serpinc1 expression. The methods include administering to the subject a therapeutically effective amount of the iRNA, e.g., dsRNA, or vector of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in Serpinc1 expression. For example, the invention provides methods for preventing bleeding in a subject suffering from a disorder that would benefit from reduction in Serpinc1 expression, e.g., a hemophilia

[0396] In another aspect, the present invention provides use of a therapeutically effective amount of an iRNA of the invention for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of Serpinc1 expression. The iRNA includes iRNA targeting a Serpinc1 gene or a pharmaceutical composition comprising an iRNA targeting a Serpinc1 gene.

[0397] In yet another aspect, the present invention provides use of an iRNA of the invention targeting a Serpinc1 gene or a pharmaceutical composition comprising an iRNA targeting a Serpinc1 gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of Serpinc1 expression.

[0398] In another aspect, the invention provides uses of an iRNA, e.g., a dsRNA, of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder, e.g., a hemophilia.

[0399] In a further aspect, the present invention provides uses of an iRNA of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of Serpinc1 expression, such as a bleeding disorder, e.g., a hemophilia. An iRNA of the invention may be administered in "naked" form, or as a "free iRNA." A naked iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

[0400] Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

[0401] Subjects that would benefit from a reduction and/or inhibition of Serpinc1gene expression are those having a bleeding disorder, e.g., an inherited bleeding disorder or an acquired bleeding disorder as described herein. In one embodiment, a subject having an inherited bleeding disorder has a hemophilia, e.g., hemophilia A, B, or C. In one embodiment, a subject having an inherited bleeding disorder, e.g., a hemophilia, is an inhibitor subject. In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C. Treatment of a subject that would benefit from a reduction and/or inhibition of Serpinc1 gene expression includes therapeutic (e.g., on-demand, e.g., the subject is bleeding (spontaneous bleeding or bleeding as a result of trauma) and failing to clot) and prophylactic (e.g., the subject is not bleeding and/or is to undergo surgery) treatment.

[0402] The invention further provides methods and uses for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of Serpinc1 expression, e.g., a subject having a bleeding disorder, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting Serpinc1 is administered in combination with, e.g., an agent useful in treating a bleeding disorder as described elsewhere herein. For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in Serpinc1 expression, e.g., a subject having a bleeding disorder, include fresh-frozen plasma (FFP); recombinant FVIIa; recombinant FIX; FXI concentrates; virus-inactivated, vWF-containing FVIII concentrates; desensitization therapy which may include large doses of FVIII or FIX, along with steroids or intravenous immunoglobulin (IVIG) and cyclophosphamide; plasmapheresis in conjunction with immunosuppression and infusion of FVIII or FIX, with or without antifibrinolytic therapy; immune tolerance induction (ITI), with or without immunosuppressive therapy (e.g., cyclophosphamide, prednisone, and/or anti-CD20); desmopressin acetate [DDAVP]; antifibrinolytics, such as aminocaproic acid and tranexamic acid; activated prothrombin complex concentrate (PCC); antihemophilic agents; corticosteroids; immunosuppressive agents; and estrogens. The iRNA and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

[0403] In one embodiment, the methods and uses include administering a composition featured herein such that expression of the target Serpinc1 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target Serpinc1 gene is decreased for an extended duration, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or longer.

[0404] Preferably, the iRNAs useful for the methods, uses, and compositions featured herein specifically target RNAs (primary or processed) of the target Serpinc1 gene. Compositions, uses, and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

[0405] Administration of the dsRNA according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a bleeding disorder. By "reduction" in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0406] Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, frequency of bleeds, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a bleeding disorder may be assessed, for example, by periodic monitoring of thrombin:anti-thrombin levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting Serpinc1 or pharmaceutical composition thereof, "effective against" a bleeding disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating bleeding disorders and the related causes.

[0407] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

[0408] Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale, as but one example the Child-Pugh score (sometimes the Child-Turcotte-Pugh score). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.

[0409] Subjects can be administered a therapeutic amount of iRNA, such as about 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, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 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, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0410] In certain embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and a lipid, subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/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 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 to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0411] For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0412] In other embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of iRNA, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, 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/kg, about 1.5 to about 45 mg/kg, 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, 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/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 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/kg, about 1.5 to about 30 mg/kg, 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, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0413] For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

[0414] The iRNA can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as weekly, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

[0415] In one embodiment, the present invention provides methods for treating a subject suffering from a bleeding disorder, e.g., a hemophilia, by subcutaneously administering to said subject compound AD-57213 at a cumulative weekly dose of about 0.5 mg/kg to about 5 mg/kg, or about 1 mg/kg to about 3 mg/kg.

[0416] In one embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 0.5 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 0.5 mg/kg as 0.5 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 0.5 mg/kg as 1 mg/kg every two weeks.

[0417] In another embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 1.5 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 1.5 mg/kg as 1.5 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 1.5 mg/kg as 3 mg/kg every two weeks.

[0418] In another embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 2 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 2 mg/kg as 2 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 2 mg/kg as 4 mg/kg every two weeks.

[0419] In yet another embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 3 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 3 mg/kg as 3 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 3 mg/kg as 6 mg/kg every two weeks.

[0420] In another embodiment, the present invention provides methods for preventing in a subject at least one symptom of a bleeding disorder, e.g., a hemophilia, by subcutaneously administering to the subject compound AD-57213 at a cumulative weekly dose of about 0.5 mg/kg to about 5 mg/kg or about 1 mg/kg to about 3 mg/kg.

[0421] In one embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 0.5 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 0.5 mg/kg as 0.5 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 0.5 mg/kg as 1 mg/kg every two weeks.

[0422] In another embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 1.5 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 1.5 mg/kg as 1.5 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 1.5 mg/kg as 3 mg/kg every two weeks.

[0423] In another embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 2 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 2 mg/kg as 2 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 2 mg/kg as 4 mg/kg every two weeks.

[0424] In yet another embodiment, the methods may include subcutaneously administering to the subject a cumulative weekly dose of about 3 mg/kg. For example, in one embodiment, the methods may include administering to the subject a cumulative weekly dose of about 3 mg/kg as 3 mg/kg every week. In another embodiment, the methods may include administering to the subject a cumulative weekly dose of about 3 mg/kg as 6 mg/kg every two weeks.

[0425] Administration of the iRNA can reduce Serpinc1 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.

[0426] In one embodiment, the treatment and/or preventive methods include subcutaneously administering to a subject compound AD-57213 at a dose sufficient to inhibit reduce Serpinc1 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about by 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%.

[0427] Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0428] Owing to the inhibitory effects on Serpinc1 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

[0429] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Example 1

iRNA Synthesis

Source of Reagents

[0430] Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts

[0431] siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), dog, mouse, and rat SERPINC1 transcripts annotated in the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/). Design used the following transcripts from the NCBI RefSeq collection: Human--NM_000488.2, NM_000488.3; Rhesus--NM_001104583.1; Dog--XM_856414.1; Mouse--NM_080844.4; Rat--NM_001012027.1. Due to high primate/canine/rodent sequence divergence, siRNA duplexes were designed in several separate batches, including but not limited to batches containing duplexes matching human and rhesus transcripts only; human, rhesus, and dog transcripts only; human, rhesus, mouse, and rat transcripts only; and mouse and rat transcripts only. All siRNA duplexes were designed that shared 100% identity the listed human transcript and other species transcripts considered in each design batch (above).

siRNA Design, Specificity, and Efficacy Prediction

[0432] The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 874 candidate human/rhesus, 67 human/rhesus/dog, 103 human/rhesus/mouse/rat, and 569 mouse/rat siRNAs were used in comprehensive searches against the appropriate transcriptomes (defined as the set of NM_ and XM_ records within the human, rhesus, dog, mouse, or rat NCBI Refseq sets) using an exhaustive "brute-force" algorithm implemented in the python script `BruteForce.py`. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential `off-target` transcript. The off-target score is weighted to emphasize differences in the `seed` region of siRNAs, in positions 2-9 from the 5'-end of the molecule.

[0433] Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5' start were used to create 2 heptamers and one octamer. `Heptamer1` was created by adding a 3'-A to the hexamer; heptamer2 was created by adding a 5'-A to the hexamer; the octomer was created by adding an A to both 5'- and 3'-ends of the hexamer. The frequency of octamers and heptamers in the human, rhesus, mouse, or rat 3'-UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the `CDS`, is clearly defined) was pre-calculated. The octamer frequency was normalized to the heptamer frequency using the median value from the range of octamer frequencies. A `mirSeedScore` was then calculated by calculating the sum of ((3.times.normalized octamer count)+(2.times.heptamer2 count)+(1.times.heptamer1 count)).

[0434] Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. The duplexes were sorted by the specificity of the antisense strand and those duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region were selected.

siRNA Sequence Selection

[0435] A total of 66 sense and 66 antisense derived human/rhesus, 6 sense and 6 antisense derived human/rhesus/mouse, 12 human/rhesus/mouse/rat, and 21 sense and 21 antisense derived mouse/rat siRNA oligos were synthesized and formed into duplexes. A detailed list of Sepinc1 sense and antisense strand sequences is shown in Tables 3 and 4.

siRNA Synthesis

[0436] I. General Small and Medium Scale RNA Synthesis Procedure

[0437] RNA oligonucleotides were synthesized at scales between 0.2-500 .mu.mol using commercially available 5'-O-(4,4'-dimethoxytrityl)-2'-O-t-butyldimethylsilyl-3'-O-(2-cyanoethyl-- N,N-diisopropyl)phosphoramidite monomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and 2-N-isobutyrylguanosine and the corresponding 2'-O-methyl and 2'-fluoro phosphoramidites according to standard solid phase oligonucleotide synthesis protocols. The amidite solutions were prepared at 0.1-0.15M concentration and 5-ethylthio-1H-tetrazole (0.25-0.6M in acetonitrile) was used as the activator. Phosphorothioate backbone modifications were introduced during synthesis using 0.2M phenylacetyl disulfide (PADS) in lutidine:acetonitrile (1:1) (v;v) or 0.1M 3-(dimethylaminomethylene) amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidation step. After completion of synthesis, the sequences were cleaved from the solid support and deprotected using methylamine followed by triethylamine.3HF to remove any 2'-O-t-butyldimethylsilyl protecting groups present.

[0438] For synthesis scales between 5-500 .mu.mol and fully 2' modified sequences (2'-fluoro and/or 2'-O-methyl or combinations thereof) the oligonucleotides where deprotected using 3:1 (v/v) ethanol and concentrated (28-32%) aqueous ammonia either at 35.degree. C. 16 h or 55.degree. C. for 5.5 h. Prior to ammonia deprotection the oligonucleotides where treated with 0.5M piperidine in acetonitrile for 20 min on the solid support. The crude oligonucleotides were analyzed by LC-MS and anion-exchange HPLC (IEX-HPLC). Purification of the oligonucleotides was carried out by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH=8.5 (buffer A) and 20 mM phosphate, 10%-15% ACN, 1M NaBr, pH=8.5 (buffer B). Fractions were analyzed for purity by analytical HPLC. The product-containing fractions with suitable purity were pooled and concentrated on a rotary evaporator prior to desalting. The samples were desalted by size exclusion chromatography and lyophilized to dryness. Equal molar amounts of sense and antisense strands were annealed in 1.times.PBS buffer to prepare the corresponding siRNA duplexes.

[0439] For small scales (0.2-1 .mu.mol), synthesis was performed on a MerMade 192 synthesizer in a 96 well format. In case of fully 2'-modified sequences (2'-fluoro and/or 2'-O-methyl or combinations thereof) the oligonucleotides where deprotected using methylamine at room temperature for 30-60 min followed by incubation at 60.degree. C. for 30 min or using 3:1 (v/v) ethanol and concentrated (28-32%) aqueous ammonia at room temperature for 30-60 min followed by incubation at 40.degree. C. for 1.5 hours. The crude oligonucleotides were then precipitated in a solution of acetonitrile:acetone (9:1) and isolated by centrifugation and decanting the supernatant. The crude oligonucleotide pellet was re-suspended in 20 mM NaOAc buffer and analyzed by LC-MS and anion exchange HPLC. The crude oligonucleotide sequences were desalted in 96 deep well plates on a 5 mL HiTrap Sephadex G25 column (GE Healthcare). In each well about 1.5 mL samples corresponding to an individual sequence was collected. These purified desalted oligonucleotides were analyzed by LC-MS and anion exchange chromatography. Duplexes were prepared by annealing equimolar amounts of sense and antisense sequences on a Tecan robot. Concentration of duplexes was adjusted to 10 .mu.M in 1.times.PBS buffer.

[0440] II. Synthesis of GalNAc-Conjugated Oligonucleotides for In Vivo Analysis

[0441] Oligonucleotides conjugated with GalNAc ligand at their 3'-terminus were synthesized at scales between 0.2-500 .mu.mol using a solid support pre-loaded with a Y-shaped linker bearing a 4,4'-dimethoxytrityl (DMT)-protected primary hydroxy group for oligonucleotide synthesis and a GalNAc ligand attached through a tether.

[0442] For synthesis of GalNAc conjugates in the scales between 5-500 .mu.mol, the above synthesis protocol for RNA was followed with the following adaptions: For polystyrene-based synthesis supports 5% dichloroacetic acid in toluene was used for DMT-cleavage during synthesis. Cleavage from the support and deprotection was performed as described above. Phosphorothioate-rich sequences (usually >5 phorphorothioates) were synthesized without removing the final 5'-DMT group ("DMT-on") and, after cleavage and deprotection as described above, purified by reverse phase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mM ammoniumacetate in 80% acetonitirile (buffer B). Fractions were analyzed for purity by analytical HPLC and/or LC-MS. The product-containing fractions with suitable purity were pooled and concentrated on a rotary evaporator. The DMT-group was removed using 20%-25% acetic acid in water until completion. The samples were desalted by size exclusion chromatography and lyophilized to dryness. Equal molar amounts of sense and antisense strands were annealed in 1.times.PBS buffer to prepare the corresponding siRNA duplexes.

[0443] For small scale synthesis of GalNAc conjugates (0.2-1 .mu.mol), including sequences with multiple phosphorothioate linkages, the protocols described above for synthesis of RNA or fully 2'-F/2'-OMe-containing sequences on MerMade platform were applied. Synthesis was performed on pre-packed columns containing GalNAc-functionalized controlled pore glass support.

Example 2

In Vitro Screening

Cell Culture and Transfections

[0444] Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37.degree. C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. For mouse cross reactive duplexes, primary mouse hepatocytes (PMH) were freshly isolated less than 1 hour prior to transfections and grown in primary hepatocyte media. For both Hep3B and PMH, transfection was carried out by adding 14.8 .mu.l of Opti-MEM plus 0.2 .mu.l of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 .mu.l of each siRNA duplex to an individual well in a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. Eighty .mu.l of complete growth media without antibiotic containing .about.2.times.10.sup.4 Hep3B cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done using 8.times. 5-fold serial dilutions over the range of 10 nM to 128 pM (see FIGS. 2A and 2B).

Free Uptake Transfection

[0445] Five .mu.l of each GalNac conjugated siRNA in PBS was combined with 4.times.10.sup.4 freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspended in 95 .mu.l of In Vitro Gro CP media (In Vitro Technologies-Celsis, Baltimore, Md.) in each well of a 96 well plate. The mixture was incubated for about 24 hrs at 37.degree. C. in an atmosphere of 5% CO.sub.2. siRNAs were tested at final concentrations of 100 nM, 10 nM and 0.1 nM for efficacy free uptake assays.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)

[0446] Cells were harvested and lysed in 150 .mu.l of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 .mu.l Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. 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, magnetic beads were washed 2 times with 150 .mu.l Wash Buffer A and mixed for 1 minute. The beads were captured again and the supernatant was removed. The beads were then washed with 150 .mu.l Wash Buffer B, captured and the supernatant was removed. The beads were next washed with 150 .mu.l Elution Buffer, captured and the supernatant removed. Finally, the beads were allowed to dry for 2 minutes. After drying, 50 .mu.l of Elution Buffer was added and mixed for 5 minutes at 70.degree. C. The beads were captured on magnet for 5 minutes. 40 .mu.l of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

[0447] A master mix of 2 .mu.l 10.times. Buffer, 0.8 .mu.l 25.times.dNTPs, 2 .mu.l Random primers, 1 .mu.l Reverse Transcriptase, 1 .mu.l RNase inhibitor and 3.2 .mu.l of H.sub.2O per reaction were added into 10 .mu.l total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25.degree. C. for 10 minutes, 37.degree. C. for 120 minutes, 85.degree. C. for 5 seconds, and 4.degree. C. hold.

[0448] Real Time PCR

[0449] Two .mu.l of cDNA were added to a master mix containing 0.5 .mu.l human GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 .mu.l human SERPINC1 TaqMan probe (Applied Biosystems cat # Hs00892758_m1) for human cells or 0.5 .mu.l mouse GAPDH TaqMan Probe (Applied Biosystems Cat #4308313), 0.5 .mu.l mouse SERPINC1 TaqMan probe (Applied Biosystems cat # Mm00446573_m1) for mouse cells and 5 .mu.l Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates. Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the .DELTA..DELTA.Ct(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

[0450] To calculate relative fold change in Serpinc1 mRNA levels, real time data were analyzed using the .DELTA..DELTA.Ct method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC.sub.50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 over the same dose range, or to its own lowest dose. Table 5 shows the results of a single dose screen in Hep3B cells and PMH cells transfected with the indicated iRNAs. Table 6 shows the results of dose response of the indicated iRNAs transfected into Hep3B and PMH cells.

[0451] The sense and antisense sequences of AD-1955 are:

TABLE-US-00002 (SEQ ID NO: 13) SENSE: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14) ANTISENSE: UCGAAGuACUcAGCGuAAGdTsdT.

TABLE-US-00003 TABLE 2 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3'-phosphate Ab beta-L-adenosine-3'-phosphate Abs beta-L-adenosine-3'-phosphorothioate Af 2'-fluoroadenosine-3'-phosphate Afs 2'-fluoroadenosine-3'-phosphorothioate As adenosine-3'-phosphorothioate C cytidine-3'-phosphate Cb beta-L-cytidine-3'-phosphate Cbs beta-L-cytidine-3'-phosphorothioate Cf 2'-fluorocytidine-3'-phosphate Cfs 2'-fluorocytidine-3'-phosphorothioate (Chd) 2'-O-hexadecyl-cytidine-3'-phosphate (Chds) 2'-O-hexadecyl-cytidine-3'-phosphorothioate Cs cytidine-3'-phosphorothioate G guanosine-3'-phosphate Gb beta-L-guanosine-3'-phosphate Gbs beta-L-guanosine-3'-phosphorothioate Gf 2'-fluoroguanosine-3'-phosphate Gfs 2'-fluoroguanosine-3'-phosphorothioate Gs guanosine-3'-phosphorothioate T 5'-methyluridine-3'-phosphate Tb beta-L-thymidine-3'-phosphate Tbs beta-L-thymidine-3'-phosphorothioate Tf 2'-fluoro-5-methyluridine-3'-phosphate Tfs 2'-fluoro-5-methyluridine-3'-phosphorothioate Ts 5-methyluridine-3'-phosphorothioate U Uridine-3'-phosphate Ub beta-L-uridine-3'-phosphate Ubs beta-L-uridine-3'-phosphorothioate Uf 2'-fluorouridine-3'-phosphate Ufs 2'-fluorouridine-3'-phosphorothioate (Uhd) 2'-O-hexadecyl-uridine-3'-phosphate (Uhds) 2'-O-hexadecyl-uridine-3'-phosphorothioate Us uridine-3'-phosphorothioate N any nucleotide (G, A, C, T or U) a 2'-O-methyladenosine-3'-phosphate as 2'-O-methyladenosine-3'- phosphorothioate c 2'-O-methylcytidine-3'-phosphate cs 2'-O-methylcytidine-3'- phosphorothioate g 2'-O-methylguanosine-3'-phosphate gs 2'-O-methylguanosine-3'- phosphorothioate t 2'-O-methyl-5-methyluridine-3'-phosphate ts 2'-O-methyl-5-methyluridine-3'-phosphorothioate u 2'-O-methyluridine-3'-phosphate us 2'-O-methyluridine-3'-phosphorothioate dA 2'-deoxyadenosine-3'-phosphate dAs 2'-deoxyadenosine-3'-phosphorothioate dC 2'-deoxycytidine-3'-phosphate dCs 2'-deoxycytidine-3'-phosphorothioate dG 2'-deoxyguanosine-3'-phosphate dGs 2'-deoxyguanosine-3'-phosphorothioate dT 2'-deoxythymidine dTs 2'-deoxythymidine-3'-phosphorothioate dU 2'-deoxyuridine s phosphorothioate linkage L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp- (GalNAc-alkyl)3 (Aeo) 2'-O-methoxyethyladenosine-3'-phosphate (Aeos) 2'-O-methoxyethyladenosine-3'-phosphorothioate (Geo) 2'-O-methoxyethylguanosine-3'-phosphate (Geos) 2'-O-methoxyethylguanosine-3'- phosphorothioate (Teo) 2'-O-methoxyethyl-5-methyluridine-3'-phosphate (Teos) 2'-O-methoxyethyl-5-methyluridine-3'- phosphorothioate (m5Ceo) 2'-O-methoxyethyl-5-methylcytidine-3'-phosphate (m5Ceos) 2'-O-methoxyethyl-5-methylcytidine-3'- phosphorothioate

TABLE-US-00004 TABLE 3 Unmodified Sense and antisense strand sequences of Serpinc1 dsRNAs (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 15-71, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 72-128, respectively, in order of appearance) Antisense Duplex Name Sense Name Sense Sequence Name Antisense Sequence AD-50475.1-UM A-104633.1 CCCUGUGGACAUCUGCACA A-104634.1 UGUGCAGAUGUCCACAGGG AD-50476.1-UM A-104649.1 CUACCACUUUCUAUCAGCA A-104650.1 UGCUGAUAGAAAGUGGUAG AD-50477.1-UM A-104665.1 CUAUCGAAAAGCCAACAAA A-104666.1 UUUGUUGGCUUUUCGAUAG AD-50478.1-UM A-104681.1 GGACUUCAAGGAAAAUGCA A-104682.1 UGCAUUUUCCUUGAAGUCC AD-50479.1-UM A-104697.1 GUUAACACCAUUUACUUCA A-104698.1 UGAAGUAAAUGGUGUUAAC AD-50480.1-UM A-104713.1 CCUGGUUUUUAUAAGAGAA A-104714.1 UUCUCUUAUAAAAACCAGG AD-50481.1-UM A-104635.1 GACAUUCCCAUGAAUCCCA A-104636.1 UGGGAUUCAUGGGAAUGUC AD-50482.1-UM A-104651.1 CACCUGGCAGAUUCCAAGA A-104652.1 UCUUGGAAUCUGCCAGGUG AD-50483.1-UM A-104667.1 CGAAAAGCCAACAAAUCCU A-104668.1 AGGAUUUGUUGGCUUUUCG AD-50484.1-UM A-104683.1 GAAAAUGCAGAGCAAUCCA A-104684.1 UGGAUUGCUCUGCAUUUUC AD-50485.1-UM A-104699.1 GGCCUGUGGAAGUCAAAGU A-104700.1 ACUUUGACUUCCACAGGCC AD-50486.1-UM A-104715.1 GAAGUUCCUCUGAACACUA A-104716.1 UAGUGUUCAGAGGAACUUC AD-50487.1-UM A-104637.1 CCAUGAAUCCCAUGUGCAU A-104638.1 AUGCACAUGGGAUUCAUGG AD-50488.1-UM A-104653.1 CAACUGAUGGAGGUAUUUA A-104654.1 UAAAUACCUCCAUCAGUUG AD-50489.1-UM A-104669.1 CCAAGUUAGUAUCAGCCAA A-104670.1 UUGGCUGAUACUAACUUGG AD-50490.1-UM A-104685.1 CGGCCAUCAACAAAUGGGU A-104686.1 ACCCAUUUGUUGAUGGCCG AD-50491.1-UM A-104701.1 GAGGACGGCUUCAGUUUGA A-104702.1 UCAAACUGAAGCCGUCCUC AD-50492.1-UM A-104717.1 CCUCUGAACACUAUUAUCU A-104718.1 AGAUAAUAGUGUUCAGAGG AD-50493.1-UM A-104639.1 CAUGAAUCCCAUGUGCAUU A-104640.1 AAUGCACAUGGGAUUCAUG AD-50494.1-UM A-104655.1 GAUGGAGGUAUUUAAGUUU A-104656.1 AAACUUAAAUACCUCCAUC AD-50495.1 UM A-104671.1 GUAUCAGCCAAUCGCCUUU A-104672.1 AAAGGCGAUUGGCUGAUAC AD-50496.1-UM A-104687.1 GGGUGUCCAAUAAGACCGA A-104688.1 UCGGUCUUAUUGGACACCC AD-50497.1-UM A-104703.1 CAGCCCUGAAAAGUCCAAA A-104704.1 UUUGGACUUUUCAGGGCUG AD-50498.1-UM A-104641.1 CCCAUGUGCAUUUACCGCU A-104642.1 AGCGGUAAAUGCACAUGGG AD-50499.1-UM A-104657.1 GUAUUUAAGUUUGACACCA A-104658.1 UGGUGUCAAACUUAAAUAC AD-50500.1-UM A-104673.1 GACAAAUCCCUUACCUUCA A-104674.1 UGAAGGUAAGGGAUUUGUC AD-50501.1-UM A-104689.1 CUGUUCUGGUGCUGGUUAA A-104690.1 UUAACCAGCACCAGAACAG AD-50502.1UM A-104705.1 CCAAACUCCCAGGUAUUGU A-104706.1 ACAAUACCUGGGAGUUUGG AD-50503.1-UM A-104643.1 CCCGCUUUGCUACCACUUU A-104644.1 AAAGUGGUAGCAAAGCGGG AD-50505.1-UM A-104675.1 CUUACCUUCAAUGAGACCU A-104676.1 AGGUCUCAUUGAAGGUAAG AD-50506.1-UM A-104691.1 CUGGUGCUGGUUAACACCA A-104692.1 UGGUGUUAACCAGCACCAG AD-50507.1-UM A-104707.1 CAAACUCCCAGGUAUUGUU A-104708.1 AACAAUACCUGGGAGUUUG AD-50508.1-UM A-104645.1 GCUUUGCUACCACUUUCUA A-104646.1 UAGAAAGUGGUAGCAAAGC AD-50510.1-UM A-104677.1 CCUACCAGGACAUCAGUGA A-104678.1 UCACUGAUGUCCUGGUAGG AD-50511.1-UM A-104693.1 GGUGCUGGUUAACACCAUU A-104694.1 AAUGGUGUUAACCAGCACC AD-50512.1-UM A-104709.1 GCCGUUCGCUAAACCCCAA A-104710.1 UUGGGGUUUAGCGAACGGC AD-50515.1-UM A-104679.1 GGACAUCAGUGAGUUGGUA A-104680.1 UACCAACUCACUGAUGUCC AD-50516.1-UM A-104695.1 GUGCUGGUUAACACCAUUU A-104696.1 AAAUGGUGUUAACCAGCAC AD-50517.1-UM A-104711.1 GCCUUUCCUGGUUUUUAUA A-104712.1 UAUAAAAACCAGGAAAGGC AD-50518.1-UM A-104719.1 CUUUUGCUAUGACCAAGCU A-104720.1 AGCUUGGUCAUAGCAAAAG AD-50523.1-UM A-104721.1 UGUACCAGGAAGGCAAGUU A-104722.1 AACUUGCCUUCCUGGUACA AD-50528.1-UM A-104723.1 ACUAUUAUCUUCAUGGGCA A-104724.1 UGCCCAUGAAGAUAAUAGU AD-50540.1-UM A-104729.1 UCAUGGGCAGAGUAGCCAA A-104730.1 UUGGCUACUCUGCCCAUGA AD-50539.1-UM A-104785.1 CCAUUUACUUCAAGGGCCU A-104786.1 AGGCCCUUGAAGUAAAUGG AD-50544.1-UM A-104787.1 UACUUCAAGGGCCUGUGGA A-104788.1 UCCACAGGCCCUUGAAGUA AD-50549.1-UM A-104789.1 ACUUCAAGGGCCUGUGGAA A-104790.1 UUCCACAGGCCCUUGAAGU AD-50514.1-UM A-104663.1 CGACUCUAUCGAAAAGCCA A-104664.1 UGGCUUUUCGAUAGAGUCG AD-50522.1-UM A-104779.1 AACUGCCGACUCUAUCGAA A-104780.1 UUCGAUAGAGUCGGCAGUU AD-50527.1-UM A-104781.1 ACUGCCGACUCUAUCGAAA A-104782.1 UUUCGAUAGAGUCGGCAGU AD-50531.1-UM A-104739.1 GACUCUAUCGAAAAGCCAA A-104740.1 UUGGCUUUUCGAUAGAGUC AD-50534.1-UM A-104769.1 UCUUCUUUGCCAAACUGAA A-104770.1 UUCAGUUUGGCAAAGAAGA AD-50538.1-UM A-104771.1 UGCCAAACUGAACUGCCGA A-104772.1 UCGGCAGUUCAGUUUGGCA AD-50543.1-UM A-104773.1 CCAAACUGAACUGCCGACU A-104774.1 AGUCGGCAGUUCAGUUUGG AD-50553.1-UM A-104777.1 ACUGAACUGCCGACUCUAU A-104778.1 AUAGAGUCGGCAGUUCAGU AD-50504.1-UM A-104659.1 GAACUGCCGACUCUAUCGA A-104660.1 UCGAUAGAGUCGGCAGUUC AD-50509.1-UM A-104661.1 CUGCCGACUCUAUCGAAAA A-104662.1 UUUUCGAUAGAGUCGGCAG AD-50529.1-UM A-104751.1 CUGGUUAACACCAUUUACU A-104752.1 AGUAAAUGGUGUUAACCAG

TABLE-US-00005 TABLE 4 Modified Sense and antisense strand sequences of Serpinc1 dsRNAs (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 129-185, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 186-242, respectively, in order of appearance) Antisense Duplex Name Sense Name Sense Sequence Name Antisense Sequence AD-50475.1 A-104633.1 cccuGuGGAcAucuGcAcAdTsdT A-104634.1 UGUGcAGAUGUCcAcAGGGdTsdT AD-50476.1 A-104649.1 cuAccAcuuucuAucAGcAdTsdT A-104650.1 UGCUGAuAGAAAGUGGuAGdTsdT AD-50477.1 A-104665.1 cuAucGAAAAGccAAcAAAdTsdT A-104666.1 UUUGUUGGCUUUUCGAuAGdTsdT AD-50478.1 A-104681.1 GGAcuucAAGGAAAAuGcAdTsdT A-104682.1 UGcAUUUUCCUUGAAGUCCdTsdT AD-50479.1 A-104697.1 GuuAAcAccAuuuAcuucAdTsdT A-104698.1 UGAAGuAAAUGGUGUuAACdTsdT AD-50480.1 A-104713.1 ccuGGuuuuuAuAAGAGAAdTsdT A-104714.1 UUCUCUuAuAAAAACcAGGdTsdT AD-50481.1 A-104635.1 GAcAuucccAuGAAucccAdTsdT A-104636.1 UGGGAUUcAUGGGAAUGUCdTsdT AD-50482.1 A-104651.1 cAccuGGcAGAuuccAAGAdTsdT A-104652.1 UCUUGGAAUCUGCcAGGUGdTsdT AD-50483.1 A-104667.1 cGAAAAGccAAcAAAuccudTsdT A-104668.1 AGGAUUUGUUGGCUUUUCGdTsdT AD-50484.1 A-104683.1 GAAAAuGcAGAGcAAuccAdTsdT A-104684.1 UGGAUUGCUCUGcAUUUUCdTsdT AD-50485.1 A-104699.1 GGccuGuGGAAGucAAAGudTsdT A-104700.1 ACUUUGACUUCcAcAGGCCdTsdT AD-50486.1 A-104715.1 GAAGuuccucuGAAcAcuAdTsdT A-104716.1 uAGUGUUcAGAGGAACUUCdTsdT AD-50487.1 A-104637.1 ccAuGAAucccAuGuGcAudTsdT A-104638.1 AUGcAcAUGGGAUUcAUGGdTsdT AD-50488.1 A-104653.1 cAAcuGAuGGAGGuAuuuAdTsdT A-104654.1 uAAAuACCUCcAUcAGUUGdTsdT AD-50489.1 A-104669.1 ccAAGuuAGuAucAGccAAdTsdT A-104670.1 UUGGCUGAuACuAACUUGGdTsdT AD-50490.1 A-104685.1 cGGccAucAAcAAAuGGGudTsdT A-104686.1 ACCcAUUUGUUGAUGGCCGdTsdT AD-50491.1 A-104701.1 GAGGAcGGcuucAGuuuGAdTsdT A-104702.1 UcAAACUGAAGCCGUCCUCdTsdT AD-50492.1 A-104717.1 ccucuGAAcAcuAuuAucudTsdT A-104718.1 AGAuAAuAGUGUUcAGAGGdTsdT AD-50493.1 A-104639.1 cAuGAAucccAuGuGcAuudTsdT A-104640.1 AAUGcAcAUGGGAUUcAUGdTsdT AD-50494.1 A-104655.1 GAuGGAGGuAuuuAAGuuudTsdT A-104656.1 AAACUuAAAuACCUCcAUCdTsdT AD-50495.1 A-104671.1 GuAucAGccAAucGccuuudTsdT A-104672.1 AAAGGCGAUUGGCUGAuACdTsdT AD-50496.1 A-104687.1 GGGuGuccAAuAAGAccGAdTsdT A-104688.1 UCGGUCUuAUUGGAcACCCdTsdT AD-50497.1 A-104703.1 cAGcccuGAAAAGuccAAAdTsdT A-104704.1 UUUGGACUUUUcAGGGCUGdTsdT AD-50498.1 A-104641.1 cccAuGuGcAuuuAccGcudTsdT A-104642.1 AGCGGuAAAUGcAcAUGGGdTsdT AD-50499.1 A-104657.1 GuAuuuAAGuuuGAcAccAdTsdT A-104658.1 UGGUGUcAAACUuAAAuACdTsdT AD-50500.1 A-104673.1 GAcAAAucccuuAccuucAdTsdT A-104674.1 UGAAGGuAAGGGAUUUGUCdTsdT AD-50501.1 A-104689.1 cuGuucuGGuGcuGGuuAAdTsdT A-104690.1 UuAACcAGcACcAGAAcAGdTsdT AD-50502.1 A-104705.1 ccAAAcucccAGGuAuuGudTsdT A-104706.1 AcAAuACCUGGGAGUUUGGdTsdT AD-50503.1 A-104643.1 cccGcuuuGcuAccAcuuudTsdT A-104644.1 AAAGUGGuAGcAAAGCGGGdTsdT AD-50505.1 A-104675.1 cuuAccuucAAuGAGAccudTsdT A-104676.1 AGGUCUcAUUGAAGGuAAGdTsdT AD-50506.1 A-104691.1 cuGGuGcuGGuuAAcAccAdTsdT A-104692.1 UGGUGUuAACcAGcACcAGdTsdT AD-50507.1 A-104707.1 cAAAcucccAGGuAuuGuudTsdT A-104708.1 AAcAAuACCUGGGAGUUUGdTsdT AD-50508.1 A-104645.1 GcuuuGcuAccAcuuucuAdTsdT A-104646.1 uAGAAAGUGGuAGcAAAGCdTsdT AD-50510.1 A-104677.1 ccuAccAGGAcAucAGuGAdTsdT A-104678.1 UcACUGAUGUCCUGGuAGGdTsdT AD-50511.1 A-104693.1 GGuGcuGGuuAAcAccAuudTsdT A-104694.1 AAUGGUGUuAACcAGcACCdTsdT AD-50512.1 A-104709.1 GccGuucGcuAAAccccAAdTsdT A-104710.1 UUGGGGUUuAGCGAACGGCdTsdT AD-50515.1 A-104679.1 GGAcAucAGuGAGuuGGuAdTsdT A-104680.1 uACcAACUcACUGAUGUCCdTsdT AD-50516.1 A-104695.1 GuGcuGGuuAAcAccAuuudTsdT A-104696.1 AAAUGGUGUuAACcAGcACdTsdT AD-50517.1 A-104711.1 GccuuuccuGGuuuuuAuAdTsdT A-104712.1 uAuAAAAACcAGGAAAGGCdTsdT AD-50518.1 A-104719.1 cuuuuGcuAuGAccAAGcudTsdT A-104720.1 AGCUUGGUcAuAGcAAAAGdTsdT AD-50523.1 A-104721.1 uGuAccAGGAAGGcAAGuudTsdT A-104722.1 AACUUGCCUUCCUGGuAcAdTsdT AD-50528.1 A-104723.1 AcuAuuAucuucAuGGGcAdTsdT A-104724.1 UGCCcAUGAAGAuAAuAGUdTsdT AD-50540.1 A-104729.1 ucAuGGGcAGAGuAGccAAdTsdT A-104730.1 UUGGCuACUCUGCCcAUGAdTsdT AD-50539.1 A-104785.1 ccAuuuAcuucAAGGGccudTsdT A-104786.1 AGGCCCUUGAAGuAAAUGGdTsdT AD-50544.1 A-104787.1 uAcuucAAGGGccuGuGGAdTsdT A-104788.1 UCcAcAGGCCCUUGAAGuAdTsdT AD-50549.1 A-104789.1 AcuucAAGGGccuGuGGAAdTsdT A-104790.1 UUCcAcAGGCCCUUGAAGUdTsdT AD-50514.1 A-104663.1 cGAcucuAucGAAAAGccAdTsdT A-104664.1 UGGCUUUUCGAuAGAGUCGdTsdT AD-50522.1 A-104779.1 AAcuGccGAcucuAucGAAdTsdT A-104780.1 UUCGAuAGAGUCGGcAGUUdTsdT AD-50527.1 A-104781.1 AcuGccGAcucuAucGAAAdTsdT A-104782.1 UUUCGAuAGAGUCGGcAGUdTsdT AD-50531.1 A-104739.1 GAcucuAucGAAAAGccAAdTsdT A-104740.1 UUGGCUUUUCGAuAGAGUCdTsdT AD-50534.1 A-104769.1 ucuucuuuGccAAAcuGAAdTsdT A-104770.1 UUcAGUUUGGcAAAGAAGAdTsdT AD-50538.1 A-104771.1 uGccAAAcuGAAcuGccGAdTsdT A-104772.1 UCGGcAGUUcAGUUUGGcAdTsdT AD-50543.1 A-104773.1 ccAAAcuGAAcuGccGAcudTsdT A-104774.1 AGUCGGcAGUUcAGUUUGGdTsdT AD-50553.1 A-104777.1 AcuGAAcuGccGAcucuAudTsdT A-104778.1 AuAGAGUCGGcAGUUcAGUdTsdT AD-50504.1 A-104659.1 GAAcuGccGAcucuAucGAdTsdT A-104660.1 UCGAuAGAGUCGGcAGUUCdTsdT AD-50509.1 A-104661.1 cuGccGAcucuAucGAAAAdTsdT A-104662.1 UUUUCGAuAGAGUCGGcAGdTsdT AD-50529.1 A-104751.1 cuGGuuAAcAccAuuuAcudTsdT A-104752.1 AGuAAAUGGUGUuAACcAGdTsdT

TABLE-US-00006 TABLE 5.sup.1 Serpinc1 single dose screen Human (Hep3B) Mouse (PMH) Duplex Name 10 nM Ave 0.1 nM Ave 10 nM Ave 0.1 nM Ave AD-50475.1 0.11 0.21 AD-50476.1 0.08 0.43 AD-50477.1 0.10 0.10 AD-50478.1 0.12 0.36 AD-50479.1 0.24 0.84 AD-50480.1 0.31 0.73 AD-50481.1 0.74 1.12 AD-50482.1 0.61 0.89 AD-50483.1 0.07 0.14 AD-50484.1 0.12 0.33 AD-50485.1 0.58 1.18 AD-50486.1 0.79 0.94 AD-50487.1 0.05 0.09 AD-50488.1 0.83 1.07 AD-50489.1 0.09 0.28 AD-50490.1 0.04 0.78 AD-50491.1 0.19 0.77 AD-50492.1 0.16 0.84 AD-50493.1 0.17 0.55 AD-50494.1 0.16 0.59 AD-50495.1 0.08 0.13 AD-50496.1 0.57 0.94 AD-50497.1 0.85 1.15 AD-50498.1 0.16 1.02 AD-50499.1 0.10 0.21 AD-50500.1 0.22 0.58 AD-50501.1 0.10 0.32 AD-50502.1 0.76 1.07 AD-50503.1 0.08 0.47 AD-50505.1 0.74 0.77 AD-50506.1 0.85 0.89 AD-50507.1 0.03 0.37 AD-50508.1 0.16 0.97 AD-50510.1 0.09 0.89 AD-50511.1 0.15 0.71 AD-50512.1 0.88 1.19 AD-50515.1 0.13 0.49 AD-50516.1 0.85 0.95 AD-50517.1 0.14 0.59 AD-50518.1 0.36 1.05 AD-50523.1 0.03 0.66 AD-50528.1 0.04 0.27 AD-50540.1 0.14 0.37 AD-50539.1 0.09 0.46 0.39 1.10 AD-50544.1 0.23 0.75 0.36 1.07 AD-50549.1 0.10 0.19 0.17 0.71 AD-50514.1 0.12 0.48 0.19 0.95 AD-50522.1 0.61 1.02 0.46 1.32 AD-50527.1 0.06 0.15 0.08 0.45 AD-50531.1 0.09 0.47 0.24 1.04 AD-50534.1 0.05 0.10 0.11 0.55 AD-50538.1 0.61 0.86 0.79 1.23 AD-50543.1 0.40 1.04 0.49 1.23 AD-50553.1 0.40 0.93 0.72 1.25 AD-50504.1 ND ND 0.92 1.25 AD-50509.1 ND ND 0.12 0.37 AD-50529.1 ND ND 0.23 0.47 .sup.1Modified.

TABLE-US-00007 TABLE 6 Serpinc1 IC.sub.50 Data Duplex Hep3B IC.sub.50 PMH IC.sub.50 Name (nM) (nM) AD-50487.1 0.003 -- AD-50477.1 0.006 -- AD-50483.1 0.011 -- AD-50475.1 0.011 -- AD-50495.1 0.017 -- AD-50476.1 0.026 -- AD-50499.1 0.027 -- AD-50478.1 0.028 -- AD-50489.1 0.029 -- AD-50501.1 0.045 -- AD-50507.1 0.052 -- AD-50484.1 0.081 -- AD-50515.1 0.185 -- AD-50540.1 0.023 -- AD-50528.1 0.056 -- AD-50549.1 0.053 ND AD-50539.1 0.170 ND AD-50534.1 0.007 ND AD-50527.1 0.028 ND AD-50514.1 0.085 ND AD-50527.1 ND 0.019 AD-50534.1 ND 0.011 AD-50509.1 ND 0.006 AD-50529.1 ND 0.021

[0452] A subset of siRNAs were also synthesized with 2'-OMe modifications, and duplexes of these siRNAs in lipofectamine formulations were used to transfect Hep3B cells. The results of the single dose screen of the modified duplexes are shown in Table 7.

TABLE-US-00008 TABLE 7 Lead Optimization (2'-OMe variants) Parent Duplex ID Ave 1 nM Ave 0.1 nM Ave 0.01 nM AD-50477 AD-50477.1 0.22 0.33 0.53 AD-50477 AD-55025.1 0.29 0.68 0.86 AD-50477 AD-55031.1 0.42 0.74 0.93 AD-50477 AD-55037.1 0.52 0.73 0.95 AD-50477 AD-55043.1 0.45 0.70 0.94 AD-50477 AD-55049.1 0.24 0.47 0.95 AD-50477 AD-55055.1 0.37 0.68 0.99 AD-50477 AD-55061.1 0.43 0.66 0.85 AD-50477 AD-55067.1 0.56 0.72 0.92 AD-50477 AD-55026.1 0.28 0.59 0.87 AD-50477 AD-55032.1 0.49 0.76 0.86 AD-50477 AD-55038.1 0.52 0.75 0.93 AD-50477 AD-55044.1 0.84 0.77 1.06 AD-50487 AD-50487.1 0.21 0.50 0.76 AD-50487 AD-55050.1 0.24 0.53 0.75 AD-50487 AD-55056.1 0.27 0.50 0.84 AD-50487 AD-55062.1 0.30 0.61 0.84 AD-50487 AD-55068.1 0.20 0.37 0.66 AD-50487 AD-55027.1 0.18 0.36 0.67 AD-50487 AD-55033.1 0.22 0.43 0.70 AD-50487 AD-55039.1 0.19 0.38 0.67 AD-50487 AD-55045.1 0.18 0.29 0.57 AD-50487 AD-55051.1 0.17 0.29 0.60 AD-50487 AD-55057.1 0.21 0.37 0.65 AD-50487 AD-55063.1 0.19 0.33 0.63 AD-50487 AD-55069.1 0.16 0.26 0.51 AD-50509 AD-50509.1 0.15 0.31 0.57 AD-50509 AD-55029.1 0.17 0.26 0.49 AD-50509 AD-55028.1 0.17 0.35 0.54 AD-50509 AD-55052.1 0.21 0.32 0.59 AD-50509 AD-55035.1 0.19 0.31 0.62 AD-50509 AD-55047.1 0.19 0.35 0.66 AD-50509 AD-55058.1 0.21 0.40 0.66 AD-50509 AD-55046.1 0.18 0.37 0.66 AD-50509 AD-55070.1 0.17 0.40 0.68 AD-50509 AD-55034.1 0.19 0.37 0.69 AD-50509 AD-55041.1 0.20 0.28 0.63 AD-50509 AD-55064.1 0.19 0.34 0.65 AD-50509 AD-55040.1 0.18 0.34 0.69 AD-50534 AD-50534.1 0.19 0.42 0.83 AD-50534 AD-55053.1 0.24 0.38 0.59 AD-50534 AD-55030.1 0.15 0.33 0.64 AD-50534 AD-55054.1 0.18 0.40 0.69 AD-50534 AD-55059.1 0.18 0.33 0.56 AD-50534 AD-55036.1 0.22 0.37 0.61 AD-50534 AD-55060.1 0.19 0.42 0.62 AD-50534 AD-55071.1 0.29 0.56 0.81 AD-50534 AD-55048.1 0.26 0.56 0.83 AD-50534 AD-55066.1 0.30 0.49 0.76 AD-50534 AD-55042.1 0.25 0.47 0.79 AD-50534 AD-55065.1 0.24 0.50 0.83

Examples 3-4

Lead Optimization and In Vivo Testing

[0453] Table 8 is a detailed list of sequences of duplex siRNAs targeting Serpinc1 that were formulated as a lipid nanoparticle (LNP) (i.e., with MC3) or conjugated to GalNAc for lead optimization and in vivo delivery.

TABLE-US-00009 TABLE 8 Sense and antisense strand sequences of Serpinc1 dsRNAs for lead optimization (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 243-510, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 511-778, respectively, in order of appearance) Duplex Name Sense Name Sense Sequence Antisense Name Antisense Sequences AD-50477.1 A-104665.1 cuAucGAAAAGccAAcAAAdTsdT A-104666.1 UUUGUUGGCUUUUCGAuAGdTsdT AD-55025.1 A-113301.1 cuAucGAAAAGccAAcAAAdTdT A-113302.1 UUUGUUGGCUUUUcGAuAGdTdT AD-55031.1 A-113301.2 cuAucGAAAAGccAAcAAAdTdT A-113303.1 UUUGUUGGCUUuUcGAuAGdTdT AD-55037.1 A-113301.3 cuAucGAAAAGccAAcAAAdTdT A-113304.1 UUUGUUGGcUUuUcGAuAGdTdT AD-55043.1 A-113301.4 cuAucGAAAAGccAAcAAAdTdT A-113305.1 UUUGuUGGcUUuUcGAuAGdTdT AD-55049.1 A-113306.1 cuAucGAAAAGcCAAcAAAdTdT A-113302.2 UUUGUUGGCUUUUcGAuAGdTdT AD-55055.1 A-113306.2 cuAucGAAAAGcCAAcAAAdTdT A-113303.2 UUUGUUGGCUUuUcGAuAGdTdT AD-55061.1 A-113306.3 cuAucGAAAAGcCAAcAAAdTdT A-113304.2 UUUGUUGGcUUuUcGAuAGdTdT AD-55067.1 A-113306.4 cuAucGAAAAGcCAAcAAAdTdT A-113305.2 UUUGuUGGcUUuUcGAuAGdTdT AD-55026.1 A-113307.1 cuAucGAAAAGcCAACAAAdTdT A-113302.3 UUUGUUGGCUUUUcGAuAGdTdT AD-55032.1 A-113307.2 cuAucGAAAAGcCAACAAAdTdT A-113303.3 UUUGUUGGCUUuUcGAuAGdTdT AD-55038.1 A-113307.3 cuAucGAAAAGcCAACAAAdTdT A-113304.3 UUUGUUGGcUUuUcGAuAGdTdT AD-55044.1 A-113307.4 cuAucGAAAAGcCAACAAAdTdT A-113305.3 UUUGuUGGcUUuUcGAuAGdTdT AD-50487.1 A-104637.1 ccAuGAAucccAuGuGcAudTsdT A-104638.1 AUGcAcAUGGGAUUcAUGGdTsdT AD-55050.1 A-113308.1 ccAuGAAucccAuGuGcAudTdT A-113309.1 AUGcAcAUGGGAUUcAuGGdTdT AD-55056.1 A-113308.2 ccAuGAAucccAuGuGcAudTdT A-113310.1 AUGCAcAUGGGAUUcAuGGdTdT AD-55062.1 A-113308.3 ccAuGAAucccAuGuGcAudTdT A-113311.1 AUGCAcAUGGGAuUcAuGGdTdT AD-55068.1 A-113308.4 ccAuGAAucccAuGuGcAudTdT A-113312.1 AUGCACAUGGGAuUcAuGgdTdT AD-55027.1 A-113313.1 ccAuGAAucccAuGUGcAUdTdT A-113309.2 AUGcAcAUGGGAUUcAuGGdTdT AD-55033.1 A-113313.2 ccAuGAAucccAuGUGcAUdTdT A-113310.2 AUGCAcAUGGGAUUcAuGGdTdT AD-55039.1 A-113313.3 ccAuGAAucccAuGUGcAUdTdT A-113311.2 AUGCAcAUGGGAuUcAuGGdTdT AD-55045.1 A-113313.4 ccAuGAAucccAuGUGcAUdTdT A-113312.2 AUGCACAUGGGAuUcAuGgdTdT AD-55051.1 A-113314.1 ccAuGAAucCcAuGUGcAUdTdT A-113309.3 AUGcAcAUGGGAUUcAuGGdTdT AD-55057.1 A-113314.2 ccAuGAAucCcAuGUGcAUdTdT A-113310.3 AUGCAcAUGGGAUUcAuGGdTdT AD-55063.1 A-113314.3 ccAuGAAucCcAuGUGcAUdTdT A-113311.3 AUGCAcAUGGGAuUcAuGGdTdT AD-55069.1 A-113314.4 ccAuGAAucCcAuGUGcAUdTdT A-113312.3 AUGCACAUGGGAuUcAuGgdTdT AD-50509.1 A-104661.1 cuGccGAcucuAucGAAAAdTsdT A-104662.1 UUUUCGAuAGAGUCGGcAGdTsdT AD-55029.1 A-113321.1 cuGccGAcuCuAuCGAaAAdTdT A-113316.3 UUUUCGAuAGAGUCGGcAgdTdT AD-55028.1 A-113315.1 cuGccGAcucuAucGAAAAdTdT A-113316.1 UUUUCGAuAGAGUCGGcAgdTdT AD-55052.1 A-113320.1 cuGccGAcuCuAuCGAAAAdTdT A-113316.2 UUUUCGAuAGAGUCGGcAgdTdT AD-55035.1 A-113321.2 cuGccGAcuCuAuCGAaAAdTdT A-113317.3 UUUUCGAuAGAGUCgGcAgdTdT AD-55047.1 A-113321.4 cuGccGAcuCuAuCGAaAAdTdT A-113319.3 UUuUCGAUAGAGUCgGcAgdTdT AD-55058.1 A-113320.2 cuGccGAcuCuAuCGAAAAdTdT A-113317.2 UUUUCGAuAGAGUCgGcAgdTdT AD-55046.1 A-113315.4 cuGccGAcucuAucGAAAAdTdT A-113319.1 UUuUCGAUAGAGUCgGcAgdTdT AD-55070.1 A-113320.4 cuGccGAcuCuAuCGAAAAdTdT A-113319.2 UUuUCGAUAGAGUCgGcAgdTdT AD-55034.1 A-113315.2 cuGccGAcucuAucGAAAAdTdT A-113317.1 UUUUCGAuAGAGUCgGcAgdTdT AD-55041.1 A-113321.3 cuGccGAcuCuAuCGAaAAdTdT A-113318.3 UUuUCGAuAGAGUCgGcAgdTdT AD-55064.1 A-113320.3 cuGccGAcuCuAuCGAAAAdTdT A-113318.2 UUuUCGAuAGAGUCgGcAgdTdT AD-55040.1 A-113315.3 cuGccGAcucuAucGAAAAdTdT A-113318.1 UUuUCGAuAGAGUCgGcAgdTdT AD-50534.1 A-104769.1 ucuucuuuGccAAAcuGAAdTsdT A-104770.1 UUcAGUUUGGcAAAGAAGAdTsdT AD-55053.1 A-113322.1 ucuucuuuGccAAAcuGAAdTdT A-113323.1 UUcAGUUUGGcAAAGAAGadTdT AD-55030.1 A-113327.1 ucuucuuuGcCAAACuGAAdTdT A-113323.2 UUcAGUUUGGcAAAGAAGadTdT AD-55054.1 A-113327.5 ucuucuuuGcCAAACuGAAdTdT A-113328.1 UUCAGUUuGGcAAAGAAGadTdT AD-55059.1 A-113322.2 ucuucuuuGccAAAcuGAAdTdT A-113324.1 UUcAGUUUGGcAAAgAAGadTdT AD-55036.1 A-113327.2 ucuucuuuGcCAAACuGAAdTdT A-113324.2 UUcAGUUUGGcAAAgAAGadTdT AD-55060.1 A-113327.6 ucuucuuuGcCAAACuGAAdTdT A-113329.1 UUCAGUUuGGcAAAgAAGadTdT AD-55071.1 A-113322.4 ucuucuuuGccAAAcuGAAdTdT A-113326.1 UUcAGUUUGGCaAAgAAGadTdT AD-55048.1 A-113327.4 ucuucuuuGcCAAACuGAAdTdT A-113326.2 UUcAGUUUGGCaAAgAAGadTdT AD-55066.1 A-113327.7 ucuucuuuGcCAAACuGAAdTdT A-113330.1 UUCAGUUuGGcaAAgAAGadTdT AD-55042.1 A-113327.3 ucuucuuuGcCAAACuGAAdTdT A-113325.2 UUcAGUUUGGcaAAgAAGadTdT AD-55065.1 A-113322.3 ucuucuuuGccAAAcuGAAdTdT A-113325.1 UUcAGUUUGGcaAAgAAGadTdT AD-54944.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-54951.1 A-112997.1 UfaGfuAfuCfaGfCfCfaAfuCfgCfcUfuUfL96 A-112998.1 aAfaGfgCfgAfuUfggcUfgAfuAfcUfasAfsc AD-54942.1 A-113041.1 CfgCfuUfuGfcUfAfCfcAfcUfuUfcUfaUfL96 A-113042.1 aUfaGfaAfaGfuGfguaGfcAfaAfgCfgsGfsg AD-54948.1 A-113043.1 CfuUfuGfcUfaCfCfAfcUfuUfcUfaUfcAfL96 A-113044.1 uGfaUfaGfaAfaGfuggUfaGfcAfaAfgsCfsg AD-54957.1 A-112999.1 AfuCfgAfaAfaGfCfCfaAfcAfaAfuCfcUfL96 A-113000.1 aGfgAfuUfuGfuUfggcUfuUfuCfgAfusAfsg AD-54933.1 A-112991.1 UfcCfcAfuGfaAfUfCfcCfaUfgUfgCfaUfL96 A-112992.1 aUfgCfaCfaUfgGfgauUfcAfuGfgGfasAfsu AD-54962.1 A-113079.1 UfaAfgGfcAfuUfUfCfuUfgAfgGfuAfaAfL96 A-113080.1 uUfuAfcCfuCfaAfgaaAfuGfcCfuUfasUfsg AD-54972.1 A-113051.1 GfcAfaCfuGfaUfGfGfaGfgUfaUfuUfaAfL96 A-113052.1 uUfaAfaUfaCfcUfccaUfcAfgUfuGfcsUfsg AD-54949.1 A-113059.1 UfuUfaAfgUfuUfGfAfcAfcCfaUfaUfcUfL96 A-113060.1 aGfaUfaUfgGfuGfucaAfaCfuUfaAfasUfsa AD-54936.1 A-113039.1 CfcAfuGfaAfuCfCfCfaUfgUfgCfaUfuUfL96 A-113040.1 aAfaUfgCfaCfaUfgggAfuUfcAfuGfgsGfsa AD-54971.1 A-113035.1 UfgCfuGfgUfuAfAfCfaCfcAfuUfuAfcUfL96 A-113036.1 aGfuAfaAfuGfgUfguuAfaCfcAfgCfasCfsc AD-54955.1 A-113061.1 UfcUfaUfcGfaAfAfAfgCfcAfaCfaAfaUfL96 A-113062.1 aUfuUfgUfuGfgCfuuuUfcGfaUfaGfasGfsu AD-54953.1 A-113029.1 UfuCfcCfgCfuUfUfGfcUfaCfcAfcUfuUfL96 A-113030.1 aAfaGfuGfgUfaGfcaaAfgCfgGfgAfasUfsu AD-54937.1 A-113055.1 GfaUfgGfaGfgUfAfUfuUfaAfgUfuUfgAfL96 A-113056.1 uCfaAfaCfuUfaAfauaCfcUfcCfaUfcsAfsg AD-54967.1 A-113065.1 AfgAfcAfaAfuCfCfCfuUfaCfcUfuCfaAfL96 A-113066.1 uUfgAfaGfgUfaAfgggAfuUfuGfuCfusCfsc AD-54935.1 A-113023.1 UfgCfuAfcCfaCfUfUfuCfuAfuCfaGfcAfL96 A-113024.1 uGfcUfgAfuAfgAfaagUfgGfuAfgCfasAfsa AD-54976.1 A-113037.1 UfgUfaUfuCfcAfAfUfgUfgAfuAfgGfaAfL96 A-113038.1 uUfcCfuAfuCfaCfauuGfgAfaUfaCfasUfsg AD-54965.1 A-113033.1 AfaCfuGfcCfgAfCfUfcUfaUfcGfaAfaAfL96 A-113034.1 uUfuUfcGfaUfaGfaguCfgGfcAfgUfusCfsa AD-54959.1 A-113031.1 GfcCfgAfcUfcUfAfUfcGfaAfaAfgCfcAfL96 A-113032.1 uGfgCfuUfuUfcGfauaGfaGfuCfgGfcsAfsg AD-54943.1 A-113057.1 UfaUfuUfaAfgUfUfUfgAfcAfcCfaUfaUfL96 A-113058.1 aUfaUfgGfuGfuCfaaaCfuUfaAfaUfasCfsc AD-54956.1 A-113077.1 UfgGfgCfcUfuGfUfCfgAfuCfuGfuUfcAfL96 A-113078.1 uGfaAfcAfgAfuCfgacAfaGfgCfcCfasUfsg AD-54973.1 A-113067.1 CfaAfaUfcCfcUfUfAfcCfuUfcAfaUfgAfL96 A-113068.1 uCfaUfuGfaAfgGfuaaGfgGfaUfuUfgsUfsc AD-54975.1 A-113021.1 UfcCfaAfaCfuCfCfCfaGfgUfaUfuGfuUfL96 A-113022.1 aAfcAfaUfaCfcUfgggAfgUfuUfgGfasCfsu AD-54963.1 A-113001.1 GfaAfcUfgCfcGfAfCfuCfuAfuCfgAfaAfL96 A-113002.1 uUfuCfgAfuAfgAfgucGfgCfaGfuUfcsAfsg AD-54978.1 A-113069.1 UfcAfaCfaAfaUfGfGfgUfgUfcCfaAfuAfL96 A-113070.1 uAfuUfgGfaCfaCfccaUfuUfgUfuGfasUfsg AD-54952.1 A-113013.1 CfaCfuGfuUfcUfGfGfuGfcUfgGfuUfaAfL96 A-113014.1 uUfaAfcCfaGfcAfccaGfaAfcAfgUfgsAfsg AD-54950.1 A-113075.1 GfgAfcGfgCfuUfCfAfgUfuUfgAfaGfgAfL96 A-113076.1 uCfcUfuCfaAfaCfugaAfgCfcGfuCfcsUfsc AD-54964.1 A-113017.1 CfuGfgAfcUfuCfAfAfgGfaAfaAfuGfcAfL96 A-113018.1 uGfcAfuUfuUfcCfuugAfaGfuCfcAfgsGfsg AD-54974.1 A-113005.1 AfgGfuAfuUfuAfAfGfuUfuGfaCfaCfcAfL96 A-113006.1 uGfgUfgUfcAfaAfcuuAfaAfuAfcCfusCfsc

AD-54969.1 A-113003.1 UfuAfcUfuCfaAfGfGfgCfcUfgUfgGfaAfL96 A-113004.1 uUfcCfaCfaGfgCfccuUfgAfaGfuAfasAfsu AD-54961.1 A-113063.1 UfuUfuUfgGfaGfAfCfaAfaUfcCfcUfuAfL96 A-113064.1 uAfaGfgGfaUfuUfgucUfcCfaAfaAfasGfsg AD-54968.1 A-113081.1 GfaUfuGfcUfgGfCfCfgUfuCfgCfuAfaAfL96 A-113082.1 uUfuAfgCfgAfaCfggcCfaGfcAfaUfcsAfsc AD-54947.1 A-113027.1 CfcGfaCfuCfuAfUfCfgAfaAfaGfcCfaAfL96 A-113028.1 uUfgGfcUfuUfuCfgauAfgAfgUfcGfgsCfsa AD-54941.1 A-113025.1 CfaCfcAfuUfuAfCfUfuCfaAfgGfgCfcUfL96 A-113026.1 aGfgCfcCfuUfgAfaguAfaAfuGfgUfgsUfsu AD-54966.1 A-113049.1 CfaAfgCfuGfgGfUfGfcCfuGfuAfaUfgAfL96 A-113050.1 uCfaUfuAfcAfgGfcacCfcAfgCfuUfgsGfsu AD-54940.1 A-113009.1 AfcAfcUfaUfuAfUfCfuUfcAfuGfgGfcAfL96 A-113010.1 uGfcCfcAfuGfaAfgauAfaUfaGfuGfusUfsc AD-54958.1 A-113015.1 AfgGfaAfaAfuGfCfAfgAfgCfaAfuCfcAfL96 A-113016.1 uGfgAfuUfgCfuCfugcAfuUfuUfcCfusUfsg AD-54938.1 A-113071.1 UfcUfgGfuGfcUfGfGfuUfaAfcAfcCfaUfL96 A-113072.1 aUfgGfuGfuUfaAfccaGfcAfcCfaGfasAfsc AD-54934.1 A-113007.1 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uUfgAfaGfuAfaAfuggUfgUfUfAfAfCfcsAfsg AD-56442.2 A-113073.19 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115917.1 uUfgAfaGfuAfaAfuggUfgUfuAfAfCfCfsAfsg AD-56448.2 A-113073.20 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115918.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfCfsAfsGf AD-56454.2 A-113073.21 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115919.1 uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56460.2 A-113073.22 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115920.1 uUfgaaguAfaAfuggUfgUfuAfaCfcsAfsg AD-56420.2 A-113073.23 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115921.1 uUfgAfaguaaAfuggUfgUfuAfaCfcsAfsg AD-56426.2 A-113073.24 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115922.1 uUfgAfaGfuaaauggUfgUfuAfaCfcsAfsg AD-56432.2 A-113073.25 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115923.1 uUfgAfaGfuAfaauggugUfuAfaCfcsAfsg AD-56437.2 A-113073.26 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115924.1 uUfgAfaGfuAfaAfugguguuAfaCfcsAfsg AD-56443.2 A-113073.27 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115925.1 uUfgAfaGfuAfaAfuggUfguuaaCfcsAfsg AD-56449.2 A-113073.28 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115926.1 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg AD-56455.2 A-113073.29 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115927.1 uUfgAfaGfuAfaAfuggUfgUfuAfaccsasg AD-56461.2 A-115888.2 GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96 A-115919.2 uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56421.2 A-115889.2 GfgUfuAfaCfaCfCfAfuUfuAfCfUfUfCfaAfL96 A-115920.2 uUfgaaguAfaAfuggUfgUfuAfaCfcsAfsg AD-56427.2 A-115890.2 GfgUfuAfaCfaCfCfAfuUfUfAfCfUfuCfaAfL96 A-115921.2 uUfgAfaguaaAfuggUfgUfuAfaCfcsAfsg AD-56433.2 A-115891.2 GfgUfuAfaCfaCfCfAfUfUfUfAfcUfuCfaAfL96 A-115922.2 uUfgAfaGfuaaauggUfgUfuAfaCfcsAfsg AD-56438.2 A-115892.2 GfgUfuAfaCfAfCfCfAfUfUfuAfcUfuCfaAfL96 A-115923.2 uUfgAfaGfuAfaauggugUfuAfaCfcsAfsg AD-56444.2 A-115893.2 GfgUfuAfAfCfAfCfCfAfuUfuAfcUfuCfaAfL96 A-115924.2 uUfgAfaGfuAfaAfugguguuAfaCfcsAfsg AD-56450.2 A-115894.2 GfgUfUfAfAfCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115925.2 uUfgAfaGfuAfaAfuggUfguuaaCfcsAfsg AD-56456.2 A-115895.2 GfGfUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115926.2 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg AD-56462.2 A-115896.2 GfgUfuAfaCfaCfCfAfuUfuAfcUfucaaL96 A-115907.2 UfUfGfAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56422.2 A-115897.2 GfgUfuAfaCfaCfCfAfuUfuAfcuucaAfL96 A-115908.2 uUfGfAfAfGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56428.2 A-115898.2 GfgUfuAfaCfaCfCfAfuUfuacuuCfaAfL96 A-115909.2 uUfgAfAfGfUfAfaAfuggUfgUfuAfaCfcsAfsg AD-56434.2 A-115899.2 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 A-115910.2 uUfgAfaGfUfAfAfAfuggUfgUfuAfaCfcsAfsg AD-56439.2 A-115900.2 GfgUfuAfaCfaCfcauuuAfcUfuCfaAfL96 A-115911.2 uUfgAfaGfuAfAfAfUfGfgUfgUfuAfaCfcsAfsg AD-56445.2 A-115901.2 GfgUfuAfaCfaccauUfuAfcUfuCfaAfL96 A-115912.2 uUfgAfaGfuAfaAfUfGfGfUfgUfuAfaCfcsAfsg AD-56451.2 A-115902.2 GfgUfuAfacaccAfuUfuAfcUfuCfaAfL96 A-115913.2 uUfgAfaGfuAfaAfuGfGfUfGfUfuAfaCfcsAfsg AD-56457.2 A-115903.2 GfgUfuAfacaCfCfAfuuuAfcUfuCfaAfL96 A-115914.2 uUfgAfaGfuAfAfAfuggUfGfUfuAfaCfcsAfsg AD-56463.2 A-115904.2 GfgUfuaacaCfCfAfuUfuAfcUfuCfaAfL96 A-115915.2 uUfgAfaGfuAfaAfuggUfGfUfUfAfaCfcsAfsg AD-56423.2 A-115905.2 GfguuaaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115916.2 uUfgAfaGfuAfaAfuggUfgUfUfAfAfCfcsAfsg AD-56429.2 A-115906.2 gguuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115917.2 uUfgAfaGfuAfaAfuggUfgUfuAfAfCfCfsAfsg AD-56440.2 A-113073.31 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115928.1 uUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfsg AD-56446.2 A-115929.1 GfgUfuAfaCfacCfAfuUfuAfcUfuCfaAfL96 A-115928.2 uUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfsg AD-56452.2 A-113073.32 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115930.1 UUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfsg AD-56458.2 A-115929.2 GfgUfuAfaCfacCfAfuUfuAfcUfuCfaAfL96 A-115930.2 UUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfsg AD-56464.2 A-113073.33 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115931.1 uUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg AD-56424.2 A-115929.3 GfgUfuAfaCfacCfAfuUfuAfcUfuCfaAfL96 A-115931.2 uUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg AD-56430.2 A-115932.1 GfgUfuAfaCfaCCfAfuUfuAfcUfuCfaAfL96 A-115931.3 uUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg AD-56435.2 A-113073.34 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115933.1 UUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg AD-56441.2 A-115929.4 GfgUfuAfaCfacCfAfuUfuAfcUfuCfaAfL96 A-115933.2 UUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg AD-56447.2 A-115932.2 GfgUfuAfaCfaCCfAfuUfuAfcUfuCfaAfL96 A-115933.3 UUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg AD-56453.2 A-115934.1 GfgUfuAfaCfaUfCfAfuUfuAfcUfuCfaAfL96 A-113074.39 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56459.2 A-115935.1 GfgUfuAfaCfaAfCfAfuUfuAfcUfuCfaAfL96 A-113074.40 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56465.2 A-115936.1 GfgUfuAfaCfaCfUfAfuUfuAfcUfuCfaAfL96 A-113074.41 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56471.2 A-115937.1 GfgUfuAfaCfaCfAfAfuUfuAfcUfuCfaAfL96 A-113074.42 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56477.2 A-113073.35 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115938.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfc AD-56483.3 A-113073.36 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115939.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfsc AD-56488.2 A-113073.37 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115940.1 uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc AD-56483.4 A-113073.38 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115939.2 uUfgAfaGfuAfaAfuggUfgUfuAfaCfsc AD-56497.2 A-115941.1 UfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115940.2 uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc AD-56502.2 A-115941.2 UfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115942.1 uUfgAfaGfuAfaAfuggUfgUfuAfa AD-56466.2 A-115941.3 UfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115943.1 uUfgAfaGfuAfaAfuggUfgUfuAfsa AD-56472.2 A-115941.4 UfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115944.1 uUfgAfaGfuAfaAfuggUfgUfusAfsa AD-56478.2 A-115945.1 AfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115943.2 uUfgAfaGfuAfaAfuggUfgUfuAfsa AD-56484.2 A-115945.2 AfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115944.2 uUfgAfaGfuAfaAfuggUfgUfusAfsa AD-56493.2 A-113073.39 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115948.1 uUfgAf(Aeo)GfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56498.2 A-113073.40 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115949.1

uUfgAfa(Geo)uAfaAfuggUfgUfuAfaCfcsAfsg AD-56467.2 A-113073.42 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115951.1 uUfgAfaGfu(Aeo)aAfuggUfgUfuAfaCfcsAfsg AD-56473.2 A-113073.43 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115952.1 uUfgAfaGfuAf(Aeo)AfuggUfgUfuAfaCfcsAfsg AD-56479.2 A-113073.44 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115953.1 uUfgAfaGfuAfa(Aeo)uggUfgUfuAfaCfcsAfsg AD-56485.2 A-113073.45 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115954.1 uUfgAfaGfuAfaAfugg(Teo)gUfuAfaCfcsAfsg AD-56490.2 A-113073.46 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115955.1 uUfgAfaGfuAfaAfuggUf(Geo)UfuAfaCfcsAfsg AD-56494.2 A-113073.47 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115956.1 uUfgAfaGfuAfaAfuggUfg(Teo)uAfaCfcsAfsg AD-56499.2 A-113073.48 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115957.1 uUfgAfaGfuAfaAfuggUfgUf(Teo)AfaCfcsAfsg AD-56504.2 A-113073.49 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115958.1 uUfgAfaGfuAfaAfuggUfgUfu(Aeo)aCfcsAfsg AD-56468.2 A-113073.50 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115959.1 uUfgAfaGfuAfaAfuggUfgUfuAf(Aeo)CfcsAfsg AD-56474.2 A-113073.51 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115960.1 uUfgAfaGfuAfaAfuggUfgUfuAfa(m5Ceo)csAfsg AD-56480.2 A-113073.52 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115961.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCf(m5Ceos)Afsg AD-56486.2 A-113073.53 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115962.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs(Aeos)(Geo) AD-56491.2 A-113073.54 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-115963.1 uUfgAfaGfuAfaAf(Teo)(Geo)(Geo)UfgUfusAfsa AD-56495.2 A-115964.1 GfgUfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 A-113074.43 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56500.2 A-115965.1 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 A-113074.44 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56505.2 A-115966.1 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 A-113074.45 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56469.2 A-115967.1 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAf(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 A-113074.46 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56475.2 A-115968.1 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(m5Ceo)Uf(Teo) A-113074.47 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg Cf(Aeo)AfL96 AD-56481.2 A-115969.1 Gf(Geo)Uf(Teo)AfaCfaCfCfAf(Teo)Uf(Teo)Af(m5Ceo)Uf A-113074.48 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg (Teo)Cf(Aeo)AfL96 AD-56487.2 A-115970.1 Gf(Geo)Uf(Teo)Af(Aeo)Cf(Aeo)CfCfAf(Teo)Uf(Teo)Af(m5 A-113074.49 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg Ceo)Uf(Teo)Cf(Aeo)AfL96

Example 3

LNP-Mediated Delivery of siRNAs

[0454] Based on the in vitro single dose and IC.sub.50 results described above, modified AD-50509 was selected for formulation in a lipid nanoparticle (LNP). In order to determine an effective dose for LNP-mediated delivery of AD-50509, CD1 mice were intravenously injected with a single dose of an LNP formulation (AF-011) of AD-50509 siRNA at 0.003, 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg. Animals were sacrificed 48 hours later and the level of Serpinc1 mRNA relative to GAPDH and the level of Serpinc1 protein were determined as described herein. As shown in FIGS. 3A and 3B, the maximum Serpinc1 mRNA silencing of 85% with AF-011-AD-50509 was achieved with an ED.sub.50 of about 0.1 mg/kg (FIG. 3A) and the maximum Serpinc1 protein silencing of 90% was achieved with an ED.sub.50 of about 0.05 mg/kg (FIG. 3B).

[0455] The duration of silencing of an LNP formulation of AD-50509 siRNA (AF-011-50509) was determined in CD1 mice following a single 1 mg/kg intravenous injection of the siRNA. Animals were sacrificed at Day 1, 2, 3, 7, 14, 21, or 28 after administration and the relative level of Serpinc1 mRNA and the level of Serpinc1 protein were determined. FIG. 4A demonstrates that AF-011 formulated AD-50509 achieved Serpinc1 mRNA silencing of about 90% within 24 hours of administration and that there was approximately a 50% recovery in the relative amount of Serpinc1 mRNA by about two weeks after administration. FIG. 4B demonstrates that AF-011 formulated AD-50509 achieved Serpinc1 protein silencing of about 90% within about 72 hours of administration and that there was approximately a 50% recovery in the relative amount of Serpinc1 protein by about two weeks after administration. Serpinc1 activity was also determined by measuring Factor Xa activity using a commercially available kit (Aniara) in CD1 mice following a single 1 mg/kg intravenous injection of the LNP formulated AD-50509 siRNA. Animals were sacrificed at Day 1, 2, 3, 7, 14, 21, or 28 after administration and the relative activity level of Serpinc1 protein and the relative Serpinc1 protein level were determined. FIG. 4C shows that there is good correlation between the level of Serpinc1 protein level and Serpinc1 activity.

Example 4

GalNAc-conjugated siRNAs

[0456] Forty-four modified Serpinc1 siRNA duplexes were conjugated with a trivalent GALNAc at the 3-end of the sense strand. These duplexes were assayed for efficacy in single dose free uptake of the conjugated duplexes in Cynomolgus monkey hepatocytes. Table 9 shows the results of these assays.

TABLE-US-00010 TABLE 9 GalNAc Free-Uptake Single Dose Duplex ID Ave 100 nM Ave 10 nM Ave 0.1 nM AD-54944.1 0.4 0.53 0.94 AD-54951.1 0.37 0.56 1 AD-54942.1 0.38 0.58 1.01 AD-54948.1 0.36 0.6 0.96 AD-54957.1 0.47 0.61 1 AD-54933.1 0.51 0.65 0.96 AD-54962.1 0.48 0.66 0.95 AD-54972.1 0.49 0.66 1.05 AD-54949.1 0.49 0.71 0.96 AD-54936.1 0.54 0.72 1.07 AD-54971.1 0.49 0.72 1 AD-54955.1 0.52 0.74 0.98 AD-54953.1 0.63 0.76 1.07 AD-54937.1 0.64 0.81 0.94 AD-54967.1 0.74 0.82 1.02 AD-54935.1 0.68 0.83 0.99 AD-54976.1 0.7 0.85 1.04 AD-54965.1 0.7 0.86 0.97 AD-54959.1 0.79 0.86 0.95 AD-54943.1 0.75 0.86 0.94 AD-54956.1 0.86 0.87 0.95 AD-54973.1 0.96 0.89 1 AD-54975.1 0.67 0.89 0.99 AD-54963.1 0.73 0.9 0.96 AD-54978.1 0.85 0.9 0.98 AD-54952.1 0.59 0.91 1.11 AD-54950.1 0.89 0.91 0.95 AD-54964.1 0.87 0.93 1.01 AD-54974.1 0.83 0.93 0.96 AD-54969.1 0.87 0.94 0.94 AD-54961.1 0.74 0.94 1.07 AD-54968.1 0.89 0.95 0.91 AD-54947.1 0.92 0.96 0.94 AD-54941.1 0.91 0.96 1 AD-54966.1 0.93 0.97 1.06 AD-54940.1 0.86 0.99 1.03 AD-54958.1 0.97 0.99 1.06 AD-54938.1 0.93 0.99 1.05 AD-54934.1 0.92 1 0.96 AD-54939.1 0.84 1.02 1.02 AD-54960.1 0.98 1.03 1.02 AD-54954.1 1.04 1.03 1.01 AD-54970.1 1.03 1.06 1.01 AD-54946.1 0.83 1.17 1.1

[0457] These duplexes were also assayed for dose response in free uptake and transfection assays.

[0458] Table 10 shows the results of these assays and the rank order of the duplexes for both free uptake and transfection. The 5 duplexes with the best IC.sub.50 are shaded in light gray and the bottom 5 duplexes are shaded in dark gray. The IC.sub.50 rank order of the duplexes is well conserved between free uptake and transfection-mediated uptake of GalNAc conjugates.

TABLE-US-00011 TABLE 10 Dose Response of GalNAc-conjugated duplexes: Free uptake and Transfection ##STR00020##

Example 5

AD-54944 Optimization

[0459] As described in Example 4 above, AD-54944 was among the most active GalNAc-conjugated siRNA duplex as determined by both free uptake and transfection assays and was, thus, selected for further optimization and in vivo testing.

[0460] Twenty-nine compounds were prepared based on the same AD-54944 parent sequence and screened for in vivo efficacy using a single 10 mg/kg dose. Animals (C57BL/6) were injected subcutaneously at Day 0 and sacrificed at Day 3. Serum Serpinc1 protein levels were determined by ELISA assay and the level of Serpinc1 mRNA was determined by QRT-PCR using liver samples from the animals. Tables 11 and 12 show the sequences of the duplexes and the results of the single dose screen with these duplexes as a percent knock-down of Serpinc1 protein levels from PBS. FIG. 5 shows the results of the single dose screen as a percent knock-down of Serpince mRNA and protein levels from PBS.

TABLE-US-00012 TABLE 11 AD-54944 optimized sequences and protein levels. (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 779-808, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 809-838, respectively, in order of appearance) Duplex Name Sense Sequence Antisense Sequence % PBS Std Dev AD-54944 (new) GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 66.8 4.8 AD-56345 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 83.6 19.2 AD-56351 GfguuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 71.7 7.5 AD-56363 GfguuAfaCfaCfCfAfuUfuacUfuCfaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 57.9 7.6 AD-56334 GfguuAfacaCfCfAfuuuacUfucaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 59.4 6.0 AD-56346 GfguuaacaCfCfAfuuuacuucaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 82.3 13.2 AD-56352 GfgUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 52.2 3.6 AD-56410 GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 55.0 2.5 AD-56405 GfGfUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 60.6 3.1 AD-56383 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 UfUfGfAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 66.7 7.6 AD-56730 GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfCfsAfsGf 64.4 3.4 AD-56380 GfgUfUfAfaCfaCfCfAfuUfUfAfcUfUfCfAfAfL96 uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsAfsg 75.7 5.0 AD-56397 GfGfUfUfAfAfCfAfCfCfAfuUfUfAfCfUfUfCfAfAfL96 uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAfsg 76.9 0.8 AD-56434 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgAfaGfUfAfAfAfuggUfgUfuAfaCfcsAfsg 52.0 5.1 AD-56488 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc 85.3 16.6 AD-56486 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs(Aeos)(Geo) 67.7 0.5 AD-56505 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 59.7 0.2 AD-56736 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 uUfgAfaGfuAfaAfuggUf(Geo)UfuAfaCfcsAfsg 49.7 1.2 AD-56738 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 65.0 3.6 AD-56739 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(m5Ceo)Uf(Teo)CfAeo)AfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 57.6 2.1 AD-56740 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 85.5 14.8 AD-56743 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 48.3 3.9 AD-56745 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAf(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 54.4 9.9 AD-56454 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsg 72.0 11.0 AD-56449 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg 54.4 15.9 AD-56746 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg 64.7 17.2 AD-56333 GfguuAfacaCfCfAfuuuacuucaaL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 71.5 11.3 AD-56382 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 65.7 2.7 AD-56748 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 99.4 19.1 AD-54944 (original) GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 46.9 1.7 control 96.0 8.5 PBS 100.0 4.7

TABLE-US-00013 TABLE 12 AD-54944 optimized sequences and protein levels. (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 839-868, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 869-898, respectively, in order of appearance) Duplex Name Sense Sequence Antisense Sequence % PBS Std Dev AD-54944 (original) GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 46.9 1.7 AD-56743 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 48.3 3.9 AD-56736 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 uUfgAfaGfuAfaAfuggUf(Geo)UfuAfaCfcsAfsg 49.7 1.2 AD-56434 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgAfaGfUfAfAfAfuggUfgUfuAfaCfcsAfsg 52.0 5.1 AD-56352 GfgUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 52.2 3.6 AD-56745 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAf(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 54.4 9.9 AD-56449 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg 54.4 15.9 AD-56410 GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 55.0 2.5 AD-56739 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 57.6 2.1 AD-56363 GfguuAfaCfaCfCfAfuUfuacUfuCfaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 57.9 7.6 AD-56334 GfguuAfacaCfCfAfuuuacUfucaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 59.4 6.0 AD-56505 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 59.7 0.2 AD-56405 GfGfUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 60.6 3.1 AD-56730 GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfCfsAfsGf 64.4 3.4 AD-56746 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg 64.7 17.2 AD-56738 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 65.0 3.6 AD-56382 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 65.7 2.7 AD-56383 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 UfUfGfAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 66.7 7.6 AD-54944 (new) GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 66.8 4.8 AD-56486 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs(Aeos)(Geo) 67.7 0.5 AD-56333 GfguuAfacaCfCfAfuuuacuucaaL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 71.5 11.3 AD-56351 GfguuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 71.7 7.5 AD-56454 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsg 72.0 11.0 AD-56380 GfgUfUfAfaCfaCfCfAfuUfUfAfcUfUfCfAfAfL96 uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsAfsg 75.7 5.0 AD-56397 GfGfUfUfAfAfCfAfCfCfAfuUfUfAfCfUfUfCfAfAfL96 uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAfsg 76.9 0.8 AD-56346 GfguuaacaCfCfAfuuuacuucaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 82.3 13.2 AD-56345 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 83.6 19.2 AD-56488 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc 85.3 16.6 AD-56740 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo)AfL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 85.5 14.8 96.0 8.5 AD-56748 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 99.4 19.1 PBS 100.0 4.7

[0461] The in vivo dose response of AD-54944 conjugated to GalNAc was determined by administering a single subcutaneous dose to C57BL/6J mice (n=5). AD-54944 conjugated to GalNAc was also administered subcutaneously as a repeat daily dose of 5 mg/kg to C57BL/6J mice (n=5) over a 5 day period. Animals were sacrificed 72 hours after administration and Serpinc1 protein and activity levels were determined in liver and serum samples as described above.

[0462] As shown in FIG. 6, a single subcutaneous dose of AD-54944 conjugated to GalNAc resulted in a protein EC.sub.50 of about 10 mg/kg and a 5.times.5 mg/kg daily, repeat dose resulted in about a 75% protein silencing.

[0463] Additional repeat-dosing of AD-54944 conjugated to GalNAc in C57BL/6J mice was also performed over an 8 week period to determine the efficacy and duration of silencing. FIGS. 7A and 7B show the results of these studies.

Example 6

Dose Duration of a Split-Dose of AD-54944

[0464] In order to further evaluate compound AD-54944 knock-down of Serpinc1 expression and activity, a split-dosing experiment was performed. C57BL/6 mice were subcutaneously administered GalNAc-conjugated AD-54944 and the effect of a 3 times per week, 1/3 dose of AD-54944 was compared to the effect of a 1 time per week fully concentrated dose of AD-54944. A summary of the study design is presented in Table 13. Serum Serpinc1 protein levels were determined at Days 0, 3, 7, 10, 14, 17, 21, 24, 29, 31, and 35.

TABLE-US-00014 TABLE 13 Study Design of Split- Dosing Test Dose Experiment Group compound (mg/kg) Frequency 1 AD-54944 1.25 3x/week (M, W, 2 2.5 F) 3 5 4 10 5 3.75 1x/week 6 7.5 (Monday) 7 15 8 30 9 PBS --

[0465] The results of the one-time per week split-dose screen as a percent knock-down of Serpinc1 protein levels from pre-dose levels are shown in FIG. 8 and the results of the three-time per week screen as a percent knock-down of Serpinc1 protein levels from pre-dose levels are shown in FIG. 9. The results demonstrate that there is a dose response effect with AD-54944 conjugated to GalNAc in both groups and that dosages at both 30 mg/kg one time per week and at 10 mg/kg three times per week lead to long-term silencing of Serpinc1.

Example 7

Further Optimization of AD-54944

[0466] In order to further improve the efficacy of AD-54944, additional compounds were prepared based on the AD-54944 parent sequence. In general, the modifications included the addition of phosphorothioate linkages, C16(hexadecyl) modifications, 5'-end-caps, and 2'-methyls. The new compounds were screened for in vivo efficacy using both a single 3 mg/kg dose and a single 10 mg/kg dose. Animals (C57BL/6) were injected subcutaneously at Day 0 and sacrificed at Day 3. Serum Serpinc1 protein levels were determined by ELISA assay. The ELISA assay was performed using an Antithrombin III Mouse ELISA kit purchased from Abcam. Briefly, serum was diluted (e.g., about 1:10,000) and used accordingly to manufacturers instructions. The plates were read at 450 nm at the end of the assay.

[0467] Table 14 shows the sequences of the duplexes and the results of the single dose screens with these duplexes as a percent knock-down of Serpinc1 protein levels from PBS. FIGS. 10A and 10B show the results of the single dose screen as a percent knock-down of Serpinc1 protein levels from PBS. As can be seen in Table 14 and FIG. 10, compound AD-56813 emerged as a new lead based on the level of knock-down of Serpinc1 protein levels.

[0468] Further compounds were prepared based on the AD-56813 parent sequence in which the number of 2'-methoxyethyl and phosphorothioate linkages were reduced in order to determine the minimum chemical modifications required for stability of the compounds which maintained activity of the compounds. The new compounds were screened for in vivo efficacy using both a single 3 mg/kg dose and a single 10 mg/kg dose. Animals (C57BL/6) were injected subcutaneously at Day 0 and sacrificed at Day 3. Serpinc1 (AT3) activity and serum Serpinc1 protein levels were determined by ELISA assay. The ELISA assay was performed as described above. Serpinc1 activity was determined using a BIOPHEN (anti-Factor Xa) activity assay kit. Briefly, serum samples were diluted from about 1:20 to about 1:60 and processed according to the manufacturers' instructions. The plates were read at 450 nm at the end of the assay.

[0469] The sequences of the duplexes that were newly prepared and the results of the single dose screens with these duplexes as a percent knock-down of Serpinc1 protein levels from PBS are shown in Table 15. FIG. 11 shows the results of the single dose screen as a percent knock-down of Serpinc1 protein levels from PBS and FIG. 12 shows the results of the single dose screen as a percent knock-down of Serpinc1 activity from PBS.

[0470] As can be seen in Table 15 and FIGS. 11 and 12, although the number of modifications to the compound was dramatically reduced, compound AD-57213 maintained knockdown of Serpinc1 expression and activity and, thus, emerged as a new lead. A single 10 mg/kg dose of AD-57213 led to an ED.sub.90 and a single 3 mg/kg dose led to an ED.sub.50.

TABLE-US-00015 TABLE 14 AD-54944 optimized sequences and protein levels. (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 899-919, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 920-940, respectively, in order of appearance) 3 mpk 10 mpk Sense % % Duplex ID strand Sense sequence Antisense Antisense sequence PBS stdev PBS stdev AD-56813.2 A-116280.6 Gfs(Geo)UfsuAfaCfsaCfsCfsAfuUfsuAfcUfsuCfs A-116278.6 usUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs(Aeos) 0.62 0.04 0.26 0.04 (Aeo)AfL96 (Geo) AD-56789.2 A-116276.12 GfgUfsuAfaCfsaCfsCfsAfuUfsuAfcUfsuCfsaAfL A-116275.7 uUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscsAfsg 0.74 0.11 0.44 0.11 96 AD-56741 A-115968.11 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(mSCeo) A-115861.11 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 0.52 0.09 Uf(Teo)Cf(Aeo)AfL96 AD- A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.93 0.10 0.63 0.10 54944/parent AD-56743 A-115965.5 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo) A-115861.11 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 0.71 0.24 AfL96 AD-56836.2 A-115966.25 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)Cf A-116284.3 uUfgAfaGf(Uhd)AfaAfuggUfgUfuAfaCfcsasg 1.01 0.11 0.73 0.11 (Aeo)AfL96 AD-56797.2 A-115968.8 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(mSCeo) A-116250.6 uUfgAfaGfuAfaAfuggUf(Geo)UfuAfaCfcs(Aeos) 0.87 0.25 0.74 0.25 Uf(Teo)Cf(Aeo)AfL96 (Geo) AD-56801.2 A-116277.6 Gf(Geo)UfsuAfaCfsaCfsCfsAfuUfsuAfcUfsuCfs A-116279.9 uUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs(Aeos) 0.83 0.08 0.77 0.08 (Aeo)AfL96 (Geo) AD-56831.2 A-116290.3 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Uhd)AfcUf(Teo) A-115962.21 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs(Aeos)(Geo) 1.11 0.09 0.78 0.09 Cf(Aeo)AfL96 AD-56830.2 A-116283.3 Gfs(Geo)UfsuAfaCfsaCfCfAfuUfsuAfcUfsuCfs(Aeo) A-116279.12 uUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs(Aeos) 1.01 0.13 0.79 0.13 AfL96 (Geo) AD-56761.2 A-116247.13 gguuaacaCfCfAfuuuacu(Uhd)caaL96 A-116244.6 uUfgaaGfuAfaAfuggUfguuaaccsasg 0.82 0.26 0.83 0.26 AD-56735.2 A-115966.29 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)C A-115962.18 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs(Aeos)(Geo) 1.20 0.15 0.90 0.15 f(Aeo)AfL96 AD-56872 A-115965.5 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf(Aeo) A-116392.1 uUfgaaGfuAfaAfuggUfguuAfaCfcs(Aeos)(Geo) 0.93 0.24 AfL96 AD-56820.2 A-116317.4 gUf(Uhd)AfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-116297.10 uUfgAfaGfuAfaAfuggUfgUfuAfaCfs(m5Ceos)(Aeo) 1.05 0.10 0.94 0.10 AD-56793.2 A-116382.1 gdGuudAdAcadCdCauudTdAcu(Uhd)dCadAL96 A-116373.11 udTdGadAgdTadAdAuggdTdGuudAdAcdCsasg 0.98 0.08 0.99 0.08 PBS/Control 1.00 0.04 1.00 0.04 AD-56873 A-115968.11 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af(m5Ceo) A-116392.1 uUfgaaGfuAfaAfuggUfguuAfaCfcs(Aeos)(Geo) 1.01 0.18 Uf(Teo)Cf(Aeo)AfL96 AD-54965 A-113033.1 AfaCfuGfcCfgAfCfUfcUfaUfcGfaAfaAfL96 A-113034.1 uUfuUfcGfaUfaGfaguCfgGfcAfgUfusCfsa 0.89 0.07 1.03 0.07 AD-56787.2 A-116381.1 gguuaacaccdAudTudAcdT(Uhd)dCasaL96 A-116373.10 udTdGadAgdTadAdAuggdTdGuudAdAcdCsasg 1.03 0.18 1.04 0.18 AD-56840.2 A-116317.5 gUf(Uhd)AfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-116344.5 uUfgAfaGfuAfaAfuggUfgUfuAfaCfs(Chds)(Aeo) 1.38 0.14 1.05 0.14 AD-56834.2 A-116333.3 GbguuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 A-116334.3 uUfgaaGfuAfaAfuggUfguuAfaCfcsasGb 1.17 0.14 1.06 0.14 AD-56841.2 A-115966.26 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf(Teo)C A-116285.3 uUfgAfaGf(Uhd)AfaAfuggUfgUfuAfaCfcs(Aeos) 1.15 0.21 1.09 0.21 f(Aeo)AfL96 (Geo)

TABLE-US-00016 TABLE 15 AD-56813 optimized sequences and protein and activity levels. (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 941-959, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 960-978, respectively, in order of appearance) ELISA Activity Duplex 10 mpk 3 mpk 10 mpk 3 mpk name Sense ID Sense (5' to 3) AS ID Antisense (5' to 3) Av SD Av SD Av SD Av SD AD-57213.1 A-116858.1 GfsgsUfuAfaCfaCfCfAfuUfuA A-116861.1 usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg 0.098 0.021 0.264 0.032 0.099 0.021 0.255 0.030 fcUfuCfaAfL96 AD-57214.1 A-116859.1 GfsgsUfuAfaCfaCfCfAfuUf A-116861.1 usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg 0.140 0.027 0.200 0.132 0.121 0.028 0.123 0.114 (Uhd)AfcUfuCfaAfL96 AD-57205.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfu A-116861.1 usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg 0.147 0.100 0.592 0.094 0.145 0.043 0.542 0.139 AfcUfuCfaAfL96 AD-56813.2 A-116280.6 Gfs(Geo)UfsuAfaCfsaCfsCfs A-116278.6 usUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs 0.161 0.047 0.500 0.033 0.179 0.055 0.583 0.065 AfuUfsuAfcUfsuCfs(Aeo) (Aeos)(Geo) AfL96 AD-57212.1 A-116276.12 GfgUfsuAfaCfsaCfsCfsAfuUfs A-116860.1 usUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs 0.164 0.026 0.355 0.007 0.198 0.011 0.395 0.023 uAfcUfsuCfsaAfL96 asg AD-57204.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAf A-116860.1 usUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs 0.170 0.127 0.659 0.004 0.121 0.094 0.608 0.060 cUfuCfaAfL96 asg AD-56741.2 A-115968 Gf(Geo)Uf(Teo)AfaCfaCfCfA A-115861 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 0.347 0.098 0.666 0.036 0.419 0.126 0.880 0.070 fuUf(Teo)Af(m5Ceo)Uf(Teo) Cf(Aeo)AfL96 AD-57203.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAf A-116278.6 usUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscs 0.359 0.053 0.829 0.056 0.358 0.064 0.796 0.030 cUfuCfaAfL96 (Aeos)(Geo) AD-56765.2 A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAfc A-116275.7 uUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscsA 0.366 0.053 0.668 0.040 0.385 0.041 0.791 0.047 UfuCfaAfL96 fsg AD-56789.2 A-116276.12 GfgUfsuAfaCfsaCfsCfsAfuUf A-116275.7 uUfsgAfaGfuAfaAfuggUfsgUfsuAfaCfscsA 0.401 0.057 0.700 0.059 0.406 0.078 0.785 0.059 suAfcUfsuCfsaAfL96 fsg AD-57211.1 A-116858.1 GfsgsUfuAfaCfaCfCfAfuUfuA A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.446 0.023 0.760 0.142 0.536 0.027 0.831 0.117 fcUfuCfaAfL96 AD-57210.1 A-116857.1 GfsgsUfuAfaCfaCfsCfsAfuUf A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.476 0.028 0.734 0.053 0.547 0.041 0.826 0.065 uAfcUfuCfaAfL96 AD-57208.1 A-116276.12 GfgUfsuAfaCfsaCfsCfsAfuUf A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.542 0.016 0.915 0.085 0.486 0.006 0.831 0.034 suAfcUfsuCfsaAfL96 AD-56475.3 A-115968 Gf(Geo)Uf(Teo)AfaCfaCfCf A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.574 0.058 0.932 0.127 0.415 0.019 0.735 0.123 AfuUf(Teo)Af(m5Ceo)U f(Teo)Cf(Aeo)AfL96 AD-57209.1 A-116280.6 Gfs(Geo)UfsuAfaCfsaCfsCfsA A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.588 0.135 0.966 0.100 0.460 0.136 0.823 0.026 fuUfsuAfcUfsuCfs(Aeo)AfL96 AD- A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAfcU A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.617 0.025 0.715 0.071 0.667 0.016 0.835 0.083 54944.13 fuCfaAfL96 AD-57206.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfu A-116870.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcAfsg 0.681 0.097 0.811 0.110 0.707 0.068 0.940 0.149 AfcUfuCfaAfL96 AD-57208.2 A-116276.12 GfgUfsuAfaCfsaCfsCfsAfuUf A-113074.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 0.736 0.109 0.714 0.101 0.729 0.122 0.817 0.090 suAfcUfsuCfsaAfL96 AD-57207.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfuAf A-116871.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcAfg 0.934 0.043 0.975 0.035 0.984 0.104 1.009 0.066 cUfuCfaAfL96 1.000 0.029 1.000 1.000 0.077 1.000

Example 8

Serpinc1 Knock-Down in Hemophilic Mice

[0471] Male and female mice having a targeted deletion of Factor VIII (C57BL/6/129 hybrids) and recapitulating the hemophilia A phenotype and control or wild-type (C57BL/6 female) mice were subcutaneously injected with a single dose of compound AD-57213 conjugated to GalNAc at 30 mg/kg, 10 mg/kg, 3 mg/kg, or 1 mg/kg at Day 0, animals were sacrificed at Day 3 and Serpinc1 activity was determined as described above. FIG. 13 shows that, not only does a single dose of AD-57213 effectively knock-down Serpinc1 activity, but there is also a dose response to AD-57213.

[0472] To investigate the impact of antithrombin reduction on thrombin generation in a hemophilia setting, thrombin generation studies were performed on Factor IX (FIX) and Antithrombin- (AT-) depleted human plasma. FIX depletion recapitulates the hemophilia B phenotype AT was subsequently added back to the plasma samples at various levels (1 IU/ml=100%) to generate FIX-depleted plasma samples with different levels of antithrombin (0-100%). Control plasma was generated by adding back 1 IU/ml antithrombin and 5 .mu.g/ml FIX (100%) to the double-depleted plasma. FIG. 31A depicts thrombin generation in FIX- and AT-depleted human plasma (tissue factor=5 pM). FIG. 31B depicts the peak thrombin in FIX- and AT-depleted human plasma (tissue factor=5 pM). Therefore, antithrombin reduction increases thrombin generation in Factor IX-depleted human plasma in vitro.

Example 9

Dose Duration of AD-57213

[0473] In order to evaluate the duration of anti-thrombin silencing in Hemophilia A mice (B6; 129S4-F8.sup.tml/Kaz/J; Jackson Labs) following a single dose of AD-57213 conjugated to GalNAc, mice were subcutaneously injected with compounds AD-57214, AD-57205, or AD-57213 or PBS. Whole blood was collected retroorbitally and assayed for Serpinc1 mRNA levels and Serpinc1 activity. The results of the single dose screen for compound AD-57213 administered at 10 mg/kg, 3 mg/kg, or 1 mg/kg as a percent knock-down of Serpinc1 activity from PBS at Days 3, 7, 10, 14, 17, 21, 28, and 36 are depicted in FIG. 14. FIG. 16 shows the results of the single dose screen for compounds AD-57213, AD-57205, and AD-57214 administered at the doses indicated in the Figures as a percent knock-down of Serpinc1 activity from PBS at Days 3, 7, 10, 14, 17, 21, and 25.

[0474] Liver mRNA, AT antigen in serum and AT activity were measured in hemophilia A mice (B6; 129S4-F8.sup.tml/Kaz/J; Jackson labs) injected subcutaneously with AD-57213 at a dose of 30 mg/kg, 10 mg/kg, 3 mg/kg, 1 mg/kg, or PBS at Day 0. Animals were sacrificed on Day 3 post-injection as described above. FIG. 15 shows the results of the single dose screen as a percent knock-down of Serpinc1 mRNA levels from PBS, as a percent knock-down of Serpinc1 antigen levels from PBS, and as a percent knock-down of Serpinc1 activity from PBS at Day 3 for AD-57213.

[0475] As evidenced by FIGS. 14-16, administration of compound AD-57213 leads to potent, dose-dependent suppression of Serpinc1 in HA mice with a single dose ED.sub.50 of less than 1 mg/kg on Day 7. Serpinc1 suppression was durable and correlated with the maximal level of antithrombin suppression achieved. A single dose of 1 mg/kg led to the maintenance of 50% suppression for about 15 days, while a dose of 10 mg/kg led to greater than 80% suppression maintained for 28 days.

Example 10

Dose Duration of a Split-Dose of AD-57213

[0476] In order to further evaluate compound AD-57213 knock-down of Serpinc1 expression and activity, a split-dosing experiment was performed. C57BL/6 mice were subcutaneously administered GalNAc-conjugated AD-57213 and the effect of a 3 times per week, 1/3 dose of AD-57213 was compared to the effect of a 1 time per week fully concentrated dose of AD-57213. A summary of the study design is presented in Table 16. Serum Serpinc1 protein levels were determined.

TABLE-US-00017 TABLE 16 Study Design of Split-Dosing Experiment Group Test compound Dose (mg/kg) Frequency 1 AD-57213 3 q1w 2 1.5 (Monday) 3 0.75 4 1 t.i.w. 5 0.5 (M, W, F) 6 0.25 7 PBS -- q1w (q1w: Once a week)

[0477] The results of the once a week (q1w) dosing as a percent knock-down of Serpinc1 protein levels from PBS are shown in FIG. 17 and the results of the three-time per week (t.i.w.) dosing as a percent knock-down of Serpinc1 protein levels from PBS are shown in FIG. 18.

[0478] As shown in FIGS. 17 and 18, repeat dosing of compound AD-57213 led to a dose-dependent, durable response, with some additive effect. Animals dosed with 3 mg/kg reached the nadir levels of >95% knock-down after 2 weekly doses whereas the lower two dose groups attained nadir after 3 weekly doses (.about.90% knock-down for 1.5 mg/kg and .about.80% for 0.75 mg/kg).

[0479] To further study the different dosing regimens, in different groups, the same weekly dose was split and dosed three times a week, e.g., 1.5 mg/kg q1w was compared with 0.5 mg/kg tiw. As shown in FIG. 19, the cumulative weekly dose gave the same level of knock-down. For example, Serpinc1 levels achieved with 1.5 mg/kg (q1w) were equivalent to Serpinc1 levels achieved with 0.5 mg/kg administered three times a week.

Example 11

Non-Human Primate Dosing of Serpinc1 siRNAs

[0480] Compound AD-57213 was tested for efficacy in non-human primates as outlined in Table 17. Serum Serpinc1 protein levels were determined at Days -14, -8, -4, Day 1 at 4 hours post-dosing, Days 2, 4, 8, 11, 15, 22, 29, 37, 44, 51, and 58.

TABLE-US-00018 TABLE 17 Non-Human Primate Dosing Experiment. Group Dose Route of Test Num- level admin- Article ber n (mg/kg) istration Rationale AD54944 1 3 10 SC Parent compound AD- 2* 3 30 SC Dose response Similar to 3 .times. 57213 3 3 10 SC for the lead 10 mpk 4 3 3 SC Compare 5 3 1 SC compounds at 10 mpk Dose curve AD- 5 3 10 SC Same potency Has less PS 57205 as 57213 at 10 mpk in mice LNP- 7 3 0.3 IV Test target Positive control 55029 for target knock-down and assays

[0481] FIG. 20 shows the results of the single dose screen for all compounds tested as a group average of the relative serum Serpinc1 levels compared to pre-dosing Serpinc1 levels and demonstrates that all of the siRNAs tested effectively knock-down Serpinc1 protein levels.

[0482] FIG. 21 shows the results of the single dose screen for compoundAD-57213 as a group average of the relative serum Serpinc1 levels compared to pre-dosing Serpinc1 levels.

[0483] FIG. 22 shows the results of the single dose screen for all compounds tested as a group average of the relative serum Serpinc1 levels compared to pre-dosing Serpinc1 levels on Day 8.

[0484] Overall, the results demonstrate that there is dose-dependent knock-down of Serpinc1 protein levels with AD-57213 in non-human primates and that both AD-57213 and AD-57205 show improved potency over the parent compound AD-52444.

Example 12

Non-Human Primate Dosing of a Therapeutic Serpinc1 siRNA

[0485] Compound AD-57213 was tested for efficacy in non-human primates. Cynomolgus monkeys were administered compound AD-57213 as outlined in Table 18 below. Plasma was collected at various time points after administration of AD-57213 and analyzed for antithrombin protein (Serpinc1) levels by ELISA.

TABLE-US-00019 TABLE 18 Study Design: AD-57213 pharmacology in nonhuman primates Group Test Dose Level Route of Sample Number Article N (mg/kg) Administration Collection 1 AD-57213 3 1 SC Plasma for AT 2 3 3 SC protein and 3 3 10 SC thrombin 4 3 30 SC generation

[0486] FIG. 23 shows the results of the single dose screen for compound AD-57213 as a group average of the relative serum Serpinc1 levels compared to the average of three pre-dose measurements. The results demonstrate dose dependent Serpinc1 silencing with approximately 50, 70, 80 and >90% silencing at 1, 3, 10 and 30 mg/kg, respectively. Data points represent group mean and error bars represent standard deviation.

[0487] FIGS. 24A-D show the relationship between relative serum Serpinc1 level and fold change in peak plasma thrombin level at a single A) 1 mg/kg, B) 3 mg/kg, C) 10 mg/kg, and D) 30 mg/kg dose of compound AD57213. Serpinc1 levels are represented relative to the average of three pre-dose measurements. Thrombin generation curves were generated from plasma samples collected at various time points using a Calibrated Automated Thrombinoscope (tissue factor=1 pM). Fold change in peak thrombin was calculated relative to the average peak thrombin value for two pre-dose values for each animal. Data points represent group mean and error bars represent standard deviation. FIG. 25 shows a consolidated scatterplot of fold change increase in peak thrombin as a function of relative Serpinc1 silencing.

[0488] Animals were also administered three weekly AD-57213 doses of 30 mg/kg and the Serpinc1 protein and mRNA levels were determined in blood samples collected from the animals. The results of these studies are presented in FIGS. 26A (Serpinc1 protein levels relative to prebleed levels) and 26B (Serpinc1 mRNA levels relative to GAPDH).

[0489] Overall, the results demonstrate that there is a durable, dose-dependent inhibition of Serpinc1 protein levels with compound AD-57213 in non-human primates that results in up to a 4-fold increase in thrombin generation.

Example 13

Repeat Administration of a Serpinc1 SiRNA in Non-Human Primate Dosing

[0490] Compound AD-57213 was tested for efficacy and to evaluate the cumulative effect of the compound in non-human primates with a repeat administration protocol. Cynomolgus monkeys were administered compound AD-57213 at 0.5 mg/kg q1w; 1 mg/kg q2w (every other week), for 2months and 1.5 mg/kg q1w; 3 mg/kg q2w for 6 weeks. Serum was collected at various time points as illustrated in FIG. 27 and analyzed for antithrombin protein level (SerpinC1) by ELISA. Antithrombin levels were represented relative to the average of three pre-dose measurements.

[0491] The first two dose groups with 0.5 mg/kg weekly cumulative dose led to 80% decrease in AT levels after 5 weeks. The latter two groups with 1.5 mg/kg the cumulative weekly dose led to >95% maximum knockdown. FIG. 27A shows the data from latter two groups. Animals receiving the 3 mg/kg dose were euthanized on Day 54 and animals receiving the 1.5 mg/kg q1w dose were administered an additional 6th weekly dose on day 36 and are being monitored for recovery to base line levels. FIG. 27B shows AT levels after 0.5 mg/kg cumulative weekly dose.

[0492] The data demonstrate that dose dependent antithrombin silencing was observed with all dosing regimens and achieved a steady-state level of suppression by day 25.

Example 14

Correction in Hemostasis Following Administration of Compound AD-57213 in Hemophilic Mice

[0493] Hemophilic animals have less thrombin generation potential and cannot form stable clots. Reduction of antithrombin protein in these animals should help rebalance the hemostasis, increase the endogenous thrombin generating potential, and enable clot formation. This hypothesis was tested in hemophilia A and hemophilia B mice in the microvessel laser injury model accompanied with intravital imaging. Mice were injected with compound AD-57213 and injury was induced 10 days post-treatment. Accumulation of platelets and fibrin at the site of injury were visualized, recorded and quantified. FIG. 28A shows the median values of platelet accululation over time after laser surgery and FIG. 28B shows the median fibrin values from all inflicted injuries over time after laser surgery. As demonstrated by FIGS. 28A and 28B, compound AD-57213 injected at 1 mg/kg or 30 mg/kg led to platelet and fibrin deposition leading to clot formation in 100% of the injuries. Table 19 summarizes the results from two separate experiments with HA and HB animals.

TABLE-US-00020 TABLE 19 Percent Stable AT Animals Injuries Thrombus mRNA Group (N) (N) (N) in liver WT 2 10 10 100% HA + PBS 2 13 0 100% HA + 1 mg/kg 4 20 20 50% ALN-AT3 HA + 30 mg/kg 4 20 20 5% ALN-AT3 HB + PBS 2 6 0 100% HB + 30 mg/kg 2 6 6 5% ALNAT3

Example 15

In Vivo Efficacy of AD-57213 LNP Formulation

[0494] Compounds AD-55029 (unconjugated) and AD-57213 (conjugated to GalNAc) were formulated in a lipid nucleic acid particle (AF-11) and wild-type animals were administered doses of 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg of these LNP formulated compounds. Luciferase (AF11-1955) was used as control.

[0495] Both compounds led to >95% knock-down at 0.3 mg/kg but the levels were maintained by AF11-57213 for 15 days versus 8 days by AF11-55029 (see FIG. 29). A similar difference in duration of action between the two compounds was observed at the lower doses.

Example 16

Design, Synthesis, and in Vitro Screening of Additional siRNAs

[0496] siRNA Design

[0497] SiRNA duplexes, 19 nucleotides long for both the sense and antisense strand, were designed using the human SERPINC1 mRNA sequence set forth in GenBank Accession No. NM_000488.3. One thousand five hundred and eighty-one duplexes were initially identified that did not contain repeats longer than 7 nucleotides, spanning the entire 1599 nucleotide transcript. All 1581 duplexes were then scored for predicted efficacy according to a linear model that evaluates the nucleotide pair at each duplex position, and the dose and cell line to be used for screening. The duplexes were also matched against all transcripts in the human RefSeq collection using a custom brute force algorithm, and scored for lowest numbers of mismatches (per strand) to transcripts other than SERPINC1. Duplexes to be synthesized and screened were then selected from the 1581, according to the following scheme: Beginning at the 5' end of the transcript, a duplex was selected within a "window" of every 10.+-.2 nucleotides that

[0498] 1) had the highest predicted efficacy,

[0499] 2) had at least one mismatch in both strands to all transcripts other than SERPINC1,

[0500] 3) had not already been synthesized and screened as part of other duplex sets.

[0501] If no duplex could be identified within a given window that satisfied all criteria, that window was skipped. One hundred and sixty-four duplexes were identified that satisfied these criteria.

[0502] A detailed list of Sepinc1 sense and antisense strand sequences is shown in Tables 20 and 21.

TABLE-US-00021 TABLE 20 AT3 (SERPINC1) unmodified sequences (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 979-1141, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 1142-1304, respectively, in order of appearance) Duplex Sense Oligo Antis Oligo Position in Name Name Sense Sequence Name Antisense Sequence NM_000488.3 AD-59267.1 A-120250.1 GGAGAAGAAGGCAACUGAG A-120251.1 CUCAGUUGCCUUCUUCUCC 293-311 AD-59268.1 A-120266.1 UGACCAAGCUGGGUGCCUG A-120267.1 CAGGCACCCAGCUUGGUCA 481-499 AD-59269.1 A-120282.1 GGUUAACACCAUUUACUUC A-120283.1 GAAGUAAAUGGUGUUAACC 860-878 AD-59270.1 A-120298.1 GCUGGUUAACACCAUUUAC A-120299.1 GUAAAUGGUGUUAACCAGC 857-875 AD-59271.1 A-120314.1 UAAUGACACCCUCCAGCAA A-120315.1 UUGCUGGAGGGUGUCAUUA 500-518 AD-59272.1 A-120330.1 CGUGUUCAGCAUCUAUGAU A-120331.1 AUCAUAGAUGCUGAACACG 952-970 AD-59273.1 A-120252.1 UUGAGGACGGCUUCAGUUU A-120253.1 AAACUGAAGCCGUCCUCAA 1189-1207 AD-59274.1 A-120268.1 CGGCGUGUCUGGGAACUGU A-120269.1 ACAGUUCCCAGACACGCCG 351-369 AD-59275.1 A-120284.1 UUAACACCAUUUACUUCAA A-120285.1 UUGAAGUAAAUGGUGUUAA 862-880 AD-59276.1 A-120300.1 CCCUGAAAAGUCCAAACUC A-120301.1 GAGUUUGGACUUUUCAGGG 1250-1268 AD-59277.1 A-120316.1 CGAGAUGACCUCUAUGUCU A-120317.1 AGACAUAGAGGUCAUCUCG 1290-1308 AD-59278.1 A-120332.1 UCUACAAGGCUGAUGGAGA A-120333.1 UCUCCAUCAGCCUUGUAGA 931-949 AD-59279.1 A-120254.1 AGCUCACUGUUCUGGUGCU A-120255.1 AGCACCAGAACAGUGAGCU 841-859 AD-59280.1 A-120270.1 AGGAGCAGCUGCAAGACAU A-120271.1 AUGUCUUGCAGCUGCUCCU 1210-1228 AD-59281.1 A-120286.1 GCCACCAACCGGCGUGUCU A-120287.1 AGACACGCCGGUUGGUGGC 342-360 AD-59282.1 A-120302.1 CAGAACAGAAGAUCCCGGA A-120303.1 UCCGGGAUCUUCUGUUCUG 322-340 AD-59283.1 A-120318.1 CCUUGUCGAUCUGUUCAGC A-120319.1 GCUGAACAGAUCGACAAGG 1232-1250 AD-59284.1 A-120334.1 AGGCAAGUUCCGUUAUCGG A-120335.1 CCGAUAACGGAACUUGCCU 980-998 AD-59285.1 A-120256.1 UUUUGUCCUUGCUGCUCAU A-120257.1 AUGAGCAGCAAGGACAAAA 172-190 AD-59286.1 A-120272.1 AGACCUACCAGGACAUCAG A-120273.1 CUGAUGUCCUGGUAGGUCU 682-700 AD-59287.1 A-120288.1 AACUGAACUGCCGACUCUA A-120289.1 UAGAGUCGGCAGUUCAGUU 589-607 AD-59288.1 A-120304.1 CAUUUACUUCAAGGGCCUG A-120305.1 CAGGCCCUUGAAGUAAAUG 869-887 AD-59289.1 A-120320.1 CCCUGGACUUCAAGGAAAA A-120321.1 UUUUCCUUGAAGUCCAGGG 730-748 AD-59290.1 A-120336.1 AGCUGCAAGUACCGCUGUU A-120337.1 AACAGCGGUACUUGCAGCU 1361-1379 AD-59291.1 A-120258.1 ACACAAGGAAGGAACUGUU A-120259.1 AACAGUUCCUUCCUUGUGU 913-931 AD-59292.1 A-120274.1 GCAACUGAGGAUGAGGGCU A-120275.1 AGCCCUCAUCCUCAGUUGC 303-321 AD-59293.1 A-120290.1 GUAGCCAACCCUUGUGUUA A-120291.1 UAACACAAGGGUUGGCUAC 1491-1509 AD-59294.1 A-120306.1 GUUUGUGAACAGAAGUAAA A-120307.1 UUUACUUCUGUUCACAAAC 1550-1568 AD-59295.1 A-120322.1 GGGUGACUUUCAAGGCCAA A-120323.1 UUGGCCUUGAAAGUCACCC 1411-1429 AD-59296.1 A-120338.1 UUAUCGGCGCGUGGCUGAA A-120339.1 UUCAGCCACGCGCCGAUAA 992-1010 AD-59297.1 A-120260.1 CCACUUCUUCUUUGCCAAA A-120261.1 UUUGGCAAAGAAGAAGUGG 572-590 AD-59298.1 A-120276.1 AACACCAUUUACUUCAAGG A-120277.1 CCUUGAAGUAAAUGGUGUU 864-882 AD-59299.1 A-120292.1 GAUGGAGAGUCGUGUUCAG A-120293.1 CUGAACACGACUCUCCAUC 942-960 AD-59300.1 A-120308.1 CACCAUUUACUUCAAGGGC A-120309.1 GCCCUUGAAGUAAAUGGUG 866-884 AD-59301.1 A-120324.1 UUUACUUCAAGGGCCUGUG A-120325.1 CACAGGCCCUUGAAGUAAA 871-889 AD-59302.1 A-120340.1 UAAGAGAAGUUCCUCUGAA A-120341.1 UUCAGAGGAACUUCUCUUA 1450-1468 AD-59303.1 A-120262.1 GCGGGACAUUCCCAUGAAU A-120263.1 AUUCAUGGGAAUGUCCCGC 251-269 AD-59304.1 A-120278.1 UGCCCCACCCUGUCCUCUG A-120279.1 CAGAGGACAGGGUGGGGCA 21-Mar AD-59305.1 A-120294.1 UGGUUAACACCAUUUACUU A-120295.1 AAGUAAAUGGUGUUAACCA 859-877 AD-59306.1 A-120310.1 CGGAUUGCCUCAGAUCACA A-120311.1 UGUGAUCUGAGGCAAUCCG 62-80 AD-59307.1 A-120326.1 CCAGGACAUCAGUGAGUUG A-120327.1 CAACUCACUGAUGUCCUGG 689-707 AD-59308.1 A-120342.1 CCAGUUUUCAGGCGGAUUG A-120343.1 CAAUCCGCCUGAAAACUGG 50-68 AD-59309.1 A-120264.1 CACCAUAUCUGAGAAAACA A-120265.1 UGUUUUCUCAGAUAUGGUG 542-560 AD-59310.1 A-120280.1 AAGUAAAAAUAAAUACAAA A-120281.1 UUUGUAUUUAUUUUUACUU 1562-1580 AD-59311.1 A-120296.1 GCACCCAGGUGCUUGAGUU A-120297.1 AACUCAAGCACCUGGGUGC 1012-1030 AD-59312.1 A-120312.1 UAACACCAUUUACUUCAAG A-120313.1 CUUGAAGUAAAUGGUGUUA 863-881 AD-59313.1 A-120328.1 CCUUCAAAGGUGAUGACAU A-120329.1 AUGUCAUCACCUUUGAAGG 1033-1051 AD-59314.1 A-120344.1 CAAGGCCAAUUCCCGCUUU A-120345.1 AAAGCGGGAAUUGGCCUUG 371-389 AD-59315.1 A-120360.1 UCAGUUUGAAGGAGCAGCU A-120361.1 AGCUGCUCCUUCAAACUGA 1201-1219 AD-59316.1 A-120376.1 GGGACUGCGUGACCUGUCA A-120377.1 UGACAGGUCACGCAGUCCC 199-217 AD-59317.1 A-120392.1 UCAGCCAAUCGCCUUUUUG A-120393.1 CAAAAAGGCGAUUGGCUGA 639-657 AD-59318.1 A-120408.1 CCUCGGAAGCCAUCAAUGA A-120409.1 UCAUUGAUGGCUUCCGAGG 823-841 AD-59319.1 A-120424.1 GACAAUGAUAACAUUUUCC A-120425.1 GGAAAAUGUUAUCAUUGUC 429-447 AD-59320.1 A-120346.1 CUUAUUCUUUGCACCUCUU A-120347.1 AAGAGGUGCAAAGAAUAAG 1521-1539 AD-59321.1 A-120362.1 AUUGCUGGCCGUUCGCUAA A-120363.1 UUAGCGAACGGCCAGCAAU 1383-1401 AD-59322.1 A-120378.1 AAAUGAAGAAGGCAGUGAA A-120379.1 UUCACUGCCUUCUUCAUUU 1340-1358 AD-59323.1 A-120394.1 UGAGUUGGUAUAUGGAGCC A-120395.1 GGCUCCAUAUACCAACUCA 701-719 AD-59324.1 A-120410.1 UCCAGCAACUGAUGGAGGU A-120411.1 ACCUCCAUCAGUUGCUGGA 511-529 AD-59325.1 A-120426.1 AACUGUAACCUCUGGAAAA A-120427.1 UUUUCCAGAGGUUACAGUU 140-158 AD-59326.1 A-120348.1 UCAACAAAUGGGUGUCCAA A-120349.1 UUGGACACCCAUUUGUUGA 772-790 AD-59327.1 A-120364.1 GAUUAGCGGCCAUGUAUUC A-120365.1 GAAUACAUGGCCGCUAAUC 109-127 AD-59328.1 A-120380.1 GUGCUUGAGUUGCCCUUCA A-120381.1 UGAAGGGCAACUCAAGCAC 1020-1038 AD-59329.1 A-120396.1 UGCAGAAGGCCGAGAUGAC A-120397.1 GUCAUCUCGGCCUUCUGCA 1280-1298 AD-59330.1 A-120412.1 CUAUUUUUGGUUUGUGAAC A-120413.1 GUUCACAAACCAAAAAUAG 1541-1559 AD-59331.1 A-120428.1 CAGUGAAGCAGCUGCAAGU A-120429.1 ACUUGCAGCUGCUUCACUG 1352-1370 AD-59332.1 A-120350.1 GUGUCCAAUAAGACCGAAG A-120351.1 CUUCGGUCUUAUUGGACAC 783-801 AD-59333.1 A-120366.1 AUGAAUUGGAGGAGAUGAU A-120367.1 AUCAUCUCCUCCAAUUCAU 1141-1159 AD-59334.1 A-120382.1 AUCUAUGAUGUACCAGGAA A-120383.1 UUCCUGGUACAUCAUAGAU 962-980 AD-59335.1 A-120398.1 CCAUAAGGCAUUUCUUGAG A-120399.1 CUCAAGAAAUGCCUUAUGG 1319-1337 AD-59336.1 A-120414.1 AUGCAUUCCAUAAGGCAUU A-120415.1 AAUGCCUUAUGGAAUGCAU 1312-1330 AD-59337.1 A-120430.1 UGGUGCUGGUUAACACCAU A-120431.1 AUGGUGUUAACCAGCACCA 853-871 AD-59338.1 A-120352.1 AUCUGUUCAGCCCUGAAAA A-120353.1 UUUUCAGGGCUGAACAGAU 1240-1258 AD-59339.1 A-120368.1 AGAUGAUGCUGGUGGUCCA A-120369.1 UGGACCACCAGCAUCAUCU 1153-1171 AD-59340.1 A-120384.1 AUAUGGAGCCAAGCUCCAG A-120385.1 CUGGAGCUUGGCUCCAUAU 710-728 AD-59341.1 A-120400.1 CCGAAUCACCGAUGUCAUU A-120401.1 AAUGACAUCGGUGAUUCGG 803-821 AD-59342.1 A-120416.1 GCAGAGCAAUCCAGAGCGG A-120417.1 CCGCUCUGGAUUGCUCUGC 750-768 AD-59343.1 A-120432.1 ACACCAUUUACUUCAAGGG A-120433.1 CCCUUGAAGUAAAUGGUGU 865-883 AD-59344.1 A-120354.1 GUACCAGGAAGGCAAGUUC A-120355.1 GAACUUGCCUUCCUGGUAC 971-989 AD-59345.1 A-120370.1 UGCCCAAGCCUGAGAAGAG A-120371.1 CUCUUCUCAGGCUUGGGCA 1069-1087 AD-59346.1 A-120386.1 UUCUUUGCCAAACUGAACU A-120387.1 AGUUCAGUUUGGCAAAGAA 579-597 AD-59347.1 A-120402.1 UGGCCAAGGUAGAGAAGGA A-120403.1 UCCUUCUCUACCUUGGCCA

1090-1108 AD-59348.1 A-120418.1 UGCUGCAAGAGUGGCUGGA A-120419.1 UCCAGCCACUCUUGCAGCA 1123-1141 AD-59349.1 A-120434.1 CCAUGUGCAUUUACCGCUC A-120435.1 GAGCGGUAAAUGCACAUGG 271-289 AD-59350.1 A-120356.1 AGACAUGGGCCUUGUCGAU A-120357.1 AUCGACAAGGCCCAUGUCU 1223-1241 AD-59351.1 A-120372.1 UUUUGGAGACAAAUCCCUU A-120373.1 AAGGGAUUUGUCUCCAAAA 653-671 AD-59352.1 A-120388.1 GGAUGAGGGCUCAGAACAG A-120389.1 CUGUUCUGAGCCCUCAUCC 311-329 AD-59353.1 A-120404.1 CGGCUUUUGCUAUGACCAA A-120405.1 UUGGUCAUAGCAAAAGCCG 469-487 AD-59354.1 A-120420.1 AGCUCCAGCCCCUGGACUU A-120421.1 AAGUCCAGGGGCUGGAGCU 721-739 AD-59355.1 A-120436.1 CUGAUCAGAUCCACUUCUU A-120437.1 AAGAAGUGGAUCUGAUCAG 562-580 AD-59356.1 A-120358.1 UGCUGGUGGUCCACAUGCC A-120359.1 GGCAUGUGGACCACCAGCA 1159-1177 AD-59357.1 A-120374.1 GCGAGAUUUAGAGGAAAGA A-120375.1 UCUUUCCUCUAAAUCUCGC 30-48 AD-59358.1 A-120390.1 CUGCUCAUUGGCUUCUGGG A-120391.1 CCCAGAAGCCAAUGAGCAG 183-201 AD-59359.1 A-120406.1 CCUUCAAUGAGACCUACCA A-120407.1 UGGUAGGUCUCAUUGAAGG 673-691 AD-59360.1 A-120422.1 ACCAUUUACUUCAAGGGCC A-120423.1 GGCCCUUGAAGUAAAUGGU 867-885 AD-59587.1 A-120438.1 GCACCUCUUCCUAUUUUUG A-120439.1 CAAAAAUAGGAAGAGGUGC 1531-1549 AD-59588.1 A-120454.1 GUGGCUGAAGGCACCCAGG A-120455.1 CCUGGGUGCCUUCAGCCAC 1002-1020 AD-59589.1 A-120470.1 UCCGCAUUGAGGACGGCUU A-120471.1 AAGCCGUCCUCAAUGCGGA 1183-1201 AD-59590.1 A-120486.1 UGGACAUCUGCACAGCCAA A-120487.1 UUGGCUGUGCAGAUGUCCA 229-247 AD-59591.1 A-120502.1 GUCCAAACUCCCAGGUAUU A-120503.1 AAUACCUGGGAGUUUGGAC 1259-1277 AD-59592.1 A-120518.1 AGAAGGAACUCACCCCAGA A-120519.1 UCUGGGGUGAGUUCCUUCU 1102-1120 AD-59593.1 A-120440.1 UCUUGAGGUAAAUGAAGAA A-120441.1 UUCUUCAUUUACCUCAAGA 1331-1349 AD-59594.1 A-120456.1 AGCCCUGUGGACAUCUGCA A-120457.1 UGCAGAUGUCCACAGGGCU 222-240 AD-59595.1 A-120472.1 CAGAGCGGCCAUCAACAAA A-120473.1 UUUGUUGAUGGCCGCUCUG 761-779 AD-59596.1 A-120488.1 AUUUAAGUUUGACACCAUA A-120489.1 UAUGGUGUCAAACUUAAAU 530-548 AD-59597.1 A-120504.1 UGAGAAGAGCCUGGCCAAG A-120505.1 CUUGGCCAGGCUCUUCUCA 1079-1097 AD-59598.1 A-120520.1 UCACCAUGGUCCUCAUCUU A-120521.1 AAGAUGAGGACCAUGGUGA 1051-1069 AD-59599.1 A-120442.1 GGAAGGAACUGUUCUACAA A-120443.1 UUGUAGAACAGUUCCUUCC 919-937 AD-59600.1 A-120458.1 CUGGUUUUUAUAAGAGAAG A-120459.1 CUUCUCUUAUAAAAACCAG 1440-1458 AD-59601.1 A-120474.1 CUGGGUGCCUGUAAUGACA A-120475.1 UGUCAUUACAGGCACCCAG 489-507 AD-59602.1 A-120490.1 GUACCGCUGUUGUGAUUGC A-120491.1 GCAAUCACAACAGCGGUAC 1369-1387 AD-59603.1 A-120506.1 UCUAUCAGCACCUGGCAGA A-120507.1 UCUGCCAGGUGCUGAUAGA 400-418 AD-59604.1 A-120522.1 CUGGCAGAUUCCAAGAAUG A-120523.1 CAUUCUUGGAAUCUGCCAG 411-429 AD-59605.1 A-120444.1 CGAUGUCAUUCCCUCGGAA A-120445.1 UUCCGAGGGAAUGACAUCG 812-830 AD-59606.1 A-120460.1 GCUUCUGGGACUGCGUGAC A-120461.1 GUCACGCAGUCCCAGAAGC 193-211 AD-59607.1 A-120476.1 CCUGUCACGGGAGCCCUGU A-120477.1 ACAGGGCUCCCGUGACAGG 211-229 AD-59608.1 A-120492.1 AUUUACUUCAAGGGCCUGU A-120493.1 ACAGGCCCUUGAAGUAAAU 870-888 AD-59609.1 A-120508.1 UGCUACCACUUUCUAUCAG A-120509.1 CUGAUAGAAAGUGGUAGCA 389-407 AD-59610.1 A-120524.1 GGAACUGUCCAAGGCCAAU A-120525.1 AUUGGCCUUGGACAGUUCC 362-380 AD-59611.1 A-120446.1 ACAAAUCCUCCAAGUUAGU A-120447.1 ACUAACUUGGAGGAUUUGU 619-637 AD-59612.1 A-120462.1 AUUUACCGCUCCCCGGAGA A-120463.1 UCUCCGGGGAGCGGUAAAU 279-297 AD-59613.1 A-120478.1 ACCCCUGAGUAUCUCCACG A-120479.1 CGUGGAGAUACUCAGGGGU 452-470 AD-59614.1 A-120494.1 CACUAUCUCCACUUGCCCA A-120495.1 UGGGCAAGUGGAGAUAGUG 79-97 AD-59615.1 A-120510.1 AAAUACAAACUACUUCCAU A-120511.1 AUGGAAGUAGUUUGUAUUU 1572-1590 AD-59616.1 A-120526.1 CUGGUUAACACCAUUUACU A-120527.1 AGUAAAUGGUGUUAACCAG 858-876 AD-59617.1 A-120448.1 UCAUCUUGCCCAAGCCUGA A-120449.1 UCAGGCUUGGGCAAGAUGA 1063-1081 AD-59618.1 A-120464.1 CCUCAGAUCACACUAUCUC A-120465.1 GAGAUAGUGUGAUCUGAGG 69-87 AD-59619.1 A-120480.1 CUGUCCUCUGGAACCUCUG A-120481.1 CAGAGGUUCCAGAGGACAG 30-Dec AD-59620.1 A-120496.1 CCCUGUGGAAGAUUAGCGG A-120497.1 CCGCUAAUCUUCCACAGGG 99-117 AD-59621.1 A-120512.1 AUCUCCACGGCUUUUGCUA A-120513.1 UAGCAAAAGCCGUGGAGAU 462-480 AD-59622.1 A-120528.1 GUGGCUGGAUGAAUUGGAG A-120529.1 CUCCAAUUCAUCCAGCCAC 1133-1151 AD-59623.1 A-120450.1 CAAGUUAGUAUCAGCCAAU A-120451.1 AUUGGCUGAUACUAACUUG 629-647 AD-59624.1 A-120466.1 GUGAUGACAUCACCAUGGU A-120467.1 ACCAUGGUGAUGUCAUCAC 1042-1060 AD-59625.1 A-120482.1 UUCCAAGAAUGACAAUGAU A-120483.1 AUCAUUGUCAUUCUUGGAA 419-437 AD-59626.1 A-120498.1 UGAUGGAGGUAUUUAAGUU A-120499.1 AACUUAAAUACCUCCAUCA 520-538 AD-59627.1 A-120514.1 GUAUUCCAAUGUGAUAGGA A-120515.1 UCCUAUCACAUUGGAAUAC 122-140 AD-59628.1 A-120530.1 CAGCCAAGCCGCGGGACAU A-120531.1 AUGUCCCGCGGCUUGGCUG 241-259 AD-59629.1 A-120452.1 CAUGCCCCGCUUCCGCAUU A-120453.1 AAUGCGGAAGCGGGGCAUG 1172-1190 AD-59630.1 A-120468.1 AUGUGAUAGGAACUGUAAC A-120469.1 GUUACAGUUCCUAUCACAU 130-148 AD-59631.1 A-120484.1 ACAAAUCCCUUACCUUCAA A-120485.1 UUGAAGGUAAGGGAUUUGU 661-679 AD-59632.1 A-120500.1 AGGUUUAUCUUUUGUCCUU A-120501.1 AAGGACAAAAGAUAAACCU 163-181 AD-59633.1 A-120516.1 AGAUCCCGGAGGCCACCAA A-120517.1 UUGGUGGCCUCCGGGAUCU 331-349 AD-59634.1 A-120532.1 ACAUUUUCCUGUCACCCCU A-120533.1 AGGGGUGACAGGAAAAUGU 439-457 AD-59635.1 A-120548.1 GGCCUUUCCUGGUUUUUAU A-120549.1 AUAAAAACCAGGAAAGGCC 1432-1450 AD-59636.1 A-120564.1 UUGUGUUAAGUAAAAUGUU A-120565.1 AACAUUUUACUUAACACAA 1502-1520 AD-59637.1 A-120534.1 CAAGGAAAAUGCAGAGCAA A-120535.1 UUGCUCUGCAUUUUCCUUG 740-758 AD-59638.1 A-120550.1 CUGAGAAAACAUCUGAUCA A-120551.1 UGAUCAGAUGUUUUCUCAG 550-568 AD-59639.1 A-120566.1 UUACUUCAAGGGCCUGUGG A-120567.1 CCACAGGCCCUUGAAGUAA 872-890 AD-59640.1 A-120536.1 ACCCCAACAGGGUGACUUU A-120537.1 AAAGUCACCCUGUUGGGGU 1402-1420 AD-59641.1 A-120552.1 CCAGGUAUUGUUGCAGAAG A-120553.1 CUUCUGCAACAAUACCUGG 1269-1287 AD-59642.1 A-120568.1 GCCUGUGGAAGUCAAAGUU A-120569.1 AACUUUGACUUCCACAGGC 883-901 AD-59643.1 A-120538.1 UGGAACCUCUGCGAGAUUU A-120539.1 AAAUCUCGCAGAGGUUCCA 20-38 AD-59644.1 A-120554.1 AGCCCUGAGAACACAAGGA A-120555.1 UCCUUGUGUUCUCAGGGCU 903-921 AD-59645.1 A-120570.1 CUCUAUGUCUCAGAUGCAU A-120571.1 AUGCAUCUGAGACAUAGAG 1299-1317 AD-59646.1 A-120540.1 AAGACCGAAGGCCGAAUCA A-120541.1 UGAUUCGGCCUUCGGUCUU 792-810 AD-59647.1 A-120556.1 UAAAAUGUUCUUAUUCUUU A-120557.1 AAAGAAUAAGAACAUUUUA 1512-1530 AD-59648.1 A-120572.1 UCUGGAAAAAGGAAGGUUU A-120573.1 AAACCUUCCUUUUUCCAGA 150-168 AD-59649.1 A-120542.1 UUCAAGGCCAACAGGCCUU A-120543.1 AAGGCCUGUUGGCCUUGAA 1419-1437 AD-59650.1 A-120558.1 GAAGUCAAAGUUCAGCCCU A-120559.1 AGGGCUGAACUUUGACUUC 890-908 AD-59651.1 A-120574.1 CCAUCAAUGAGCUCACUGU A-120575.1 ACAGUGAGCUCAUUGAUGG 832-850 AD-59652.1 A-120544.1 CUCUGAACACUAUUAUCUU A-120545.1 AAGAUAAUAGUGUUCAGAG 1462-1480 AD-59653.1 A-120560.1 UGCUGGUUAACACCAUUUA A-120561.1 UAAAUGGUGUUAACCAGCA 856-874 AD-59654.1 A-120546.1 AGAGGAAAGAACCAGUUUU A-120547.1 AAAACUGGUUCUUUCCUCU 39-57 AD-59655.1 A-120562.1 UGCCCAGCCCUGUGGAAGA A-120563.1 UCUUCCACAGGGCUGGGCA 92-110

TABLE-US-00022 TABLE 21 AT3 (SERPINC1) modified sequences (The "Sense Sequence" column sequences are disclosed as SEQ ID NOS 1305-1467, respectively, in order of appearance, and the "Antisense Sequence" column sequences are disclosed as SEQ ID NOS 1468-1630, respectively, in order of appearance) Sense Oligo Antis Oligo Duplex Name Name oligoSeq Name oligoSeq AD-59267.1 A-120250.1 GGAGAAGAAGGCAACUGAGdTdT A-120251.1 CUCAGUUGCCUUCUUCUCCdTdT AD-59268.1 A-120266.1 UGACCAAGCUGGGUGCCUGdTdT A-120267.1 CAGGCACCCAGCUUGGUCAdTdT AD-59269.1 A-120282.1 GGUUAACACCAUUUACUUCdTdT A-120283.1 GAAGUAAAUGGUGUUAACCdTdT AD-59270.1 A-120298.1 GCUGGUUAACACCAUUUACdTdT A-120299.1 GUAAAUGGUGUUAACCAGCdTdT AD-59271.1 A-120314.1 UAAUGACACCCUCCAGCAAdTdT A-120315.1 UUGCUGGAGGGUGUCAUUAdTdT AD-59272.1 A-120330.1 CGUGUUCAGCAUCUAUGAUdTdT A-120331.1 AUCAUAGAUGCUGAACACGdTdT AD-59273.1 A-120252.1 UUGAGGACGGCUUCAGUUUdTdT A-120253.1 AAACUGAAGCCGUCCUCAAdTdT AD-59274.1 A-120268.1 CGGCGUGUCUGGGAACUGUdTdT A-120269.1 ACAGUUCCCAGACACGCCGdTdT AD-59275.1 A-120284.1 UUAACACCAUUUACUUCAAdTdT A-120285.1 UUGAAGUAAAUGGUGUUAAdTdT AD-59276.1 A-120300.1 CCCUGAAAAGUCCAAACUCdTdT A-120301.1 GAGUUUGGACUUUUCAGGGdTdT AD-59277.1 A-120316.1 CGAGAUGACCUCUAUGUCUdTdT A-120317.1 AGACAUAGAGGUCAUCUCGdTdT AD-59278.1 A-120332.1 UCUACAAGGCUGAUGGAGAdTdT A-120333.1 UCUCCAUCAGCCUUGUAGAdTdT AD-59279.1 A-120254.1 AGCUCACUGUUCUGGUGCUdTdT A-120255.1 AGCACCAGAACAGUGAGCUdTdT AD-59280.1 A-120270.1 AGGAGCAGCUGCAAGACAUdTdT A-120271.1 AUGUCUUGCAGCUGCUCCUdTdT AD-59281.1 A-120286.1 GCCACCAACCGGCGUGUCUdTdT A-120287.1 AGACACGCCGGUUGGUGGCdTdT AD-59282.1 A-120302.1 CAGAACAGAAGAUCCCGGAdTdT A-120303.1 UCCGGGAUCUUCUGUUCUGdTdT AD-59283.1 A-120318.1 CCUUGUCGAUCUGUUCAGCdTdT A-120319.1 GCUGAACAGAUCGACAAGGdTdT AD-59284.1 A-120334.1 AGGCAAGUUCCGUUAUCGGdTdT A-120335.1 CCGAUAACGGAACUUGCCUdTdT AD-59285.1 A-120256.1 UUUUGUCCUUGCUGCUCAUdTdT A-120257.1 AUGAGCAGCAAGGACAAAAdTdT AD-59286.1 A-120272.1 AGACCUACCAGGACAUCAGdTdT A-120273.1 CUGAUGUCCUGGUAGGUCUdTdT AD-59287.1 A-120288.1 AACUGAACUGCCGACUCUAdTdT A-120289.1 UAGAGUCGGCAGUUCAGUUdTdT AD-59288.1 A-120304.1 CAUUUACUUCAAGGGCCUGdTdT A-120305.1 CAGGCCCUUGAAGUAAAUGdTdT AD-59289.1 A-120320.1 CCCUGGACUUCAAGGAAAAdTdT A-120321.1 UUUUCCUUGAAGUCCAGGGdTdT AD-59290.1 A-120336.1 AGCUGCAAGUACCGCUGUUdTdT A-120337.1 AACAGCGGUACUUGCAGCUdTdT AD-59291.1 A-120258.1 ACACAAGGAAGGAACUGUUdTdT A-120259.1 AACAGUUCCUUCCUUGUGUdTdT AD-59292.1 A-120274.1 GCAACUGAGGAUGAGGGCUdTdT A-120275.1 AGCCCUCAUCCUCAGUUGCdTdT AD-59293.1 A-120290.1 GUAGCCAACCCUUGUGUUAdTdT A-120291.1 UAACACAAGGGUUGGCUACdTdT AD-59294.1 A-120306.1 GUUUGUGAACAGAAGUAAAdTdT A-120307.1 UUUACUUCUGUUCACAAACdTdT AD-59295.1 A-120322.1 GGGUGACUUUCAAGGCCAAdTdT A-120323.1 UUGGCCUUGAAAGUCACCCdTdT AD-59296.1 A-120338.1 UUAUCGGCGCGUGGCUGAAdTdT A-120339.1 UUCAGCCACGCGCCGAUAAdTdT AD-59297.1 A-120260.1 CCACUUCUUCUUUGCCAAAdTdT A-120261.1 UUUGGCAAAGAAGAAGUGGdTdT AD-59298.1 A-120276.1 AACACCAUUUACUUCAAGGdTdT A-120277.1 CCUUGAAGUAAAUGGUGUUdTdT AD-59299.1 A-120292.1 GAUGGAGAGUCGUGUUCAGdTdT A-120293.1 CUGAACACGACUCUCCAUCdTdT AD-59300.1 A-120308.1 CACCAUUUACUUCAAGGGCdTdT A-120309.1 GCCCUUGAAGUAAAUGGUGdTdT AD-59301.1 A-120324.1 UUUACUUCAAGGGCCUGUGdTdT A-120325.1 CACAGGCCCUUGAAGUAAAdTdT AD-59302.1 A-120340.1 UAAGAGAAGUUCCUCUGAAdTdT A-120341.1 UUCAGAGGAACUUCUCUUAdTdT AD-59303.1 A-120262.1 GCGGGACAUUCCCAUGAAUdTdT A-120263.1 AUUCAUGGGAAUGUCCCGCdTdT AD-59304.1 A-120278.1 UGCCCCACCCUGUCCUCUGdTdT A-120279.1 CAGAGGACAGGGUGGGGCAdTdT AD-59305.1 A-120294.1 UGGUUAACACCAUUUACUUdTdT A-120295.1 AAGUAAAUGGUGUUAACCAdTdT AD-59306.1 A-120310.1 CGGAUUGCCUCAGAUCACAdTdT A-120311.1 UGUGAUCUGAGGCAAUCCGdTdT AD-59307.1 A-120326.1 CCAGGACAUCAGUGAGUUGdTdT A-120327.1 CAACUCACUGAUGUCCUGGdTdT AD-59308.1 A-120342.1 CCAGUUUUCAGGCGGAUUGdTdT A-120343.1 CAAUCCGCCUGAAAACUGGdTdT AD-59309.1 A-120264.1 CACCAUAUCUGAGAAAACAdTdT A-120265.1 UGUUUUCUCAGAUAUGGUGdTdT AD-59310.1 A-120280.1 AAGUAAAAAUAAAUACAAAdTdT A-120281.1 UUUGUAUUUAUUUUUACUUdTdT AD-59311.1 A-120296.1 GCACCCAGGUGCUUGAGUUdTdT A-120297.1 AACUCAAGCACCUGGGUGCdTdT AD-59312.1 A-120312.1 UAACACCAUUUACUUCAAGdTdT A-120313.1 CUUGAAGUAAAUGGUGUUAdTdT AD-59313.1 A-120328.1 CCUUCAAAGGUGAUGACAUdTdT A-120329.1 AUGUCAUCACCUUUGAAGGdTdT AD-59314.1 A-120344.1 CAAGGCCAAUUCCCGCUUUdTdT A-120345.1 AAAGCGGGAAUUGGCCUUGdTdT AD-59315.1 A-120360.1 UCAGUUUGAAGGAGCAGCUdTdT A-120361.1 AGCUGCUCCUUCAAACUGAdTdT AD-59316.1 A-120376.1 GGGACUGCGUGACCUGUCAdTdT A-120377.1 UGACAGGUCACGCAGUCCCdTdT AD-59317.1 A-120392.1 UCAGCCAAUCGCCUUUUUGdTdT A-120393.1 CAAAAAGGCGAUUGGCUGAdTdT AD-59318.1 A-120408.1 CCUCGGAAGCCAUCAAUGAdTdT A-120409.1 UCAUUGAUGGCUUCCGAGGdTdT AD-59319.1 A-120424.1 GACAAUGAUAACAUUUUCCdTdT A-120425.1 GGAAAAUGUUAUCAUUGUCdTdT AD-59320.1 A-120346.1 CUUAUUCUUUGCACCUCUUdTdT A-120347.1 AAGAGGUGCAAAGAAUAAGdTdT AD-59321.1 A-120362.1 AUUGCUGGCCGUUCGCUAAdTdT A-120363.1 UUAGCGAACGGCCAGCAAUdTdT AD-59322.1 A-120378.1 AAAUGAAGAAGGCAGUGAAdTdT A-120379.1 UUCACUGCCUUCUUCAUUUdTdT AD-59323.1 A-120394.1 UGAGUUGGUAUAUGGAGCCdTdT A-120395.1 GGCUCCAUAUACCAACUCAdTdT AD-59324.1 A-120410.1 UCCAGCAACUGAUGGAGGUdTdT A-120411.1 ACCUCCAUCAGUUGCUGGAdTdT AD-59325.1 A-120426.1 AACUGUAACCUCUGGAAAAdTdT A-120427.1 UUUUCCAGAGGUUACAGUUdTdT AD-59326.1 A-120348.1 UCAACAAAUGGGUGUCCAAdTdT A-120349.1 UUGGACACCCAUUUGUUGAdTdT AD-59327.1 A-120364.1 GAUUAGCGGCCAUGUAUUCdTdT A-120365.1 GAAUACAUGGCCGCUAAUCdTdT AD-59328.1 A-120380.1 GUGCUUGAGUUGCCCUUCAdTdT A-120381.1 UGAAGGGCAACUCAAGCACdTdT AD-59329.1 A-120396.1 UGCAGAAGGCCGAGAUGACdTdT A-120397.1 GUCAUCUCGGCCUUCUGCAdTdT AD-59330.1 A-120412.1 CUAUUUUUGGUUUGUGAACdTdT A-120413.1 GUUCACAAACCAAAAAUAGdTdT AD-59331.1 A-120428.1 CAGUGAAGCAGCUGCAAGUdTdT A-120429.1 ACUUGCAGCUGCUUCACUGdTdT AD-59332.1 A-120350.1 GUGUCCAAUAAGACCGAAGdTdT A-120351.1 CUUCGGUCUUAUUGGACACdTdT AD-59333.1 A-120366.1 AUGAAUUGGAGGAGAUGAUdTdT A-120367.1 AUCAUCUCCUCCAAUUCAUdTdT AD-59334.1 A-120382.1 AUCUAUGAUGUACCAGGAAdTdT A-120383.1 UUCCUGGUACAUCAUAGAUdTdT AD-59335.1 A-120398.1 CCAUAAGGCAUUUCUUGAGdTdT A-120399.1 CUCAAGAAAUGCCUUAUGGdTdT AD-59336.1 A-120414.1 AUGCAUUCCAUAAGGCAUUdTdT A-120415.1 AAUGCCUUAUGGAAUGCAUdTdT AD-59337.1 A-120430.1 UGGUGCUGGUUAACACCAUdTdT A-120431.1 AUGGUGUUAACCAGCACCAdTdT AD-59338.1 A-120352.1 AUCUGUUCAGCCCUGAAAAdTdT A-120353.1 UUUUCAGGGCUGAACAGAUdTdT AD-59339.1 A-120368.1 AGAUGAUGCUGGUGGUCCAdTdT A-120369.1 UGGACCACCAGCAUCAUCUdTdT AD-59340.1 A-120384.1 AUAUGGAGCCAAGCUCCAGdTdT A-120385.1 CUGGAGCUUGGCUCCAUAUdTdT AD-59341.1 A-120400.1 CCGAAUCACCGAUGUCAUUdTdT A-120401.1 AAUGACAUCGGUGAUUCGGdTdT AD-59342.1 A-120416.1 GCAGAGCAAUCCAGAGCGGdTdT A-120417.1 CCGCUCUGGAUUGCUCUGCdTdT AD-59343.1 A-120432.1 ACACCAUUUACUUCAAGGGdTdT A-120433.1 CCCUUGAAGUAAAUGGUGUdTdT AD-59344.1 A-120354.1 GUACCAGGAAGGCAAGUUCdTdT A-120355.1 GAACUUGCCUUCCUGGUACdTdT AD-59345.1 A-120370.1 UGCCCAAGCCUGAGAAGAGdTdT A-120371.1 CUCUUCUCAGGCUUGGGCAdTdT AD-59346.1 A-120386.1 UUCUUUGCCAAACUGAACUdTdT A-120387.1 AGUUCAGUUUGGCAAAGAAdTdT

AD-59347.1 A-120402.1 UGGCCAAGGUAGAGAAGGAdTdT A-120403.1 UCCUUCUCUACCUUGGCCAdTdT AD-59348.1 A-120418.1 UGCUGCAAGAGUGGCUGGAdTdT A-120419.1 UCCAGCCACUCUUGCAGCAdTdT AD-59349.1 A-120434.1 CCAUGUGCAUUUACCGCUCdTdT A-120435.1 GAGCGGUAAAUGCACAUGGdTdT AD-59350.1 A-120356.1 AGACAUGGGCCUUGUCGAUdTdT A-120357.1 AUCGACAAGGCCCAUGUCUdTdT AD-59351.1 A-120372.1 UUUUGGAGACAAAUCCCUUdTdT A-120373.1 AAGGGAUUUGUCUCCAAAAdTdT AD-59352.1 A-120388.1 GGAUGAGGGCUCAGAACAGdTdT A-120389.1 CUGUUCUGAGCCCUCAUCCdTdT AD-59353.1 A-120404.1 CGGCUUUUGCUAUGACCAAdTdT A-120405.1 UUGGUCAUAGCAAAAGCCGdTdT AD-59354.1 A-120420.1 AGCUCCAGCCCCUGGACUUdTdT A-120421.1 AAGUCCAGGGGCUGGAGCUdTdT AD-59355.1 A-120436.1 CUGAUCAGAUCCACUUCUUdTdT A-120437.1 AAGAAGUGGAUCUGAUCAGdTdT AD-59356.1 A-120358.1 UGCUGGUGGUCCACAUGCCdTdT A-120359.1 GGCAUGUGGACCACCAGCAdTdT AD-59357.1 A-120374.1 GCGAGAUUUAGAGGAAAGAdTdT A-120375.1 UCUUUCCUCUAAAUCUCGCdTdT AD-59358.1 A-120390.1 CUGCUCAUUGGCUUCUGGGdTdT A-120391.1 CCCAGAAGCCAAUGAGCAGdTdT AD-59359.1 A-120406.1 CCUUCAAUGAGACCUACCAdTdT A-120407.1 UGGUAGGUCUCAUUGAAGGdTdT AD-59360.1 A-120422.1 ACCAUUUACUUCAAGGGCCdTdT A-120423.1 GGCCCUUGAAGUAAAUGGUdTdT AD-59587.1 A-120438.1 GCACCUCUUCCUAUUUUUGdTdT A-120439.1 CAAAAAUAGGAAGAGGUGCdTdT AD-59588.1 A-120454.1 GUGGCUGAAGGCACCCAGGdTdT A-120455.1 CCUGGGUGCCUUCAGCCACdTdT AD-59589.1 A-120470.1 UCCGCAUUGAGGACGGCUUdTdT A-120471.1 AAGCCGUCCUCAAUGCGGAdTdT AD-59590.1 A-120486.1 UGGACAUCUGCACAGCCAAdTdT A-120487.1 UUGGCUGUGCAGAUGUCCAdTdT AD-59591.1 A-120502.1 GUCCAAACUCCCAGGUAUUdTdT A-120503.1 AAUACCUGGGAGUUUGGACdTdT AD-59592.1 A-120518.1 AGAAGGAACUCACCCCAGAdTdT A-120519.1 UCUGGGGUGAGUUCCUUCUdTdT AD-59593.1 A-120440.1 UCUUGAGGUAAAUGAAGAAdTdT A-120441.1 UUCUUCAUUUACCUCAAGAdTdT AD-59594.1 A-120456.1 AGCCCUGUGGACAUCUGCAdTdT A-120457.1 UGCAGAUGUCCACAGGGCUdTdT AD-59595.1 A-120472.1 CAGAGCGGCCAUCAACAAAdTdT A-120473.1 UUUGUUGAUGGCCGCUCUGdTdT AD-59596.1 A-120488.1 AUUUAAGUUUGACACCAUAdTdT A-120489.1 UAUGGUGUCAAACUUAAAUdTdT AD-59597.1 A-120504.1 UGAGAAGAGCCUGGCCAAGdTdT A-120505.1 CUUGGCCAGGCUCUUCUCAdTdT AD-59598.1 A-120520.1 UCACCAUGGUCCUCAUCUUdTdT A-120521.1 AAGAUGAGGACCAUGGUGAdTdT AD-59599.1 A-120442.1 GGAAGGAACUGUUCUACAAdTdT A-120443.1 UUGUAGAACAGUUCCUUCCdTdT AD-59600.1 A-120458.1 CUGGUUUUUAUAAGAGAAGdTdT A-120459.1 CUUCUCUUAUAAAAACCAGdTdT AD-59601.1 A-120474.1 CUGGGUGCCUGUAAUGACAdTdT A-120475.1 UGUCAUUACAGGCACCCAGdTdT AD-59602.1 A-120490.1 GUACCGCUGUUGUGAUUGCdTdT A-120491.1 GCAAUCACAACAGCGGUACdTdT AD-59603.1 A-120506.1 UCUAUCAGCACCUGGCAGAdTdT A-120507.1 UCUGCCAGGUGCUGAUAGAdTdT AD-59604.1 A-120522.1 CUGGCAGAUUCCAAGAAUGdTdT A-120523.1 CAUUCUUGGAAUCUGCCAGdTdT AD-59605.1 A-120444.1 CGAUGUCAUUCCCUCGGAAdTdT A-120445.1 UUCCGAGGGAAUGACAUCGdTdT AD-59606.1 A-120460.1 GCUUCUGGGACUGCGUGACdTdT A-120461.1 GUCACGCAGUCCCAGAAGCdTdT AD-59607.1 A-120476.1 CCUGUCACGGGAGCCCUGUdTdT A-120477.1 ACAGGGCUCCCGUGACAGGdTdT AD-59608.1 A-120492.1 AUUUACUUCAAGGGCCUGUdTdT A-120493.1 ACAGGCCCUUGAAGUAAAUdTdT AD-59609.1 A-120508.1 UGCUACCACUUUCUAUCAGdTdT A-120509.1 CUGAUAGAAAGUGGUAGCAdTdT AD-59610.1 A-120524.1 GGAACUGUCCAAGGCCAAUdTdT A-120525.1 AUUGGCCUUGGACAGUUCCdTdT AD-59611.1 A-120446.1 ACAAAUCCUCCAAGUUAGUdTdT A-120447.1 ACUAACUUGGAGGAUUUGUdTdT AD-59612.1 A-120462.1 AUUUACCGCUCCCCGGAGAdTdT A-120463.1 UCUCCGGGGAGCGGUAAAUdTdT AD-59613.1 A-120478.1 ACCCCUGAGUAUCUCCACGdTdT A-120479.1 CGUGGAGAUACUCAGGGGUdTdT AD-59614.1 A-120494.1 CACUAUCUCCACUUGCCCAdTdT A-120495.1 UGGGCAAGUGGAGAUAGUGdTdT AD-59615.1 A-120510.1 AAAUACAAACUACUUCCAUdTdT A-120511.1 AUGGAAGUAGUUUGUAUUUdTdT AD-59616.1 A-120526.1 CUGGUUAACACCAUUUACUdTdT A-120527.1 AGUAAAUGGUGUUAACCAGdTdT AD-59617.1 A-120448.1 UCAUCUUGCCCAAGCCUGAdTdT A-120449.1 UCAGGCUUGGGCAAGAUGAdTdT AD-59618.1 A-120464.1 CCUCAGAUCACACUAUCUCdTdT A-120465.1 GAGAUAGUGUGAUCUGAGGdTdT AD-59619.1 A-120480.1 CUGUCCUCUGGAACCUCUGdTdT A-120481.1 CAGAGGUUCCAGAGGACAGdTdT AD-59620.1 A-120496.1 CCCUGUGGAAGAUUAGCGGdTdT A-120497.1 CCGCUAAUCUUCCACAGGGdTdT AD-59621.1 A-120512.1 AUCUCCACGGCUUUUGCUAdTdT A-120513.1 UAGCAAAAGCCGUGGAGAUdTdT AD-59622.1 A-120528.1 GUGGCUGGAUGAAUUGGAGdTdT A-120529.1 CUCCAAUUCAUCCAGCCACdTdT AD-59623.1 A-120450.1 CAAGUUAGUAUCAGCCAAUdTdT A-120451.1 AUUGGCUGAUACUAACUUGdTdT AD-59624.1 A-120466.1 GUGAUGACAUCACCAUGGUdTdT A-120467.1 ACCAUGGUGAUGUCAUCACdTdT AD-59625.1 A-120482.1 UUCCAAGAAUGACAAUGAUdTdT A-120483.1 AUCAUUGUCAUUCUUGGAAdTdT AD-59626.1 A-120498.1 UGAUGGAGGUAUUUAAGUUdTdT A-120499.1 AACUUAAAUACCUCCAUCAdTdT AD-59627.1 A-120514.1 GUAUUCCAAUGUGAUAGGAdTdT A-120515.1 UCCUAUCACAUUGGAAUACdTdT AD-59628.1 A-120530.1 CAGCCAAGCCGCGGGACAUdTdT A-120531.1 AUGUCCCGCGGCUUGGCUGdTdT AD-59629.1 A-120452.1 CAUGCCCCGCUUCCGCAUUdTdT A-120453.1 AAUGCGGAAGCGGGGCAUGdTdT AD-59630.1 A-120468.1 AUGUGAUAGGAACUGUAACdTdT A-120469.1 GUUACAGUUCCUAUCACAUdTdT AD-59631.1 A-120484.1 ACAAAUCCCUUACCUUCAAdTdT A-120485.1 UUGAAGGUAAGGGAUUUGUdTdT AD-59632.1 A-120500.1 AGGUUUAUCUUUUGUCCUUdTdT A-120501.1 AAGGACAAAAGAUAAACCUdTdT AD-59633.1 A-120516.1 AGAUCCCGGAGGCCACCAAdTdT A-120517.1 UUGGUGGCCUCCGGGAUCUdTdT AD-59634.1 A-120532.1 ACAUUUUCCUGUCACCCCUdTdT A-120533.1 AGGGGUGACAGGAAAAUGUdTdT AD-59635.1 A-120548.1 GGCCUUUCCUGGUUUUUAUdTdT A-120549.1 AUAAAAACCAGGAAAGGCCdTdT AD-59636.1 A-120564.1 UUGUGUUAAGUAAAAUGUUdTdT A-120565.1 AACAUUUUACUUAACACAAdTdT AD-59637.1 A-120534.1 CAAGGAAAAUGCAGAGCAAdTdT A-120535.1 UUGCUCUGCAUUUUCCUUGdTdT AD-59638.1 A-120550.1 CUGAGAAAACAUCUGAUCAdTdT A-120551.1 UGAUCAGAUGUUUUCUCAGdTdT AD-59639.1 A-120566.1 UUACUUCAAGGGCCUGUGGdTdT A-120567.1 CCACAGGCCCUUGAAGUAAdTdT AD-59640.1 A-120536.1 ACCCCAACAGGGUGACUUUdTdT A-120537.1 AAAGUCACCCUGUUGGGGUdTdT AD-59641.1 A-120552.1 CCAGGUAUUGUUGCAGAAGdTdT A-120553.1 CUUCUGCAACAAUACCUGGdTdT AD-59642.1 A-120568.1 GCCUGUGGAAGUCAAAGUUdTdT A-120569.1 AACUUUGACUUCCACAGGCdTdT AD-59643.1 A-120538.1 UGGAACCUCUGCGAGAUUUdTdT A-120539.1 AAAUCUCGCAGAGGUUCCAdTdT AD-59644.1 A-120554.1 AGCCCUGAGAACACAAGGAdTdT A-120555.1 UCCUUGUGUUCUCAGGGCUdTdT AD-59645.1 A-120570.1 CUCUAUGUCUCAGAUGCAUdTdT A-120571.1 AUGCAUCUGAGACAUAGAGdTdT AD-59646.1 A-120540.1 AAGACCGAAGGCCGAAUCAdTdT A-120541.1 UGAUUCGGCCUUCGGUCUUdTdT AD-59647.1 A-120556.1 UAAAAUGUUCUUAUUCUUUdTdT A-120557.1 AAAGAAUAAGAACAUUUUAdTdT AD-59648.1 A-120572.1 UCUGGAAAAAGGAAGGUUUdTdT A-120573.1 AAACCUUCCUUUUUCCAGAdTdT AD-59649.1 A-120542.1 UUCAAGGCCAACAGGCCUUdTdT A-120543.1 AAGGCCUGUUGGCCUUGAAdTdT AD-59650.1 A-120558.1 GAAGUCAAAGUUCAGCCCUdTdT A-120559.1 AGGGCUGAACUUUGACUUCdTdT AD-59651.1 A-120574.1 CCAUCAAUGAGCUCACUGUdTdT A-120575.1 ACAGUGAGCUCAUUGAUGGdTdT AD-59652.1 A-120544.1 CUCUGAACACUAUUAUCUUdTdT A-120545.1 AAGAUAAUAGUGUUCAGAGdTdT AD-59653.1 A-120560.1 UGCUGGUUAACACCAUUUAdTdT A-120561.1 UAAAUGGUGUUAACCAGCAdTdT AD-59654.1 A-120546.1 AGAGGAAAGAACCAGUUUUdTdT A-120547.1 AAAACUGGUUCUUUCCUCUdTdT AD-59655.1 A-120562.1 UGCCCAGCCCUGUGGAAGAdTdT A-120563.1 UCUUCCACAGGGCUGGGCAdTdT

Cell Culture and Transfections

[0503] HepG2 cells (ATCC, Manassas, Va.) were grown to near confluence at 37.degree. C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 .mu.l of Opti-MEM plus 0.2 .mu.l of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 .mu.l of each of the 164 siRNA duplexes to an individual well in a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. 80 .mu.l of complete growth media without antibiotic containing .about.2.5.times.10.sup.4 HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 20 nM and included naive cells and cells transfected with AD-1955, a luciferase targeting siRNA as negative controls.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)

[0504] Cells were harvested and lysed in 150 .mu.l of Lysis/Binding Buffer then mixed for 5 minute at 700 rpm on a platform shaker (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 .mu.l Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 .mu.l Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 .mu.l Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 .mu.l Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 .mu.l of Elution Buffer was added and mixed for 5 minutes at 70.degree. C. Beads were captured on magnet for 5 minutes. Forty .mu.l of supernatant, containing the isolated RNA was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

[0505] A master mix of 2 .mu.l 10.times. Buffer, 0.8 .mu.l 25.times.dNTPs, 2 .mu.l Random primers, 1 .mu.l Reverse Transcriptase, 1 .mu.l RNase inhibitor and 3.2 .mu.l of H.sub.2O per reaction were added into 10 .mu.l total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25.degree. C. 10 min, 37.degree. C. 120 min, 85.degree. C. 5 sec, 4.degree. C. hold.

Real Time PCR

[0506] Two .mu.l of cDNA were added to a master mix containing 0.5 .mu.l human GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 .mu.l human SERPINC1 TaqMan probe (Applied Biosystems cat # Hs00892758_m1) and 5 .mu.l Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384-well plate (Roche cat #04887301001). Real time PCR was done in an LC480 Real Time PCR machine (Roche).

[0507] To calculate relative fold change, real time data were analyzed using the .DELTA..DELTA.Ct method and normalized to assays performed with cells transfected with 20 nM AD-1955.

[0508] Table 22 shows the results of a single dose screen in HepG2 of the indicated iRNAs.

TABLE-US-00023 TABLE 22.sup.2 20 nM single dose screen of the indicated iRNAs 20 nM Standard Duplex ID Average Deviation AD1955 100.4 9.1 AD-59267.1 9.3 2.7 AD-59268.1 72.2 20.3 AD-59269.1 6.9 0.4 AD-59270.1 18.3 5.2 AD-59271.1 27.9 7.6 AD-59272.1 13.0 1.1 AD-59273.1 79.4 10.9 AD-59274.1 5.9 1.1 AD-59275.1 16.1 5.5 AD-59276.1 6.1 2.2 AD-59277.1 4.4 0.9 AD-59278.1 9.3 0.4 AD-59279.1 12.7 4.7 AD-59280.1 4.5 1.7 AD-59281.1 15.7 5.6 AD-59282.1 25.9 6.9 AD-59283.1 27.0 18.9 AD-59284.1 8.6 3.7 AD-59285.1 11.2 3.7 AD-59286.1 15.2 5.0 AD-59287.1 6.9 1.9 AD-59288.1 74.5 16.5 AD-59289.1 3.3 1.3 AD-59290.1 13.8 2.5 AD-59291.1 9.4 2.8 AD-59292.1 9.5 3.5 AD-59293.1 2.5 1.1 AD-59294.1 4.8 1.8 AD-59295.1 11.8 7.2 AD-59296.1 32.4 4.9 AD-59297.1 78.5 105.0 AD-59298.1 76.3 10.7 AD-59299.1 4.4 0.8 AD-59300.1 32.2 10.8 AD-59301.1 48.5 15.2 AD-59302.1 7.2 3.0 AD-59303.1 17.0 2.3 AD-59304.1 87.1 16.4 AD-59305.1 4.4 0.9 AD-59306.1 35.7 10.6 AD-59307.1 6.3 0.4 AD-59308.1 65.1 9.1 AD-59309.1 7.5 2.0 AD-59310.1 27.1 9.5 AD-59311.1 8.1 1.4 AD-59312.1 84.5 8.5 AD-59313.1 17.8 0.2 AD-59314.1 21.1 0.4 AD-59315.1 85.5 29.8 AD-59316.1 13.0 1.6 AD-59317.1 64.0 10.7 AD-59318.1 7.9 2.2 AD-59319.1 31.8 4.7 AD-59320.1 5.7 2.1 AD-59321.1 3.4 0.0 AD-59322.1 9.6 1.3 AD-59323.1 100.1 4.8 AD-59324.1 40.2 2.9 AD-59325.1 5.8 0.7 AD-59326.1 20.4 10.9 AD-59327.1 5.0 2.0 AD-59328.1 8.0 2.8 AD-59329.1 54.1 5.9 AD-59330.1 21.6 12.3 AD-59331.1 4.3 2.4 AD-59332.1 12.9 3.8 AD-59333.1 26.1 1.0 AD-59334.1 41.9 4.7 AD-59335.1 12.5 1.7 AD-59336.1 13.5 1.7 AD-59337.1 78.6 3.6 AD-59338.1 17.9 12.3 AD-59339.1 5.8 4.1 AD-59340.1 92.3 10.0 AD-59341.1 8.0 1.8 AD-59342.1 11.1 1.9 AD-59343.1 43.6 4.2 AD-59344.1 6.0 2.3 AD-59345.1 23.6 3.6 AD-59346.1 41.0 3.2 AD-59347.1 12.2 1.0 AD-59348.1 30.0 5.1 AD-59349.1 14.4 0.9 AD-59350.1 9.1 1.0 AD-59351.1 10.7 1.6 AD-59352.1 1.9 0.4 AD-59353.1 4.6 0.8 AD-59354.1 30.1 0.1 AD-59355.1 12.2 2.2 AD-59356.1 63.6 11.5 AD-59357.1 112.1 20.6 AD-59358.1 23.2 3.0 AD-59359.1 7.5 0.8 AD-59360.1 19.2 0.5 AD-59587.1 8.4 2.7 AD-59588.1 24.8 5.7 AD-59589.1 4.4 1.7 AD-59590.1 13.6 1.3 AD-59591.1 3.1 0.5 AD-59592.1 12.1 2.7 AD-59593.1 7.6 3.5 AD-59594.1 7.5 1.6 AD-59595.1 20.1 2.9 AD-59596.1 7.3 1.3 AD-59597.1 61.3 6.8 AD-59598.1 24.4 1.6 AD-59599.1 4.3 0.4 AD-59600.1 30.1 2.8 AD-59601.1 11.4 0.8 AD-59602.1 6.0 0.0 AD-59603.1 19.4 2.2 AD-59604.1 5.8 0.6 AD-59605.1 6.5 0.3 AD-59606.1 9.9 0.6 AD-59607.1 28.0 2.8 AD-59608.1 66.1 8.0 AD-59609.1 84.2 9.0 AD-59610.1 8.0 1.7 AD-59611.1 6.6 1.5 AD-59612.1 31.5 2.7 AD-59613.1 27.6 1.7 AD-59614.1 20.3 0.5 AD-59615.1 12.8 1.4 AD-59616.1 6.8 0.3 AD-59617.1 13.1 0.9 AD-59618.1 5.4 0.2 AD-59619.1 59.1 5.4 AD-59620.1 6.3 1.7 AD-59621.1 18.4 1.8 AD-59622.1 9.6 1.2 AD-59623.1 8.7 2.1 AD-59624.1 13.6 0.7 AD-59625.1 12.0 1.5 AD-59626.1 14.6 1.8 AD-59627.1 7.7 6.7 AD-59628.1 7.6 0.3 AD-59629.1 55.7 8.2 AD-59630.1 20.2 5.9 AD-59631.1 12.3 0.1 AD-59632.1 3.1 0.3 AD-59633.1 20.4 2.1 AD-59634.1 3.7 0.0 AD-59635.1 16.1 1.3 AD-59636.1 13.0 1.0 AD-59637.1 14.4 4.8 AD-59638.1 7.7 1.0 AD-59639.1 70.4 5.3 AD-59640.1 2.5 0.1 AD-59641.1 18.1 2.1 AD-59642.1 5.9 1.0 AD-59643.1 70.7 17.8 AD-59644.1 17.9 4.7 AD-59645.1 2.5 0.1 AD-59646.1 19.9 0.1 AD-59647.1 74.8 12.0 AD-59648.1 6.8 0.0 AD-59649.1 95.2 6.2 AD-59650.1 71.1 1.0 AD-59651.1 5.0 0.8 AD-59652.1 5.5 1.2 AD-59653.1 15.3 1.0 AD-59654.1 67.4 0.9 AD-59655.1 10.8 1.0 Naive 94.9 14.2 .sup.2Modified.

Sequence CWU 1

1

163011599DNAHomo sapiens 1tctgccccac cctgtcctct ggaacctctg cgagatttag aggaaagaac cagttttcag 60gcggattgcc tcagatcaca ctatctccac ttgcccagcc ctgtggaaga ttagcggcca 120tgtattccaa tgtgatagga actgtaacct ctggaaaaag gaaggtttat cttttgtcct 180tgctgctcat tggcttctgg gactgcgtga cctgtcacgg gagccctgtg gacatctgca 240cagccaagcc gcgggacatt cccatgaatc ccatgtgcat ttaccgctcc ccggagaaga 300aggcaactga ggatgagggc tcagaacaga agatcccgga ggccaccaac cggcgtgtct 360gggaactgtc caaggccaat tcccgctttg ctaccacttt ctatcagcac ctggcagatt 420ccaagaatga caatgataac attttcctgt cacccctgag tatctccacg gcttttgcta 480tgaccaagct gggtgcctgt aatgacaccc tccagcaact gatggaggta tttaagtttg 540acaccatatc tgagaaaaca tctgatcaga tccacttctt ctttgccaaa ctgaactgcc 600gactctatcg aaaagccaac aaatcctcca agttagtatc agccaatcgc ctttttggag 660acaaatccct taccttcaat gagacctacc aggacatcag tgagttggta tatggagcca 720agctccagcc cctggacttc aaggaaaatg cagagcaatc cagagcggcc atcaacaaat 780gggtgtccaa taagaccgaa ggccgaatca ccgatgtcat tccctcggaa gccatcaatg 840agctcactgt tctggtgctg gttaacacca tttacttcaa gggcctgtgg aagtcaaagt 900tcagccctga gaacacaagg aaggaactgt tctacaaggc tgatggagag tcgtgttcag 960catctatgat gtaccaggaa ggcaagttcc gttatcggcg cgtggctgaa ggcacccagg 1020tgcttgagtt gcccttcaaa ggtgatgaca tcaccatggt cctcatcttg cccaagcctg 1080agaagagcct ggccaaggta gagaaggaac tcaccccaga ggtgctgcaa gagtggctgg 1140atgaattgga ggagatgatg ctggtggtcc acatgccccg cttccgcatt gaggacggct 1200tcagtttgaa ggagcagctg caagacatgg gccttgtcga tctgttcagc cctgaaaagt 1260ccaaactccc aggtattgtt gcagaaggcc gagatgacct ctatgtctca gatgcattcc 1320ataaggcatt tcttgaggta aatgaagaag gcagtgaagc agctgcaagt accgctgttg 1380tgattgctgg ccgttcgcta aaccccaaca gggtgacttt caaggccaac aggcctttcc 1440tggtttttat aagagaagtt cctctgaaca ctattatctt catgggcaga gtagccaacc 1500cttgtgttaa gtaaaatgtt cttattcttt gcacctcttc ctatttttgg tttgtgaaca 1560gaagtaaaaa taaatacaaa ctacttccat ctcacatta 159921545DNAMacaca mulatta 2ggcacgagga ccatctccac ttgcccagcc ctgtggaaga ttagcgacca tgtattccaa 60tgtgatagga accgtagcct ctggaaaaag gaaggtttat cttctgtcct tgctgctcat 120tggcctctgg gactgtatga cctgtcacgg gagccctgtg gacatctgca cagccaagcc 180gcgggacatt cccatgaatc ccatgtgcat ttaccgctcc ccggagaaga aggcaactga 240ggatgagggc tcagaacaga agatccccga ggccaccaac cggcgcgtct gggaactgtc 300caaggccaat tcccgctttg ctaccacttt ctatcagcac ctggcagatt ccaagaacga 360caaggataac attttcctgt cacccctgag tgtctccacg gcttttgcta tgaccaagct 420gggtgcctgt aatgacaccc tcaagcaact gatggaggta tttaagtttg acaccatatc 480tgagaaaaca tctgatcaga tccacttctt ctttgccaaa ctgaactgcc gactctatcg 540aaaagccaac aaatcctcca agttagtatc agccaatcgc ctttttggag acaaatccct 600taccttcaat gagacctacc aggacatcag tgagttggta tacggagcca agctccagcc 660cctggacttc aaggaaaatg cagagcaatc cagagcggcc atcaacaaat gggtgtccaa 720taagaccgaa ggccgaatca ccgatgtcat tcccccggaa gccatcaacg agctcactgt 780tctggtgctg gttaacacca tttacttcaa gggcctgtgg aagtcaaagt ttagccctga 840gaacacaagg atggaaccgt tctacaaggc tgatggagag tcgtgttcag cgtctatgat 900gtaccaggaa ggcaagttct gttatcggcg cgtggctgaa ggcacccagg tgcttgagtt 960gcccttcaag ggtgatgaca tcaccatggt gctcatcctg cccaagcctg agaagagcct 1020gaccaaggtg gagcaggaac tcaccccaga ggtgctgcag gagtggctgg atgagttgga 1080ggagatgatg ctggtggttc acatgccccg cttccgcatt gaggacggct tcagtttgaa 1140ggagcagctg caagacatgg gccttgtcga tctgttcagc cctgaaaagt ccaaactccc 1200aggtattgtt gcagaaggcc gggatgacct ctatgtctcc gatgcattcc ataaggcatt 1260tcttgaggta aatgaagaag gcagtgaagc agctgcaagt accgccattg ggattgctgg 1320ccgttcgcta aaccccaaca gggtgacctt caaggccaac aggcctttcc tggtttttat 1380aagagaagtt cctctgaaca ctattatctt catgggcaga gtagccaacc cttgtgtgag 1440ctaaactgtt cttattcttt gtacctcttc ctattttggt ttgtgaatag aagtaaaaat 1500aaatacaact actcccatct tacattaaaa aaaaaaaaaa aaaaa 154532171DNAMus musculus 3ataggtaatt ttagaaatag atctgatttg tatctgagac attttagtga agtggtgaga 60tataagacat aatcagaaga catatctacc tgaagacttt aaggggagag ctccctcccc 120cacctggcct ctggacctct cagatttagg ggaaagaacc agttttcgga gtgatcgtct 180cagtcagcac catctctgta ggagcatcgg ccatgtattc ccctggggca ggaagtgggg 240ctgctggtga gaggaagctt tgtctcctct ctctgctcct catcggtgcc ttgggctgtg 300ctatctgtca cggaaaccct gtggacgaca tctgcatagc gaagccccga gacatccccg 360tgaatccctt gtgcatttac cgctcccctg ggaagaaggc caccgaggag gatggctcag 420agcagaaggt tccagaagcc accaaccggc gggtctggga actgtccaag gccaattcgc 480gatttgccac taacttctac cagcacctgg cagactccaa gaatgacaac gacaacattt 540tcctgtcacc cttgagcatc tccactgctt ttgctatgac caagctgggt gcctgtaacg 600acactctcaa gcagctgatg gaggttttta aatttgatac catctccgag aagacatccg 660accagatcca cttcttcttt gccaaactga actgccgact ctatcgaaaa gccaacaagt 720cctctgactt ggtatcagcc aaccgccttt ttggagacaa atccctcacc ttcaacgaga 780gctatcaaga tgttagtgag gttgtctatg gagccaagct ccagcccctg gacttcaagg 840agaatccgga gcaatccaga gtgaccatca acaactgggt agctaataag actgaaggcc 900gcatcaaaga tgtcatccca cagggcgcca ttaacgagct cactgccctg gttctggtta 960acaccattta cttcaagggc ctgtggaagt caaagttcag ccctgagaac acaaggaagg 1020aaccgttcta taaggtcgat gggcagtcat gcccagtgcc tatgatgtac caggaaggca 1080aattcaaata ccggcgcgtg gcagagggca cccaggtgct agagctgccc ttcaaggggg 1140atgacatcac catggtgctc atcctgccca agcctgagaa gagcctggcc aaggtggagc 1200aggagctcac cccagagctg ctgcaggagt ggctggatga gctgtcagag actatgcttg 1260tggtccacat gccccgcttc cgcaccgagg atggcttcag tctgaaggag cagctgcaag 1320acatgggcct cattgatctc ttcagccctg aaaagtccca actcccaggg atcgttgctg 1380gaggcaggga cgacctctat gtctccgacg cattccacaa agcatttctt gaggtaaatg 1440aggaaggcag tgaagcagca gcgagtactt ctgtcgtgat tactggccgg tcactgaacc 1500ccaatagggt gaccttcaag gccaacaggc ccttcctggt tcttataagg gaagttgcac 1560tgaacactat tatattcatg gggagagtgg ctaatccttg tgtgaactaa aatattctta 1620atctttgcac cttttcctac tttggtgttt gtgaatagaa gtaaaaataa atacgactgc 1680cacctcacga gaatggactt ttccacttga agacgagaga ctggagtaca gatgctacac 1740cacttttggg caagtgaagg gggagcagcc agccacggtg gcacaaacct atatcctggt 1800gcttttgaag gtagaagcag ggcggtcagg agttaaggcc agttgaggct gggctgcaga 1860gtgaaagacc atgtctcaag atggtctttc tcctccccaa agtagaaaag aaaaccataa 1920aaacaagagg taaatatatt actatttcat cttagaggat agcaggcatc ttgaaagggt 1980agagggacct taaattctca ttattgcccc catactacaa actaaaaaac aaacccgaat 2040caatctccca taaagacaga gattcaaata agagtattaa acgttttatt tctcaaacca 2100ctcacatgca taatgttctt atacacagtg tcaaaataaa gagaaatgca tttttataca 2160aaaaaaaaaa a 217141561DNARattus norvegicus 4cggagggatt gctcagcact gtctccacgg cttctctgca gaagcgtcca ccatgtattc 60cccgggaata ggaagtgcgg ttgctggaga gaggaagctt tgtctcctct ctctgctact 120cattggtgcc ttgggctgtg ctgtctgtca tggaaaccct gtggacgaca tctgcatagc 180gaagccccga gacatccccg tgaaccccat gtgcatttac cgctcccctg cgaagaaggc 240cacggaggag gatgtcctag agcagaaggt tccggaagcc accaaccggc gggtctggga 300actgtccaag gccaattctc gatttgccac taacttctat cagcacctgg cagactccaa 360gaacgacaac gacaacattt tcctgtcacc cttgagcatc tccacggcgt ttgctatgac 420caagctgggt gcttgtaata acaccctcaa gcagctgatg gaggttttta aatttgatac 480catctccgag aagacatccg accagatcca cttcttcttt gccaaactga actgccgact 540ctatcgaaaa gccaacaagt cctctaactt ggtgtcagcc aaccgccttt ttggagacaa 600atcccttacc ttcaatgaga gctatcaaga cgttagtgag attgtctatg gagccaagct 660tcagcccctg gacttcaagg agaatccgga gcaatccaga gtgaccatca acaactgggt 720agctaataag actgaaggcc gcatcaaaga cgtcatcccc caaggagcca ttgatgagct 780cactgccctg gtgctggtta acaccattta cttcaagggc ctgtggaagt caaagttcag 840ccctgagaac acaaggaagg aaccattcca caaagttgat gggcagtcat gcctggtgcc 900catgatgtac caggaaggca aattcaaata caggcgtgtg ggagagggta cccaggtgct 960agagatgccc ttcaaggggg acgacatcac catggtgctc atcctgccca agcctgagaa 1020gagcctggct aaggtggagc aggaactcac cccggagctg ctgcaggagt ggctggatga 1080gctgtcggag gtcatgcttg tggtccacgt gccccgcttc cgcatcgagg acagcttcag 1140tctgaaggag cagctgcaag acatgggcct tgttgatctc ttcagccctg agaagtccca 1200actcccaggg atcattgctg aaggcaggga cgacctcttt gtctccgatg cattccacaa 1260agcgtttctt gaggtaaatg aggaaggcag tgaagcagca gcgagtactt ctgtcgtgat 1320tactggccgg tcactgaacc ccagtagggt gaccttcaag gccaacaggc ccttcctggt 1380tcttataagg gaagtcgcac tgaacactat tatattcatg gggagagtgt ctaatccttg 1440tgtgaactaa aatattctta atctttgcac cttttcctat ctcggtgttt gttaatggaa 1500gtaaaaataa atatgactgc cacctcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560a 156151599DNAHomo sapiens 5taatgtgaga tggaagtagt ttgtatttat ttttacttct gttcacaaac caaaaatagg 60aagaggtgca aagaataaga acattttact taacacaagg gttggctact ctgcccatga 120agataatagt gttcagagga acttctctta taaaaaccag gaaaggcctg ttggccttga 180aagtcaccct gttggggttt agcgaacggc cagcaatcac aacagcggta cttgcagctg 240cttcactgcc ttcttcattt acctcaagaa atgccttatg gaatgcatct gagacataga 300ggtcatctcg gccttctgca acaatacctg ggagtttgga cttttcaggg ctgaacagat 360cgacaaggcc catgtcttgc agctgctcct tcaaactgaa gccgtcctca atgcggaagc 420ggggcatgtg gaccaccagc atcatctcct ccaattcatc cagccactct tgcagcacct 480ctggggtgag ttccttctct accttggcca ggctcttctc aggcttgggc aagatgagga 540ccatggtgat gtcatcacct ttgaagggca actcaagcac ctgggtgcct tcagccacgc 600gccgataacg gaacttgcct tcctggtaca tcatagatgc tgaacacgac tctccatcag 660ccttgtagaa cagttccttc cttgtgttct cagggctgaa ctttgacttc cacaggccct 720tgaagtaaat ggtgttaacc agcaccagaa cagtgagctc attgatggct tccgagggaa 780tgacatcggt gattcggcct tcggtcttat tggacaccca tttgttgatg gccgctctgg 840attgctctgc attttccttg aagtccaggg gctggagctt ggctccatat accaactcac 900tgatgtcctg gtaggtctca ttgaaggtaa gggatttgtc tccaaaaagg cgattggctg 960atactaactt ggaggatttg ttggcttttc gatagagtcg gcagttcagt ttggcaaaga 1020agaagtggat ctgatcagat gttttctcag atatggtgtc aaacttaaat acctccatca 1080gttgctggag ggtgtcatta caggcaccca gcttggtcat agcaaaagcc gtggagatac 1140tcaggggtga caggaaaatg ttatcattgt cattcttgga atctgccagg tgctgataga 1200aagtggtagc aaagcgggaa ttggccttgg acagttccca gacacgccgg ttggtggcct 1260ccgggatctt ctgttctgag ccctcatcct cagttgcctt cttctccggg gagcggtaaa 1320tgcacatggg attcatggga atgtcccgcg gcttggctgt gcagatgtcc acagggctcc 1380cgtgacaggt cacgcagtcc cagaagccaa tgagcagcaa ggacaaaaga taaaccttcc 1440tttttccaga ggttacagtt cctatcacat tggaatacat ggccgctaat cttccacagg 1500gctgggcaag tggagatagt gtgatctgag gcaatccgcc tgaaaactgg ttctttcctc 1560taaatctcgc agaggttcca gaggacaggg tggggcaga 159961545DNAMacaca mulatta 6tttttttttt ttttttttta atgtaagatg ggagtagttg tatttatttt tacttctatt 60cacaaaccaa aataggaaga ggtacaaaga ataagaacag tttagctcac acaagggttg 120gctactctgc ccatgaagat aatagtgttc agaggaactt ctcttataaa aaccaggaaa 180ggcctgttgg ccttgaaggt caccctgttg gggtttagcg aacggccagc aatcccaatg 240gcggtacttg cagctgcttc actgccttct tcatttacct caagaaatgc cttatggaat 300gcatcggaga catagaggtc atcccggcct tctgcaacaa tacctgggag tttggacttt 360tcagggctga acagatcgac aaggcccatg tcttgcagct gctccttcaa actgaagccg 420tcctcaatgc ggaagcgggg catgtgaacc accagcatca tctcctccaa ctcatccagc 480cactcctgca gcacctctgg ggtgagttcc tgctccacct tggtcaggct cttctcaggc 540ttgggcagga tgagcaccat ggtgatgtca tcacccttga agggcaactc aagcacctgg 600gtgccttcag ccacgcgccg ataacagaac ttgccttcct ggtacatcat agacgctgaa 660cacgactctc catcagcctt gtagaacggt tccatccttg tgttctcagg gctaaacttt 720gacttccaca ggcccttgaa gtaaatggtg ttaaccagca ccagaacagt gagctcgttg 780atggcttccg ggggaatgac atcggtgatt cggccttcgg tcttattgga cacccatttg 840ttgatggccg ctctggattg ctctgcattt tccttgaagt ccaggggctg gagcttggct 900ccgtatacca actcactgat gtcctggtag gtctcattga aggtaaggga tttgtctcca 960aaaaggcgat tggctgatac taacttggag gatttgttgg cttttcgata gagtcggcag 1020ttcagtttgg caaagaagaa gtggatctga tcagatgttt tctcagatat ggtgtcaaac 1080ttaaatacct ccatcagttg cttgagggtg tcattacagg cacccagctt ggtcatagca 1140aaagccgtgg agacactcag gggtgacagg aaaatgttat ccttgtcgtt cttggaatct 1200gccaggtgct gatagaaagt ggtagcaaag cgggaattgg ccttggacag ttcccagacg 1260cgccggttgg tggcctcggg gatcttctgt tctgagccct catcctcagt tgccttcttc 1320tccggggagc ggtaaatgca catgggattc atgggaatgt cccgcggctt ggctgtgcag 1380atgtccacag ggctcccgtg acaggtcata cagtcccaga ggccaatgag cagcaaggac 1440agaagataaa ccttcctttt tccagaggct acggttccta tcacattgga atacatggtc 1500gctaatcttc cacagggctg ggcaagtgga gatggtcctc gtgcc 154572171DNAMus musculus 7tttttttttt ttgtataaaa atgcatttct ctttattttg acactgtgta taagaacatt 60atgcatgtga gtggtttgag aaataaaacg tttaatactc ttatttgaat ctctgtcttt 120atgggagatt gattcgggtt tgttttttag tttgtagtat gggggcaata atgagaattt 180aaggtccctc taccctttca agatgcctgc tatcctctaa gatgaaatag taatatattt 240acctcttgtt tttatggttt tcttttctac tttggggagg agaaagacca tcttgagaca 300tggtctttca ctctgcagcc cagcctcaac tggccttaac tcctgaccgc cctgcttcta 360ccttcaaaag caccaggata taggtttgtg ccaccgtggc tggctgctcc cccttcactt 420gcccaaaagt ggtgtagcat ctgtactcca gtctctcgtc ttcaagtgga aaagtccatt 480ctcgtgaggt ggcagtcgta tttattttta cttctattca caaacaccaa agtaggaaaa 540ggtgcaaaga ttaagaatat tttagttcac acaaggatta gccactctcc ccatgaatat 600aatagtgttc agtgcaactt cccttataag aaccaggaag ggcctgttgg ccttgaaggt 660caccctattg gggttcagtg accggccagt aatcacgaca gaagtactcg ctgctgcttc 720actgccttcc tcatttacct caagaaatgc tttgtggaat gcgtcggaga catagaggtc 780gtccctgcct ccagcaacga tccctgggag ttgggacttt tcagggctga agagatcaat 840gaggcccatg tcttgcagct gctccttcag actgaagcca tcctcggtgc ggaagcgggg 900catgtggacc acaagcatag tctctgacag ctcatccagc cactcctgca gcagctctgg 960ggtgagctcc tgctccacct tggccaggct cttctcaggc ttgggcagga tgagcaccat 1020ggtgatgtca tcccccttga agggcagctc tagcacctgg gtgccctctg ccacgcgccg 1080gtatttgaat ttgccttcct ggtacatcat aggcactggg catgactgcc catcgacctt 1140atagaacggt tccttccttg tgttctcagg gctgaacttt gacttccaca ggcccttgaa 1200gtaaatggtg ttaaccagaa ccagggcagt gagctcgtta atggcgccct gtgggatgac 1260atctttgatg cggccttcag tcttattagc tacccagttg ttgatggtca ctctggattg 1320ctccggattc tccttgaagt ccaggggctg gagcttggct ccatagacaa cctcactaac 1380atcttgatag ctctcgttga aggtgaggga tttgtctcca aaaaggcggt tggctgatac 1440caagtcagag gacttgttgg cttttcgata gagtcggcag ttcagtttgg caaagaagaa 1500gtggatctgg tcggatgtct tctcggagat ggtatcaaat ttaaaaacct ccatcagctg 1560cttgagagtg tcgttacagg cacccagctt ggtcatagca aaagcagtgg agatgctcaa 1620gggtgacagg aaaatgttgt cgttgtcatt cttggagtct gccaggtgct ggtagaagtt 1680agtggcaaat cgcgaattgg ccttggacag ttcccagacc cgccggttgg tggcttctgg 1740aaccttctgc tctgagccat cctcctcggt ggccttcttc ccaggggagc ggtaaatgca 1800caagggattc acggggatgt ctcggggctt cgctatgcag atgtcgtcca cagggtttcc 1860gtgacagata gcacagccca aggcaccgat gaggagcaga gagaggagac aaagcttcct 1920ctcaccagca gccccacttc ctgccccagg ggaatacatg gccgatgctc ctacagagat 1980ggtgctgact gagacgatca ctccgaaaac tggttctttc ccctaaatct gagaggtcca 2040gaggccaggt gggggaggga gctctcccct taaagtcttc aggtagatat gtcttctgat 2100tatgtcttat atctcaccac ttcactaaaa tgtctcagat acaaatcaga tctatttcta 2160aaattaccta t 217181561DNARattus norvegicus 8tttttttttt tttttttttt tttttttttt tttttgaggt ggcagtcata tttattttta 60cttccattaa caaacaccga gataggaaaa ggtgcaaaga ttaagaatat tttagttcac 120acaaggatta gacactctcc ccatgaatat aatagtgttc agtgcgactt cccttataag 180aaccaggaag ggcctgttgg ccttgaaggt caccctactg gggttcagtg accggccagt 240aatcacgaca gaagtactcg ctgctgcttc actgccttcc tcatttacct caagaaacgc 300tttgtggaat gcatcggaga caaagaggtc gtccctgcct tcagcaatga tccctgggag 360ttgggacttc tcagggctga agagatcaac aaggcccatg tcttgcagct gctccttcag 420actgaagctg tcctcgatgc ggaagcgggg cacgtggacc acaagcatga cctccgacag 480ctcatccagc cactcctgca gcagctccgg ggtgagttcc tgctccacct tagccaggct 540cttctcaggc ttgggcagga tgagcaccat ggtgatgtcg tcccccttga agggcatctc 600tagcacctgg gtaccctctc ccacacgcct gtatttgaat ttgccttcct ggtacatcat 660gggcaccagg catgactgcc catcaacttt gtggaatggt tccttccttg tgttctcagg 720gctgaacttt gacttccaca ggcccttgaa gtaaatggtg ttaaccagca ccagggcagt 780gagctcatca atggctcctt gggggatgac gtctttgatg cggccttcag tcttattagc 840tacccagttg ttgatggtca ctctggattg ctccggattc tccttgaagt ccaggggctg 900aagcttggct ccatagacaa tctcactaac gtcttgatag ctctcattga aggtaaggga 960tttgtctcca aaaaggcggt tggctgacac caagttagag gacttgttgg cttttcgata 1020gagtcggcag ttcagtttgg caaagaagaa gtggatctgg tcggatgtct tctcggagat 1080ggtatcaaat ttaaaaacct ccatcagctg cttgagggtg ttattacaag cacccagctt 1140ggtcatagca aacgccgtgg agatgctcaa gggtgacagg aaaatgttgt cgttgtcgtt 1200cttggagtct gccaggtgct gatagaagtt agtggcaaat cgagaattgg ccttggacag 1260ttcccagacc cgccggttgg tggcttccgg aaccttctgc tctaggacat cctcctccgt 1320ggccttcttc gcaggggagc ggtaaatgca catggggttc acggggatgt ctcggggctt 1380cgctatgcag atgtcgtcca cagggtttcc atgacagaca gcacagccca aggcaccaat 1440gagtagcaga gagaggagac aaagcttcct ctctccagca accgcacttc ctattcccgg 1500ggaatacatg gtggacgctt ctgcagagaa gccgtggaga cagtgctgag caatccctcc 1560g 1561916PRTUnknownDescription of Unknown Exemplary hydrophobic membrane translocation peptide 9Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 1011PRTUnknownDescription of Unknown RFGF analogue peptide 10Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro 1 5 10 1113PRTHuman immunodeficiency virus 11Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 1216PRTDrosophila sp. 12Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 1321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 13cuuacgcuga guacuucgat t 211421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14ucgaaguacu cagcguaagt t

211519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15cccuguggac aucugcaca 191619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 16cuaccacuuu cuaucagca 191719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17cuaucgaaaa gccaacaaa 191819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18ggacuucaag gaaaaugca 191919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19guuaacacca uuuacuuca 192019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20ccugguuuuu auaagagaa 192119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 21gacauuccca ugaauccca 192219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 22caccuggcag auuccaaga 192319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23cgaaaagcca acaaauccu 192419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24gaaaaugcag agcaaucca 192519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25ggccugugga agucaaagu 192619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 26gaaguuccuc ugaacacua 192719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 27ccaugaaucc caugugcau 192819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 28caacugaugg agguauuua 192919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 29ccaaguuagu aucagccaa 193019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 30cggccaucaa caaaugggu 193119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 31gaggacggcu ucaguuuga 193219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 32ccucugaaca cuauuaucu 193319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 33caugaauccc augugcauu 193419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 34gauggaggua uuuaaguuu 193519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 35guaucagcca aucgccuuu 193619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 36ggguguccaa uaagaccga 193719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37cagcccugaa aaguccaaa 193819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 38cccaugugca uuuaccgcu 193919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39guauuuaagu uugacacca 194019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40gacaaauccc uuaccuuca 194119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 41cuguucuggu gcugguuaa 194219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 42ccaaacuccc agguauugu 194319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 43cccgcuuugc uaccacuuu 194419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 44cuuaccuuca augagaccu 194519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 45cuggugcugg uuaacacca 194619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 46caaacuccca gguauuguu 194719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 47gcuuugcuac cacuuucua 194819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 48ccuaccagga caucaguga 194919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 49ggugcugguu aacaccauu 195019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 50gccguucgcu aaaccccaa 195119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 51ggacaucagu gaguuggua 195219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 52gugcugguua acaccauuu 195319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 53gccuuuccug guuuuuaua 195419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 54cuuuugcuau gaccaagcu 195519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 55uguaccagga aggcaaguu 195619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 56acuauuaucu ucaugggca 195719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 57ucaugggcag aguagccaa 195819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 58ccauuuacuu caagggccu 195919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 59uacuucaagg gccugugga 196019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 60acuucaaggg ccuguggaa 196119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 61cgacucuauc gaaaagcca 196219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 62aacugccgac ucuaucgaa 196319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 63acugccgacu cuaucgaaa 196419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 64gacucuaucg aaaagccaa 196519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 65ucuucuuugc caaacugaa 196619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 66ugccaaacug aacugccga 196719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 67ccaaacugaa cugccgacu 196819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 68acugaacugc cgacucuau 196919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 69gaacugccga cucuaucga 197019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 70cugccgacuc uaucgaaaa 197119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 71cugguuaaca ccauuuacu 197219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 72ugugcagaug uccacaggg 197319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 73ugcugauaga aagugguag 197419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 74uuuguuggcu uuucgauag 197519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 75ugcauuuucc uugaagucc 197619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 76ugaaguaaau gguguuaac 197719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 77uucucuuaua aaaaccagg 197819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 78ugggauucau gggaauguc 197919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 79ucuuggaauc ugccaggug 198019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 80aggauuuguu ggcuuuucg 198119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 81uggauugcuc ugcauuuuc 198219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 82acuuugacuu ccacaggcc 198319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 83uaguguucag aggaacuuc 198419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 84augcacaugg gauucaugg 198519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 85uaaauaccuc caucaguug 198619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 86uuggcugaua cuaacuugg 198719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 87acccauuugu ugauggccg 198819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 88ucaaacugaa gccguccuc 198919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 89agauaauagu guucagagg 199019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 90aaugcacaug ggauucaug 199119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 91aaacuuaaau accuccauc 199219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 92aaaggcgauu ggcugauac 199319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 93ucggucuuau uggacaccc 199419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 94uuuggacuuu ucagggcug 199519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 95agcgguaaau gcacauggg 199619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 96uggugucaaa cuuaaauac 199719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 97ugaagguaag ggauuuguc 199819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 98uuaaccagca ccagaacag 199919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 99acaauaccug ggaguuugg 1910019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 100aaagugguag caaagcggg 1910119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 101aggucucauu gaagguaag 1910219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 102ugguguuaac cagcaccag 1910319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 103aacaauaccu gggaguuug 1910419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 104uagaaagugg uagcaaagc 1910519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 105ucacugaugu ccugguagg 1910619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 106aaugguguua accagcacc 1910719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 107uugggguuua gcgaacggc 1910819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 108uaccaacuca cugaugucc 1910919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 109aaaugguguu aaccagcac 1911019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 110uauaaaaacc aggaaaggc 1911119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 111agcuugguca uagcaaaag 1911219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 112aacuugccuu ccugguaca 1911319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 113ugcccaugaa gauaauagu 1911419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 114uuggcuacuc ugcccauga 1911519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 115aggcccuuga aguaaaugg 1911619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 116uccacaggcc cuugaagua 1911719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 117uuccacaggc ccuugaagu 1911819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 118uggcuuuucg auagagucg 1911919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 119uucgauagag ucggcaguu 1912019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 120uuucgauaga gucggcagu 1912119RNAArtificial SequenceDescription of

Artificial Sequence Synthetic oligonucleotide 121uuggcuuuuc gauagaguc 1912219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 122uucaguuugg caaagaaga 1912319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 123ucggcaguuc aguuuggca 1912419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 124agucggcagu ucaguuugg 1912519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 125auagagucgg caguucagu 1912619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 126ucgauagagu cggcaguuc 1912719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 127uuuucgauag agucggcag 1912819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 128aguaaauggu guuaaccag 1912921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 129cccuguggac aucugcacat t 2113021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 130cuaccacuuu cuaucagcat t 2113121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 131cuaucgaaaa gccaacaaat t 2113221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 132ggacuucaag gaaaaugcat t 2113321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 133guuaacacca uuuacuucat t 2113421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 134ccugguuuuu auaagagaat t 2113521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 135gacauuccca ugaaucccat t 2113621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 136caccuggcag auuccaagat t 2113721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 137cgaaaagcca acaaauccut t 2113821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 138gaaaaugcag agcaauccat t 2113921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 139ggccugugga agucaaagut t 2114021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 140gaaguuccuc ugaacacuat t 2114121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 141ccaugaaucc caugugcaut t 2114221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 142caacugaugg agguauuuat t 2114321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 143ccaaguuagu aucagccaat t 2114421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 144cggccaucaa caaaugggut t 2114521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 145gaggacggcu ucaguuugat t 2114621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 146ccucugaaca cuauuaucut t 2114721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 147caugaauccc augugcauut t 2114821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 148gauggaggua uuuaaguuut t 2114921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 149guaucagcca aucgccuuut t 2115021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 150ggguguccaa uaagaccgat t 2115121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 151cagcccugaa aaguccaaat t 2115221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 152cccaugugca uuuaccgcut t 2115321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 153guauuuaagu uugacaccat t 2115421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 154gacaaauccc uuaccuucat t 2115521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 155cuguucuggu gcugguuaat t 2115621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 156ccaaacuccc agguauugut t 2115721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 157cccgcuuugc uaccacuuut t 2115821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 158cuuaccuuca augagaccut t 2115921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 159cuggugcugg uuaacaccat t 2116021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 160caaacuccca gguauuguut t 2116121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 161gcuuugcuac cacuuucuat t 2116221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 162ccuaccagga caucagugat t 2116321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 163ggugcugguu aacaccauut t 2116421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 164gccguucgcu aaaccccaat t 2116521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 165ggacaucagu gaguugguat t 2116621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 166gugcugguua acaccauuut t 2116721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 167gccuuuccug guuuuuauat t 2116821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 168cuuuugcuau gaccaagcut t 2116921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 169uguaccagga aggcaaguut t 2117021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 170acuauuaucu ucaugggcat t 2117121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 171ucaugggcag aguagccaat t 2117221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 172ccauuuacuu caagggccut t 2117321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 173uacuucaagg gccuguggat t 2117421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 174acuucaaggg ccuguggaat t 2117521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 175cgacucuauc gaaaagccat t 2117621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 176aacugccgac ucuaucgaat t 2117721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 177acugccgacu cuaucgaaat t 2117821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 178gacucuaucg aaaagccaat t 2117921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 179ucuucuuugc caaacugaat t 2118021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 180ugccaaacug aacugccgat t 2118121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 181ccaaacugaa cugccgacut t 2118221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 182acugaacugc cgacucuaut t 2118321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 183gaacugccga cucuaucgat t 2118421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 184cugccgacuc uaucgaaaat t 2118521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 185cugguuaaca ccauuuacut t 2118621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 186ugugcagaug uccacagggt t 2118721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 187ugcugauaga aagugguagt t 2118821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 188uuuguuggcu uuucgauagt t 2118921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 189ugcauuuucc uugaagucct t 2119021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 190ugaaguaaau gguguuaact t 2119121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 191uucucuuaua aaaaccaggt t 2119221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 192ugggauucau gggaauguct t 2119321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 193ucuuggaauc ugccaggugt t 2119421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 194aggauuuguu ggcuuuucgt t 2119521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 195uggauugcuc ugcauuuuct t 2119621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 196acuuugacuu ccacaggcct t 2119721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 197uaguguucag aggaacuuct t 2119821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 198augcacaugg gauucauggt t 2119921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 199uaaauaccuc caucaguugt t 2120021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 200uuggcugaua cuaacuuggt t 2120121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 201acccauuugu ugauggccgt t 2120221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 202ucaaacugaa gccguccuct t 2120321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 203agauaauagu guucagaggt t 2120421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 204aaugcacaug ggauucaugt t 2120521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 205aaacuuaaau accuccauct t 2120621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 206aaaggcgauu ggcugauact t 2120721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 207ucggucuuau uggacaccct t 2120821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 208uuuggacuuu ucagggcugt t 2120921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 209agcgguaaau gcacaugggt t 2121021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 210uggugucaaa cuuaaauact t 2121121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 211ugaagguaag ggauuuguct t 2121221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 212uuaaccagca ccagaacagt t 2121321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 213acaauaccug ggaguuuggt t 2121421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 214aaagugguag caaagcgggt t 2121521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 215aggucucauu gaagguaagt t 2121621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 216ugguguuaac cagcaccagt t 2121721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 217aacaauaccu gggaguuugt t 2121821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 218uagaaagugg uagcaaagct t 2121921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 219ucacugaugu ccugguaggt t 2122021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 220aaugguguua accagcacct t 2122121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 221uugggguuua

gcgaacggct t 2122221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 222uaccaacuca cugaugucct t 2122321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 223aaaugguguu aaccagcact t 2122421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 224uauaaaaacc aggaaaggct t 2122521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 225agcuugguca uagcaaaagt t 2122621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 226aacuugccuu ccugguacat t 2122721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 227ugcccaugaa gauaauagut t 2122821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 228uuggcuacuc ugcccaugat t 2122921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 229aggcccuuga aguaaauggt t 2123021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 230uccacaggcc cuugaaguat t 2123121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 231uuccacaggc ccuugaagut t 2123221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 232uggcuuuucg auagagucgt t 2123321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 233uucgauagag ucggcaguut t 2123421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 234uuucgauaga gucggcagut t 2123521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 235uuggcuuuuc gauagaguct t 2123621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 236uucaguuugg caaagaagat t 2123721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 237ucggcaguuc aguuuggcat t 2123821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 238agucggcagu ucaguuuggt t 2123921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 239auagagucgg caguucagut t 2124021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 240ucgauagagu cggcaguuct t 2124121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 241uuuucgauag agucggcagt t 2124221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 242aguaaauggu guuaaccagt t 2124321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 243cuaucgaaaa gccaacaaat t 2124421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 244cuaucgaaaa gccaacaaat t 2124521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 245cuaucgaaaa gccaacaaat t 2124621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 246cuaucgaaaa gccaacaaat t 2124721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 247cuaucgaaaa gccaacaaat t 2124821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 248cuaucgaaaa gccaacaaat t 2124921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 249cuaucgaaaa gccaacaaat t 2125021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 250cuaucgaaaa gccaacaaat t 2125121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 251cuaucgaaaa gccaacaaat t 2125221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 252cuaucgaaaa gccaacaaat t 2125321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 253cuaucgaaaa gccaacaaat t 2125421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 254cuaucgaaaa gccaacaaat t 2125521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 255cuaucgaaaa gccaacaaat t 2125621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 256ccaugaaucc caugugcaut t 2125721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 257ccaugaaucc caugugcaut t 2125821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 258ccaugaaucc caugugcaut t 2125921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 259ccaugaaucc caugugcaut t 2126021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 260ccaugaaucc caugugcaut t 2126121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 261ccaugaaucc caugugcaut t 2126221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 262ccaugaaucc caugugcaut t 2126321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 263ccaugaaucc caugugcaut t 2126421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 264ccaugaaucc caugugcaut t 2126521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 265ccaugaaucc caugugcaut t 2126621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 266ccaugaaucc caugugcaut t 2126721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 267ccaugaaucc caugugcaut t 2126821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 268ccaugaaucc caugugcaut t 2126921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 269cugccgacuc uaucgaaaat t 2127021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 270cugccgacuc uaucgaaaat t 2127121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 271cugccgacuc uaucgaaaat t 2127221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 272cugccgacuc uaucgaaaat t 2127321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 273cugccgacuc uaucgaaaat t 2127421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 274cugccgacuc uaucgaaaat t 2127521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 275cugccgacuc uaucgaaaat t 2127621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 276cugccgacuc uaucgaaaat t 2127721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 277cugccgacuc uaucgaaaat t 2127821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 278cugccgacuc uaucgaaaat t 2127921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 279cugccgacuc uaucgaaaat t 2128021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 280cugccgacuc uaucgaaaat t 2128121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 281cugccgacuc uaucgaaaat t 2128221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 282ucuucuuugc caaacugaat t 2128321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 283ucuucuuugc caaacugaat t 2128421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 284ucuucuuugc caaacugaat t 2128521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 285ucuucuuugc caaacugaat t 2128621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 286ucuucuuugc caaacugaat t 2128721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 287ucuucuuugc caaacugaat t 2128821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 288ucuucuuugc caaacugaat t 2128921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 289ucuucuuugc caaacugaat t 2129021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 290ucuucuuugc caaacugaat t 2129121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 291ucuucuuugc caaacugaat t 2129221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 292ucuucuuugc caaacugaat t 2129321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 293ucuucuuugc caaacugaat t 2129421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 294gguuaacacc auuuacuuca a 2129521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 295uaguaucagc caaucgccuu u 2129621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 296cgcuuugcua ccacuuucua u 2129721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 297cuuugcuacc acuuucuauc a 2129821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 298aucgaaaagc caacaaaucc u 2129921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 299ucccaugaau cccaugugca u 2130021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 300uaaggcauuu cuugagguaa a 2130121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 301gcaacugaug gagguauuua a 2130221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 302uuuaaguuug acaccauauc u 2130321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 303ccaugaaucc caugugcauu u 2130421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 304ugcugguuaa caccauuuac u 2130521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 305ucuaucgaaa agccaacaaa u 2130621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 306uucccgcuuu gcuaccacuu u 2130721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 307gauggaggua uuuaaguuug a 2130821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 308agacaaaucc cuuaccuuca a 2130921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 309ugcuaccacu uucuaucagc a 2131021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 310uguauuccaa ugugauagga a 2131121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 311aacugccgac ucuaucgaaa a 2131221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 312gccgacucua ucgaaaagcc a 2131321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 313uauuuaaguu ugacaccaua u 2131421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 314ugggccuugu cgaucuguuc a 2131521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 315caaaucccuu accuucaaug a 2131621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 316uccaaacucc cagguauugu u 2131721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 317gaacugccga cucuaucgaa a 2131821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 318ucaacaaaug gguguccaau a 2131921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 319cacuguucug gugcugguua a 2132021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 320ggacggcuuc aguuugaagg a 2132121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 321cuggacuuca aggaaaaugc a

2132221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 322agguauuuaa guuugacacc a 2132321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 323uuacuucaag ggccugugga a 2132421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 324uuuuuggaga caaaucccuu a 2132521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 325gauugcuggc cguucgcuaa a 2132621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 326ccgacucuau cgaaaagcca a 2132721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 327caccauuuac uucaagggcc u 2132821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 328caagcugggu gccuguaaug a 2132921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 329acacuauuau cuucaugggc a 2133021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 330aggaaaaugc agagcaaucc a 2133121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 331ucuggugcug guuaacacca u 2133221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 332agcccugugg acaucugcac a 2133321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 333cuucuucuuu gccaaacuga a 2133421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 334ucuccacggc uuuugcuaug a 2133521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 335gcaccuggca gauuccaaga a 2133621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 336cuucaugggc agaguagcca a 2133721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 337cuccaaguua guaucagcca a 2133821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 338gguuaacacc auuuacuuca a 2133921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 339gguuaacacc auuuacuuca a 2134021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 340gguuaacacc auuuacuuca a 2134121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 341gguuaacacc auuuacuuca a 2134221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 342gguuaacacc auuuacuuca a 2134321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 343gguuaacacc auuuacuuca a 2134421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 344gguuaacacc auuuacuuca a 2134521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 345gguuaacacc auuuacuuca a 2134621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 346gguuaacacc auuuacuuca a 2134721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 347gguuaacacc auuuacuuca a 2134821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 348gguuaacacc auuuacuuca a 2134921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 349gguuaacacc auuuacuuca a 2135021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 350gguuaacacc auuuacuuca a 2135121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 351gguuaacacc auuuacuuca a 2135221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 352gguuaacacc auuuacuuca a 2135321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 353gguuaacacc auuuacuuca a 2135421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 354gguuaacacc auuuacuuca a 2135521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 355gguuaacacc auuuacuuca a 2135621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 356gguuaacacc auuuacuuca a 2135721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 357gguuaacacc auuuacuuca a 2135821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 358gguuaacacc auuuacuuca a 2135921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 359gguuaacacc auuuacuuca a 2136021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 360gguuaacacc auuuacuuca a 2136121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 361gguuaacacc auuuacuuca a 2136221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 362gguuaacacc auuuacuuca a 2136321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 363gguuaacacc auuuacuuca a 2136421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 364gguuaacacc auuuacuuca a 2136521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 365gguuaacacc auuuacuuca a 2136621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 366gguuaacacc auuuacuuca a 2136721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 367gguuaacacc auuuacuuca a 2136821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 368gguuaacacc auuuacuuca a 2136921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 369gguuaacacc auuuacuuca a 2137021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 370gguuaacacc auuuacuuca a 2137121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 371gguuaacacc auuuacuuca a 2137221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 372gguuaacacc auuuacuuca a 2137321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 373gguuaacacc auuuacuuca a 2137421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 374gguuaacacc auuuacuuca a 2137521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 375gguuaacacc auuuacuuca a 2137621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 376gguuaacacc auuuacuuca a 2137721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 377gguuaacacc auuuacuuca a 2137821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 378gguuaacacc auuuacuuca a 2137921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 379gguuaacacc auuuacuuca a 2138021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 380gguuaacacc auuuacuuca a 2138121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 381gguuaacacc auuuacuuca a 2138221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 382gguuaacacc auuuacuuca a 2138321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 383gguuaacacc auuuacuuca a 2138421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 384gguuaacacc auuuacuuca a 2138521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 385gguuaacacc auuuacuuca a 2138621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 386gguuaacacc auuuacuuca a 2138721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 387gguuaacacc auuuacuuca a 2138821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 388gguuaacacc auuuacuuca a 2138921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 389gguuaacacc auuuacuuca a 2139021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 390gguuaacacc auuuacuuca a 2139121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 391gguuaacacc auuuacuuca a 2139221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 392gguuaacacc auuuacuuca a 2139321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 393gguuaacacc auuuacuuca a 2139421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 394gguuaacacc auuuacuuca a 2139521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 395gguuaacacc auuuacuuca a 2139621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 396gguuaacacc auuuacuuca a 2139721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 397gguuaacacc auuuacuuca a 2139821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 398gguuaacacc auuuacuuca a 2139921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 399gguuaacacc auuuacuuca a 2140021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 400gguuaacacc auuuacuuca a 2140121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 401gguuaacacc auuuacuuca a 2140221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 402gguuaacacc auuuacuuca a 2140321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 403gguuaacacc auuuacuuca a 2140421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 404gguuaacacc auuuacuuca a 2140521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 405gguuaacacc auuuacuuca a 2140621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 406gguuaacacc auuuacuuca a 2140721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 407gguuaacacc auuuacuuca a 2140821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 408gguuaacacc auuuacuuca a 2140921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 409gguuaacacc auuuacuuca a 2141021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 410gguuaacacc auuuacuuca a 2141121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 411gguuaacacc auuuacuuca a 2141221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 412gguuaacacc auuuacuuca a 2141321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 413gguuaacacc auuuacuuca a 2141421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 414gguuaacacc auuuacuuca a 2141521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 415gguuaacacc auuuacuuca a 2141621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 416gguuaacacc auuuacuuca a 2141721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 417gguuaacacc auuuacuuca a 2141821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 418gguuaacacc auuuacuuca a 2141921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 419gguuaacacc auuuacuuca a 2142021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 420gguuaacacc auuuacuuca a 2142121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 421gguuaacacc auuuacuuca a 2142221RNAArtificial SequenceDescription of Artificial Sequence Synthetic

oligonucleotide 422gguuaacacc auuuacuuca a 2142321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 423gguuaacacc auuuacuuca a 2142421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 424gguuaacacc auuuacuuca a 2142521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 425gguuaacacc auuuacuuca a 2142621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 426gguuaacacc auuuacuuca a 2142721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 427gguuaacacc auuuacuuca a 2142821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 428gguuaacacc auuuacuuca a 2142921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 429gguuaacacc auuuacuuca a 2143021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 430gguuaacacc auuuacuuca a 2143121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 431gguuaacacc auuuacuuca a 2143221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 432gguuaacacc auuuacuuca a 2143321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 433gguuaacacc auuuacuuca a 2143421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 434gguuaacacc auuuacuuca a 2143521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 435gguuaacacc auuuacuuca a 2143621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 436gguuaacacc auuuacuuca a 2143721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 437gguuaacacc auuuacuuca a 2143821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 438gguuaacacc auuuacuuca a 2143921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 439gguuaacacc auuuacuuca a 2144021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 440gguuaacacc auuuacuuca a 2144121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 441gguuaacacc auuuacuuca a 2144221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 442gguuaacacc auuuacuuca a 2144321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 443gguuaacacc auuuacuuca a 2144421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 444gguuaacacc auuuacuuca a 2144521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 445gguuaacacc auuuacuuca a 2144621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 446gguuaacacc auuuacuuca a 2144721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 447gguuaacacc auuuacuuca a 2144821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 448gguuaacacc auuuacuuca a 2144921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 449gguuaacacc auuuacuuca a 2145021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 450gguuaacacc auuuacuuca a 2145121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 451gguuaacacc auuuacuuca a 2145221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 452gguuaacacc auuuacuuca a 2145321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 453gguuaacacc auuuacuuca a 2145421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 454gguuaacacc auuuacuuca a 2145521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 455gguuaacacc auuuacuuca a 2145621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 456gguuaacacc auuuacuuca a 2145721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 457gguuaacacc auuuacuuca a 2145821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 458gguuaacacc auuuacuuca a 2145921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 459gguuaacacc auuuacuuca a 2146021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 460gguuaacacc auuuacuuca a 2146121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 461gguuaacacc auuuacuuca a 2146221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 462gguuaacacc auuuacuuca a 2146321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 463gguuaacacc auuuacuuca a 2146421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 464gguuaacacc auuuacuuca a 2146521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 465gguuaacacc auuuacuuca a 2146621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 466gguuaacacc auuuacuuca a 2146721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 467gguuaacacc auuuacuuca a 2146821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 468gguuaacacc auuuacuuca a 2146921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 469gguuaacacc auuuacuuca a 2147021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 470gguuaacacc auuuacuuca a 2147121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 471gguuaacacc auuuacuuca a 2147221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 472gguuaacacc auuuacuuca a 2147321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 473gguuaacacc auuuacuuca a 2147421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 474gguuaacacc auuuacuuca a 2147521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 475gguuaacauc auuuacuuca a 2147621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 476gguuaacaac auuuacuuca a 2147721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 477gguuaacacu auuuacuuca a 2147821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 478gguuaacaca auuuacuuca a 2147921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 479gguuaacacc auuuacuuca a 2148021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 480gguuaacacc auuuacuuca a 2148121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 481gguuaacacc auuuacuuca a 2148221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 482gguuaacacc auuuacuuca a 2148319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 483uuaacaccau uuacuucaa 1948419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 484uuaacaccau uuacuucaa 1948519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 485uuaacaccau uuacuucaa 1948619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 486uuaacaccau uuacuucaa 1948717RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 487aacaccauuu acuucaa 1748817RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 488aacaccauuu acuucaa 1748921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 489gguuaacacc auuuacuuca a 2149021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 490gguuaacacc auuuacuuca a 2149121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 491gguuaacacc auuuacuuca a 2149221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 492gguuaacacc auuuacuuca a 2149321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 493gguuaacacc auuuacuuca a 2149421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 494gguuaacacc auuuacuuca a 2149521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 495gguuaacacc auuuacuuca a 2149621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 496gguuaacacc auuuacuuca a 2149721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 497gguuaacacc auuuacuuca a 2149821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 498gguuaacacc auuuacuuca a 2149921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 499gguuaacacc auuuacuuca a 2150021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 500gguuaacacc auuuacuuca a 2150121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 501gguuaacacc auuuacuuca a 2150221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 502gguuaacacc auuuacuuca a 2150321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 503gguuaacacc auuuacuuca a 2150421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 504gguuaacacc auuuacutca a 2150521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 505gguuaacacc auuuacutca a 2150621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 506ggutaacacc auuuacutca a 2150721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 507ggutaacacc auuuacutca a 2150821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 508ggutaacacc auutacutca a 2150921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 509ggutaacacc atutacutca a 2151021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 510ggutaacacc atutacutca a 2151121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 511uuuguuggcu uuucgauagt t 2151221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 512uuuguuggcu uuucgauagt t 2151321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 513uuuguuggcu uuucgauagt t 2151421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 514uuuguuggcu uuucgauagt t 2151521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 515uuuguuggcu uuucgauagt t 2151621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 516uuuguuggcu uuucgauagt t 2151721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 517uuuguuggcu uuucgauagt t 2151821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 518uuuguuggcu uuucgauagt t 2151921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 519uuuguuggcu uuucgauagt t 2152021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 520uuuguuggcu uuucgauagt t 2152121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 521uuuguuggcu uuucgauagt t 2152221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 522uuuguuggcu uuucgauagt t

2152321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 523uuuguuggcu uuucgauagt t 2152421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 524augcacaugg gauucauggt t 2152521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 525augcacaugg gauucauggt t 2152621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 526augcacaugg gauucauggt t 2152721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 527augcacaugg gauucauggt t 2152821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 528augcacaugg gauucauggt t 2152921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 529augcacaugg gauucauggt t 2153021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 530augcacaugg gauucauggt t 2153121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 531augcacaugg gauucauggt t 2153221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 532augcacaugg gauucauggt t 2153321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 533augcacaugg gauucauggt t 2153421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 534augcacaugg gauucauggt t 2153521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 535augcacaugg gauucauggt t 2153621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 536augcacaugg gauucauggt t 2153721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 537uuuucgauag agucggcagt t 2153821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 538uuuucgauag agucggcagt t 2153921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 539uuuucgauag agucggcagt t 2154021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 540uuuucgauag agucggcagt t 2154121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 541uuuucgauag agucggcagt t 2154221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 542uuuucgauag agucggcagt t 2154321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 543uuuucgauag agucggcagt t 2154421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 544uuuucgauag agucggcagt t 2154521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 545uuuucgauag agucggcagt t 2154621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 546uuuucgauag agucggcagt t 2154721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 547uuuucgauag agucggcagt t 2154821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 548uuuucgauag agucggcagt t 2154921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 549uuuucgauag agucggcagt t 2155021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 550uucaguuugg caaagaagat t 2155121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 551uucaguuugg caaagaagat t 2155221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 552uucaguuugg caaagaagat t 2155321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 553uucaguuugg caaagaagat t 2155421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 554uucaguuugg caaagaagat t 2155521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 555uucaguuugg caaagaagat t 2155621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 556uucaguuugg caaagaagat t 2155721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 557uucaguuugg caaagaagat t 2155821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 558uucaguuugg caaagaagat t 2155921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 559uucaguuugg caaagaagat t 2156021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 560uucaguuugg caaagaagat t 2156121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 561uucaguuugg caaagaagat t 2156223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 562uugaaguaaa ugguguuaac cag 2356323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 563aaaggcgauu ggcugauacu aac 2356423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 564auagaaagug guagcaaagc ggg 2356523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 565ugauagaaag ugguagcaaa gcg 2356623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 566aggauuuguu ggcuuuucga uag 2356723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 567augcacaugg gauucauggg aau 2356823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 568uuuaccucaa gaaaugccuu aug 2356923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 569uuaaauaccu ccaucaguug cug 2357023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 570agauauggug ucaaacuuaa aua 2357123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 571aaaugcacau gggauucaug gga 2357223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 572aguaaauggu guuaaccagc acc 2357323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 573auuuguuggc uuuucgauag agu 2357423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 574aaagugguag caaagcggga auu 2357523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 575ucaaacuuaa auaccuccau cag 2357623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 576uugaagguaa gggauuuguc ucc 2357723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 577ugcugauaga aagugguagc aaa 2357823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 578uuccuaucac auuggaauac aug 2357923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 579uuuucgauag agucggcagu uca 2358023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 580uggcuuuucg auagagucgg cag 2358123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 581auaugguguc aaacuuaaau acc 2358223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 582ugaacagauc gacaaggccc aug 2358323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 583ucauugaagg uaagggauuu guc 2358423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 584aacaauaccu gggaguuugg acu 2358523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 585uuucgauaga gucggcaguu cag 2358623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 586uauuggacac ccauuuguug aug 2358723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 587uuaaccagca ccagaacagu gag 2358823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 588uccuucaaac ugaagccguc cuc 2358923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 589ugcauuuucc uugaagucca ggg 2359023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 590uggugucaaa cuuaaauacc ucc 2359123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 591uuccacaggc ccuugaagua aau 2359223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 592uaagggauuu gucuccaaaa agg 2359323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 593uuuagcgaac ggccagcaau cac 2359423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 594uuggcuuuuc gauagagucg gca 2359523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 595aggcccuuga aguaaauggu guu 2359623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 596ucauuacagg cacccagcuu ggu 2359723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 597ugcccaugaa gauaauagug uuc 2359823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 598uggauugcuc ugcauuuucc uug 2359923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 599augguguuaa ccagcaccag aac 2360023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 600ugugcagaug uccacagggc ucc 2360123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 601uucaguuugg caaagaagaa gug 2360223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 602ucauagcaaa agccguggag aua 2360323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 603uucuuggaau cugccaggug cug 2360423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 604uuggcuacuc ugcccaugaa gau 2360523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 605uuggcugaua cuaacuugga gga 2360623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 606uugaaguaaa ugguguuaac cag 2360723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 607uugaaguaaa ugguguuaac cag 2360823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 608uugaaguaaa ugguguuaac cag 2360923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 609uugaaguaaa ugguguuaac cag 2361023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 610uugaaguaaa ugguguuaac cag 2361123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 611uugaaguaaa ugguguuaac cag 2361223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 612uugaaguaaa ugguguuaac cag 2361323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 613uugaaguaaa ugguguuaac cag 2361423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 614uugaaguaaa ugguguuaac cag 2361523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 615uugaaguaaa ugguguuaac cag 2361623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 616uugaaguaaa ugguguuaac cag 2361723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 617uugaaguaaa ugguguuaac cag 2361823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 618uugaaguaaa ugguguuaac cag 2361923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 619uugaaguaaa ugguguuaac cag 2362023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 620uugaaguaaa ugguguuaac cag 2362123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 621uugaaguaaa ugguguuaac cag 2362223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 622uugaaguaaa ugguguuaac cag 2362323RNAArtificial SequenceDescription of Artificial Sequence Synthetic

oligonucleotide 623uugaaguaaa ugguguuaac cag 2362423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 624uugaaguaaa ugguguuaac cag 2362523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 625uugaaguaaa ugguguuaac cag 2362623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 626uugaaguaaa ugguguuaac cag 2362723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 627uugaaguaaa ugguguuaac cag 2362823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 628uugaaguaaa ugguguuaac cag 2362923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 629uugaaguaaa ugguguuaac cag 2363023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 630uugaaguaaa ugguguuaac cag 2363123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 631uugaaguaaa ugguguuaac cag 2363223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 632uugaaguaaa ugguguuaac cag 2363323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 633uugaaguaaa ugguguuaac cag 2363423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 634uugaaguaaa ugguguuaac cag 2363523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 635uugaaguaaa ugguguuaac cag 2363623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 636uugaaguaaa ugguguuaac cag 2363723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 637uugaaguaaa ugguguuaac cag 2363823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 638uugaaguaaa ugguguuaac cag 2363923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 639uugaaguaaa ugguguuaac cag 2364023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 640uugaaguaaa ugguguuaac cag 2364123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 641uugaaguaaa ugguguuaac cag 2364223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 642uugaaguaaa ugguguuaac cag 2364323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 643uugaaguaaa ugguguuaac cag 2364423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 644uugaaguaaa ugguguuaac cag 2364523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 645uugaaguaaa ugguguuaac cag 2364623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 646uugaaguaaa ugguguuaac cag 2364723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 647uugaaguaaa ugguguuaac cag 2364823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 648uugaaguaaa ugguguuaac cag 2364923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 649uugaaguaaa ugguguuaac cag 2365023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 650uugaaguaaa ugguguuaac cag 2365123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 651uugaaguaaa ugguguuaac cag 2365223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 652uugaaguaaa ugguguuaac cag 2365323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 653uugaaguaaa ugguguuaac cag 2365423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 654uugaaguaaa ugguguuaac cag 2365523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 655uugaaguaaa ugguguuaac cag 2365623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 656uugaaguaaa ugguguuaac cag 2365723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 657uugaaguaaa ugguguuaac cag 2365823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 658uugaaguaaa ugguguuaac cag 2365923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 659uugaaguaaa ugguguuaac cag 2366023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 660uugaaguaaa ugguguuaac cag 2366123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 661uugaaguaaa ugguguuaac cag 2366223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 662uugaaguaaa ugguguuaac cag 2366323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 663uugaaguaaa ugguguuaac cag 2366423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 664uugaaguaaa ugguguuaac cag 2366523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 665uugaaguaaa ugguguuaac cag 2366623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 666uugaaguaaa ugguguuaac cag 2366723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 667uugaaguaaa ugguguuaac cag 2366823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 668uugaaguaaa ugguguuaac cag 2366923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 669uugaaguaaa ugguguuaac cag 2367023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 670uugaaguaaa ugguguuaac cag 2367123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 671uugaaguaaa ugguguuaac cag 2367223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 672uugaaguaaa ugguguuaac cag 2367323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 673uugaaguaaa ugguguuaac cag 2367423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 674uugaaguaaa ugguguuaac cag 2367523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 675uugaaguaaa ugguguuaac cag 2367623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 676uugaaguaaa ugguguuaac cag 2367723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 677uugaaguaaa ugguguuaac cag 2367823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 678uugaaguaaa ugguguuaac cag 2367923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 679uugaaguaaa ugguguuaac cag 2368023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 680uugaaguaaa ugguguuaac cag 2368123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 681uugaaguaaa ugguguuaac cag 2368223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 682uugaaguaaa ugguguuaac cag 2368323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 683uugaaguaaa ugguguuaac cag 2368423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 684uugaaguaaa ugguguuaac cag 2368523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 685uugaaguaaa ugguguuaac cag 2368623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 686uugaaguaaa ugguguuaac cag 2368723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 687uugaaguaaa ugguguuaac cag 2368823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 688uugaaguaaa ugguguuaac cag 2368923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 689uugaaguaaa ugguguuaac cag 2369023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 690uugaaguaaa ugguguuaac cag 2369123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 691uugaaguaaa ugguguuaac cag 2369223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 692uugaaguaaa ugguguuaac cag 2369323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 693uugaaguaaa ugguguuaac cag 2369423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 694uugaaguaaa ugguguuaac cag 2369523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 695uugaaguaaa ugguguuaac cag 2369623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 696uugaaguaaa ugguguuaac cag 2369723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 697uugaaguaaa ugguguuaac cag 2369823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 698uugaaguaaa ugguguuaac cag 2369923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 699uugaaguaaa ugguguuaac cag 2370023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 700uugaaguaaa ugguguuaac cag 2370123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 701uugaaguaaa ugguguuaac cag 2370223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 702uugaaguaaa ugguguuaac cag 2370323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 703uugaaguaaa ugguguuaac cag 2370423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 704uugaaguaaa ugguguuaac cag 2370523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 705uugaaguaaa ugguguuaac cag 2370623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 706uugaaguaaa ugguguuaac cag 2370723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 707uugaaguaaa ugguguuaac cag 2370823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 708uugaaguaaa ugguguuaac cag 2370923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 709uugaaguaaa ugguguuaac cag 2371023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 710uugaaguaaa ugguguuaac cag 2371123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 711uugaaguaaa ugguguuaac cag 2371223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 712uugaaguaaa ugguguuaac cag 2371323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 713uugaaguaaa ugguguuaac cag 2371423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 714uugaaguaaa ugguguuaac cag 2371523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 715uugaaguaaa ugguguuaac cag 2371623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 716uugaaguaaa ugguguuaac cag 2371723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 717uugaaguaaa ugguguuaac cag 2371823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 718uugaaguaaa ugguguuaac cag 2371923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 719uugaaguaaa ugguguuaac cag 2372023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 720uugaaguaaa ugguguuaac cag 2372123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 721uugaaguaaa ugguguuaac cag 2372223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 722uugaaguaaa ugguguuaac cag 2372323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 723uugaaguaaa ugguguuaac cag

2372423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 724uugaaguaaa ugguguuaac cag 2372523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 725uugaaguaaa ugguguuaac cag 2372623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 726uugaaguaaa ugguguuaac cag 2372723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 727uugaaguaaa ugguguuaac cag 2372823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 728uugaaguaaa ugguguuaac cag 2372923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 729uugaaguaaa ugguguuaac cag 2373023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 730uugaaguaaa ugguguuaac cag 2373123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 731uugaaguaaa ugguguuaac cag 2373223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 732uugaaguaaa ugguguuaac cag 2373323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 733uugaaguaaa ugguguuaac cag 2373423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 734uugaaguaaa ugguguuaac cag 2373523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 735uugaaguaaa ugguguuaac cag 2373623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 736uugaaguaaa ugguguuaac cag 2373723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 737uugaaguaaa ugguguuaac cag 2373823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 738uugaaguaaa ugguguuaac cag 2373923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 739uugaaguaaa ugguguuaac cag 2374023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 740uugaaguaaa ugguguuaac cag 2374123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 741uugaaguaaa ugguguuaac cag 2374223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 742uugaaguaaa ugguguuaac cag 2374323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 743uugaaguaaa ugguguuaac cag 2374423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 744uugaaguaaa ugguguuaac cag 2374523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 745uugaaguaaa ugguguuaac cag 2374623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 746uugaaguaaa ugguguuaac cag 2374721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 747uugaaguaaa ugguguuaac c 2174821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 748uugaaguaaa ugguguuaac c 2174921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 749uugaaguaaa ugguguuaac c 2175021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 750uugaaguaaa ugguguuaac c 2175121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 751uugaaguaaa ugguguuaac c 2175219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 752uugaaguaaa ugguguuaa 1975319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 753uugaaguaaa ugguguuaa 1975419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 754uugaaguaaa ugguguuaa 1975519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 755uugaaguaaa ugguguuaa 1975619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 756uugaaguaaa ugguguuaa 1975723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 757uugaaguaaa ugguguuaac cag 2375823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 758uugaaguaaa ugguguuaac cag 2375923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 759uugaaguaaa ugguguuaac cag 2376023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 760uugaaguaaa ugguguuaac cag 2376123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 761uugaaguaaa ugguguuaac cag 2376223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 762uugaaguaaa uggtguuaac cag 2376323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 763uugaaguaaa ugguguuaac cag 2376423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 764uugaaguaaa uggugtuaac cag 2376523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 765uugaaguaaa uggugutaac cag 2376623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 766uugaaguaaa ugguguuaac cag 2376723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 767uugaaguaaa ugguguuaac cag 2376823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 768uugaaguaaa ugguguuaac cag 2376923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 769uugaaguaaa ugguguuaac cag 2377023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 770uugaaguaaa ugguguuaac cag 2377119DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 771uugaaguaaa tgguguuaa 1977223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 772uugaaguaaa ugguguuaac cag 2377323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 773uugaaguaaa ugguguuaac cag 2377423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 774uugaaguaaa ugguguuaac cag 2377523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 775uugaaguaaa ugguguuaac cag 2377623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 776uugaaguaaa ugguguuaac cag 2377723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 777uugaaguaaa ugguguuaac cag 2377823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 778uugaaguaaa ugguguuaac cag 2377921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 779gguuaacacc auuuacuuca a 2178021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 780gguuaacacc auuuacuuca a 2178121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 781gguuaacacc auuuacuuca a 2178221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 782gguuaacacc auuuacuuca a 2178321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 783gguuaacacc auuuacuuca a 2178421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 784gguuaacacc auuuacuuca a 2178521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 785gguuaacacc auuuacuuca a 2178621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 786gguuaacacc auuuacuuca a 2178721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 787gguuaacacc auuuacuuca a 2178821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 788gguuaacacc auuuacuuca a 2178921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 789gguuaacacc auuuacuuca a 2179021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 790gguuaacacc auuuacuuca a 2179121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 791gguuaacacc auuuacuuca a 2179221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 792gguuaacacc auuuacuuca a 2179321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 793gguuaacacc auuuacuuca a 2179421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 794gguuaacacc auuuacuuca a 2179521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 795ggutaacacc auuuacutca a 2179621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 796ggutaacacc auutacutca a 2179721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 797ggutaacacc auuuacutca a 2179821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 798ggutaacacc auutacutca a 2179921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 799ggutaacacc auuuacutca a 2180021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 800gguuaacacc auuuacutca a 2180121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 801ggutaacacc auuuacutca a 2180221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 802gguuaacacc auuuacuuca a 2180321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 803gguuaacacc auuuacuuca a 2180421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 804gguuaacacc auuuacutca a 2180521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 805gguuaacacc auuuacuuca a 2180621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 806gguuaacacc auuuacuuca a 2180721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 807gguuaacacc auuuacuuca a 2180821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 808gguuaacacc auuuacuuca a 2180923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 809uugaaguaaa ugguguuaac cag 2381023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 810uugaaguaaa ugguguuaac cag 2381123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 811uugaaguaaa ugguguuaac cag 2381223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 812uugaaguaaa ugguguuaac cag 2381323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 813uugaaguaaa ugguguuaac cag 2381423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 814uugaaguaaa ugguguuaac cag 2381523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 815uugaaguaaa ugguguuaac cag 2381623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 816uugaaguaaa ugguguuaac cag 2381723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 817uugaaguaaa ugguguuaac cag 2381823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 818uugaaguaaa ugguguuaac cag 2381923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 819uugaaguaaa ugguguuaac cag 2382023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 820uugaaguaaa ugguguuaac cag 2382123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 821uugaaguaaa ugguguuaac cag 2382223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 822uugaaguaaa ugguguuaac cag 2382321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 823uugaaguaaa ugguguuaac c 2182423RNAArtificial SequenceDescription of

Artificial Sequence Synthetic oligonucleotide 824uugaaguaaa ugguguuaac cag 2382523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 825uugaaguaaa ugguguuaac cag 2382623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 826uugaaguaaa ugguguuaac cag 2382723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 827uugaaguaaa ugguguuaac cag 2382823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 828uugaaguaaa ugguguuaac cag 2382923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 829uugaaguaaa ugguguuaac cag 2383023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 830uugaaguaaa ugguguuaac cag 2383123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 831uugaaguaaa ugguguuaac cag 2383223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 832uugaaguaaa ugguguuaac cag 2383323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 833uugaaguaaa ugguguuaac cag 2383423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 834uugaaguaaa ugguguuaac cag 2383523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 835uugaaguaaa ugguguuaac cag 2383623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 836uugaaguaaa ugguguuaac cag 2383723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 837uugaaguaaa ugguguuaac cag 2383823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 838uugaaguaaa ugguguuaac cag 2383921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 839gguuaacacc auuuacuuca a 2184021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 840gguuaacacc auuuacutca a 2184121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 841ggutaacacc auutacutca a 2184221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 842gguuaacacc auuuacuuca a 2184321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 843gguuaacacc auuuacuuca a 2184421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 844ggutaacacc auuuacutca a 2184521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 845gguuaacacc auuuacuuca a 2184621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 846gguuaacacc auuuacuuca a 2184721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 847ggutaacacc auutacutca a 2184821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 848gguuaacacc auuuacuuca a 2184921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 849gguuaacacc auuuacuuca a 2185021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 850ggutaacacc auuuacutca a 2185121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 851gguuaacacc auuuacuuca a 2185221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 852gguuaacacc auuuacuuca a 2185321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 853gguuaacacc auuuacutca a 2185421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 854ggutaacacc auuuacutca a 2185521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 855gguuaacacc auuuacuuca a 2185621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 856gguuaacacc auuuacuuca a 2185721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 857gguuaacacc auuuacuuca a 2185821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 858gguuaacacc auuuacuuca a 2185921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 859gguuaacacc auuuacuuca a 2186021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 860gguuaacacc auuuacuuca a 2186121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 861gguuaacacc auuuacuuca a 2186221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 862gguuaacacc auuuacuuca a 2186321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 863gguuaacacc auuuacuuca a 2186421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 864gguuaacacc auuuacuuca a 2186521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 865gguuaacacc auuuacuuca a 2186621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 866gguuaacacc auuuacuuca a 2186721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 867ggutaacacc auuuacutca a 2186821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 868gguuaacacc auuuacuuca a 2186923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 869uugaaguaaa ugguguuaac cag 2387023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 870uugaaguaaa ugguguuaac cag 2387123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 871uugaaguaaa ugguguuaac cag 2387223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 872uugaaguaaa ugguguuaac cag 2387323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 873uugaaguaaa ugguguuaac cag 2387423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 874uugaaguaaa ugguguuaac cag 2387523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 875uugaaguaaa ugguguuaac cag 2387623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 876uugaaguaaa ugguguuaac cag 2387723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 877uugaaguaaa ugguguuaac cag 2387823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 878uugaaguaaa ugguguuaac cag 2387923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 879uugaaguaaa ugguguuaac cag 2388023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 880uugaaguaaa ugguguuaac cag 2388123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 881uugaaguaaa ugguguuaac cag 2388223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 882uugaaguaaa ugguguuaac cag 2388323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 883uugaaguaaa ugguguuaac cag 2388423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 884uugaaguaaa ugguguuaac cag 2388523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 885uugaaguaaa ugguguuaac cag 2388623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 886uugaaguaaa ugguguuaac cag 2388723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 887uugaaguaaa ugguguuaac cag 2388823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 888uugaaguaaa ugguguuaac cag 2388923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 889uugaaguaaa ugguguuaac cag 2389023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 890uugaaguaaa ugguguuaac cag 2389123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 891uugaaguaaa ugguguuaac cag 2389223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 892uugaaguaaa ugguguuaac cag 2389323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 893uugaaguaaa ugguguuaac cag 2389423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 894uugaaguaaa ugguguuaac cag 2389523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 895uugaaguaaa ugguguuaac cag 2389621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 896uugaaguaaa ugguguuaac c 2189723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 897uugaaguaaa ugguguuaac cag 2389823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 898uugaaguaaa ugguguuaac cag 2389921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 899gguuaacacc auuuacuuca a 2190021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 900gguuaacacc auuuacuuca a 2190121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 901ggutaacacc auutacutca a 2190221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 902gguuaacacc auuuacuuca a 2190321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 903gguuaacacc auuuacutca a 2190421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 904ggutaacacc auuuacutca a 2190521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 905ggutaacacc auutacutca a 2190621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 906gguuaacacc auuuacuuca a 2190721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 907ggutaacacc auuuacutca a 2190821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 908gguuaacacc auuuacuuca a 2190921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 909gguuaacacc auuuacuuca a 2191021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 910ggutaacacc auuuacutca a 2191121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 911gguuaacacc auuuacutca a 2191220RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 912guuaacacca uuuacuucaa 2091321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 913gguuaacacc auutacuuca a 2191421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 914ggutaacacc auutacutca a 2191521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 915aacugccgac ucuaucgaaa a 2191621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 916gguuaacacc autuactuca a 2191720RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 917guuaacacca uuuacuucaa 2091821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 918gguuaacacc auuuacuuca a 2191921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 919ggutaacacc auuuacutca a 2192023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 920uugaaguaaa ugguguuaac cag 2392123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 921uugaaguaaa ugguguuaac cag 2392223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 922uugaaguaaa ugguguuaac cag 2392323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 923uugaaguaaa ugguguuaac cag 2392423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 924uugaaguaaa

ugguguuaac cag 2392523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 925uugaaguaaa ugguguuaac cag 2392623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 926uugaaguaaa ugguguuaac cag 2392723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 927uugaaguaaa ugguguuaac cag 2392823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 928uugaaguaaa ugguguuaac cag 2392923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 929uugaaguaaa ugguguuaac cag 2393023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 930uugaaguaaa ugguguuaac cag 2393123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 931uugaaguaaa ugguguuaac cag 2393223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 932uugaaguaaa ugguguuaac cag 2393322RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 933uugaaguaaa ugguguuaac ca 2293423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 934utgaagtaaa uggtguuaac cag 2393523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 935uugaaguaaa ugguguuaac cag 2393623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 936uuuucgauag agucggcagu uca 2393723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 937utgaagtaaa uggtguuaac cag 2393822RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 938uugaaguaaa ugguguuaac ca 2293923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 939uugaaguaaa ugguguuaac cag 2394023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 940uugaaguaaa ugguguuaac cag 2394121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 941gguuaacacc auuuacuuca a 2194221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 942gguuaacacc auuuacuuca a 2194321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 943gguuaacacc auuuacuuca a 2194421RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 944gguuaacacc auuuacuuca a 2194521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 945gguuaacacc auuuacuuca a 2194621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 946gguuaacacc auuuacuuca a 2194721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 947ggutaacacc auutacutca a 2194821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 948gguuaacacc auuuacuuca a 2194921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 949gguuaacacc auuuacuuca a 2195021RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 950gguuaacacc auuuacuuca a 2195121RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 951gguuaacacc auuuacuuca a 2195221RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 952gguuaacacc auuuacuuca a 2195321RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 953gguuaacacc auuuacuuca a 2195421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 954ggutaacacc auutacutca a 2195521RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 955gguuaacacc auuuacuuca a 2195621RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 956gguuaacacc auuuacuuca a 2195721RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 957gguuaacacc auuuacuuca a 2195821RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 958gguuaacacc auuuacuuca a 2195921RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 959gguuaacacc auuuacuuca a 2196023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 960uugaaguaaa ugguguuaac cag 2396123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 961uugaaguaaa ugguguuaac cag 2396223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 962uugaaguaaa ugguguuaac cag 2396323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 963uugaaguaaa ugguguuaac cag 2396423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 964uugaaguaaa ugguguuaac cag 2396523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 965uugaaguaaa ugguguuaac cag 2396623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 966uugaaguaaa ugguguuaac cag 2396723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 967uugaaguaaa ugguguuaac cag 2396823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 968uugaaguaaa ugguguuaac cag 2396923RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 969uugaaguaaa ugguguuaac cag 2397023RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 970uugaaguaaa ugguguuaac cag 2397123RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 971uugaaguaaa ugguguuaac cag 2397223RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 972uugaaguaaa ugguguuaac cag 2397323RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 973uugaaguaaa ugguguuaac cag 2397423RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 974uugaaguaaa ugguguuaac cag 2397523RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 975uugaaguaaa ugguguuaac cag 2397623RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 976uugaaguaaa ugguguuaac cag 2397723RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 977uugaaguaaa ugguguuaac cag 2397823RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 978uugaaguaaa ugguguuaac cag 2397919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 979ggagaagaag gcaacugag 1998019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 980ugaccaagcu gggugccug 1998119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 981gguuaacacc auuuacuuc 1998219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 982gcugguuaac accauuuac 1998319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 983uaaugacacc cuccagcaa 1998419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 984cguguucagc aucuaugau 1998519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 985uugaggacgg cuucaguuu 1998619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 986cggcgugucu gggaacugu 1998719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 987uuaacaccau uuacuucaa 1998819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 988cccugaaaag uccaaacuc 1998919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 989cgagaugacc ucuaugucu 1999019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 990ucuacaaggc ugauggaga 1999119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 991agcucacugu ucuggugcu 1999219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 992aggagcagcu gcaagacau 1999319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 993gccaccaacc ggcgugucu 1999419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 994cagaacagaa gaucccgga 1999519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 995ccuugucgau cuguucagc 1999619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 996aggcaaguuc cguuaucgg 1999719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 997uuuuguccuu gcugcucau 1999819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 998agaccuacca ggacaucag 1999919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 999aacugaacug ccgacucua 19100019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1000cauuuacuuc aagggccug 19100119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1001cccuggacuu caaggaaaa 19100219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1002agcugcaagu accgcuguu 19100319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1003acacaaggaa ggaacuguu 19100419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1004gcaacugagg augagggcu 19100519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1005guagccaacc cuuguguua 19100619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1006guuugugaac agaaguaaa 19100719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1007gggugacuuu caaggccaa 19100819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1008uuaucggcgc guggcugaa 19100919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1009ccacuucuuc uuugccaaa 19101019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1010aacaccauuu acuucaagg 19101119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1011gauggagagu cguguucag 19101219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1012caccauuuac uucaagggc 19101319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1013uuuacuucaa gggccugug 19101419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1014uaagagaagu uccucugaa 19101519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1015gcgggacauu cccaugaau 19101619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1016ugccccaccc uguccucug 19101719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1017ugguuaacac cauuuacuu 19101819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1018cggauugccu cagaucaca 19101919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1019ccaggacauc agugaguug 19102019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1020ccaguuuuca ggcggauug 19102119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1021caccauaucu gagaaaaca 19102219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1022aaguaaaaau aaauacaaa 19102319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1023gcacccaggu gcuugaguu 19102419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1024uaacaccauu uacuucaag

19102519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1025ccuucaaagg ugaugacau 19102619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1026caaggccaau ucccgcuuu 19102719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1027ucaguuugaa ggagcagcu 19102819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1028gggacugcgu gaccuguca 19102919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1029ucagccaauc gccuuuuug 19103019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1030ccucggaagc caucaauga 19103119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1031gacaaugaua acauuuucc 19103219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1032cuuauucuuu gcaccucuu 19103319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1033auugcuggcc guucgcuaa 19103419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1034aaaugaagaa ggcagugaa 19103519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1035ugaguuggua uauggagcc 19103619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1036uccagcaacu gauggaggu 19103719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1037aacuguaacc ucuggaaaa 19103819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1038ucaacaaaug gguguccaa 19103919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1039gauuagcggc cauguauuc 19104019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1040gugcuugagu ugcccuuca 19104119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1041ugcagaaggc cgagaugac 19104219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1042cuauuuuugg uuugugaac 19104319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1043cagugaagca gcugcaagu 19104419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1044guguccaaua agaccgaag 19104519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1045augaauugga ggagaugau 19104619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1046aucuaugaug uaccaggaa 19104719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1047ccauaaggca uuucuugag 19104819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1048augcauucca uaaggcauu 19104919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1049uggugcuggu uaacaccau 19105019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1050aucuguucag cccugaaaa 19105119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1051agaugaugcu gguggucca 19105219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1052auauggagcc aagcuccag 19105319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1053ccgaaucacc gaugucauu 19105419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1054gcagagcaau ccagagcgg 19105519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1055acaccauuua cuucaaggg 19105619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1056guaccaggaa ggcaaguuc 19105719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1057ugcccaagcc ugagaagag 19105819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1058uucuuugcca aacugaacu 19105919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1059uggccaaggu agagaagga 19106019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1060ugcugcaaga guggcugga 19106119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1061ccaugugcau uuaccgcuc 19106219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1062agacaugggc cuugucgau 19106319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1063uuuuggagac aaaucccuu 19106419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1064ggaugagggc ucagaacag 19106519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1065cggcuuuugc uaugaccaa 19106619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1066agcuccagcc ccuggacuu 19106719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1067cugaucagau ccacuucuu 19106819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1068ugcugguggu ccacaugcc 19106919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1069gcgagauuua gaggaaaga 19107019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1070cugcucauug gcuucuggg 19107119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1071ccuucaauga gaccuacca 19107219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1072accauuuacu ucaagggcc 19107319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1073gcaccucuuc cuauuuuug 19107419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1074guggcugaag gcacccagg 19107519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1075uccgcauuga ggacggcuu 19107619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1076uggacaucug cacagccaa 19107719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1077guccaaacuc ccagguauu 19107819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1078agaaggaacu caccccaga 19107919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1079ucuugaggua aaugaagaa 19108019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1080agcccugugg acaucugca 19108119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1081cagagcggcc aucaacaaa 19108219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1082auuuaaguuu gacaccaua 19108319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1083ugagaagagc cuggccaag 19108419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1084ucaccauggu ccucaucuu 19108519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1085ggaaggaacu guucuacaa 19108619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1086cugguuuuua uaagagaag 19108719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1087cugggugccu guaaugaca 19108819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1088guaccgcugu ugugauugc 19108919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1089ucuaucagca ccuggcaga 19109019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1090cuggcagauu ccaagaaug 19109119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1091cgaugucauu cccucggaa 19109219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1092gcuucuggga cugcgugac 19109319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1093ccugucacgg gagcccugu 19109419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1094auuuacuuca agggccugu 19109519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1095ugcuaccacu uucuaucag 19109619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1096ggaacugucc aaggccaau 19109719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1097acaaauccuc caaguuagu 19109819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1098auuuaccgcu ccccggaga 19109919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1099accccugagu aucuccacg 19110019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1100cacuaucucc acuugccca 19110119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1101aaauacaaac uacuuccau 19110219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1102cugguuaaca ccauuuacu 19110319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1103ucaucuugcc caagccuga 19110419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1104ccucagauca cacuaucuc 19110519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1105cuguccucug gaaccucug 19110619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1106cccuguggaa gauuagcgg 19110719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1107aucuccacgg cuuuugcua 19110819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1108guggcuggau gaauuggag 19110919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1109caaguuagua ucagccaau 19111019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1110gugaugacau caccauggu 19111119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1111uuccaagaau gacaaugau 19111219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1112ugauggaggu auuuaaguu 19111319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1113guauuccaau gugauagga 19111419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1114cagccaagcc gcgggacau 19111519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1115caugccccgc uuccgcauu 19111619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1116augugauagg aacuguaac 19111719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1117acaaaucccu uaccuucaa 19111819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1118agguuuaucu uuuguccuu 19111919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1119agaucccgga ggccaccaa 19112019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1120acauuuuccu gucaccccu 19112119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1121ggccuuuccu gguuuuuau 19112219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1122uuguguuaag uaaaauguu 19112319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1123caaggaaaau gcagagcaa 19112419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1124cugagaaaac aucugauca 19112519RNAArtificial SequenceDescription of Artificial Sequence

Synthetic oligonucleotide 1125uuacuucaag ggccugugg 19112619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1126accccaacag ggugacuuu 19112719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1127ccagguauug uugcagaag 19112819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1128gccuguggaa gucaaaguu 19112919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1129uggaaccucu gcgagauuu 19113019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1130agcccugaga acacaagga 19113119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1131cucuaugucu cagaugcau 19113219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1132aagaccgaag gccgaauca 19113319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1133uaaaauguuc uuauucuuu 19113419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1134ucuggaaaaa ggaagguuu 19113519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1135uucaaggcca acaggccuu 19113619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1136gaagucaaag uucagcccu 19113719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1137ccaucaauga gcucacugu 19113819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1138cucugaacac uauuaucuu 19113919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1139ugcugguuaa caccauuua 19114019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1140agaggaaaga accaguuuu 19114119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1141ugcccagccc uguggaaga 19114219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1142cucaguugcc uucuucucc 19114319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1143caggcaccca gcuugguca 19114419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1144gaaguaaaug guguuaacc 19114519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1145guaaauggug uuaaccagc 19114619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1146uugcuggagg gugucauua 19114719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1147aucauagaug cugaacacg 19114819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1148aaacugaagc cguccucaa 19114919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1149acaguuccca gacacgccg 19115019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1150uugaaguaaa ugguguuaa 19115119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1151gaguuuggac uuuucaggg 19115219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1152agacauagag gucaucucg 19115319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1153ucuccaucag ccuuguaga 19115419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1154agcaccagaa cagugagcu 19115519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1155augucuugca gcugcuccu 19115619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1156agacacgccg guugguggc 19115719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1157uccgggaucu ucuguucug 19115819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1158gcugaacaga ucgacaagg 19115919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1159ccgauaacgg aacuugccu 19116019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1160augagcagca aggacaaaa 19116119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1161cugauguccu gguaggucu 19116219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1162uagagucggc aguucaguu 19116319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1163caggcccuug aaguaaaug 19116419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1164uuuuccuuga aguccaggg 19116519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1165aacagcggua cuugcagcu 19116619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1166aacaguuccu uccuugugu 19116719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1167agcccucauc cucaguugc 19116819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1168uaacacaagg guuggcuac 19116919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1169uuuacuucug uucacaaac 19117019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1170uuggccuuga aagucaccc 19117119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1171uucagccacg cgccgauaa 19117219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1172uuuggcaaag aagaagugg 19117319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1173ccuugaagua aaugguguu 19117419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1174cugaacacga cucuccauc 19117519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1175gcccuugaag uaaauggug 19117619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1176cacaggcccu ugaaguaaa 19117719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1177uucagaggaa cuucucuua 19117819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1178auucauggga augucccgc 19117919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1179cagaggacag gguggggca 19118019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1180aaguaaaugg uguuaacca 19118119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1181ugugaucuga ggcaauccg 19118219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1182caacucacug auguccugg 19118319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1183caauccgccu gaaaacugg 19118419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1184uguuuucuca gauauggug 19118519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1185uuuguauuua uuuuuacuu 19118619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1186aacucaagca ccugggugc 19118719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1187cuugaaguaa augguguua 19118819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1188augucaucac cuuugaagg 19118919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1189aaagcgggaa uuggccuug 19119019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1190agcugcuccu ucaaacuga 19119119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1191ugacagguca cgcaguccc 19119219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1192caaaaaggcg auuggcuga 19119319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1193ucauugaugg cuuccgagg 19119419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1194ggaaaauguu aucauuguc 19119519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1195aagaggugca aagaauaag 19119619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1196uuagcgaacg gccagcaau 19119719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1197uucacugccu ucuucauuu 19119819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1198ggcuccauau accaacuca 19119919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1199accuccauca guugcugga 19120019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1200uuuuccagag guuacaguu 19120119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1201uuggacaccc auuuguuga 19120219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1202gaauacaugg ccgcuaauc 19120319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1203ugaagggcaa cucaagcac 19120419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1204gucaucucgg ccuucugca 19120519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1205guucacaaac caaaaauag 19120619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1206acuugcagcu gcuucacug 19120719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1207cuucggucuu auuggacac 19120819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1208aucaucuccu ccaauucau 19120919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1209uuccugguac aucauagau 19121019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1210cucaagaaau gccuuaugg 19121119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1211aaugccuuau ggaaugcau 19121219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1212augguguuaa ccagcacca 19121319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1213uuuucagggc ugaacagau 19121419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1214uggaccacca gcaucaucu 19121519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1215cuggagcuug gcuccauau 19121619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1216aaugacaucg gugauucgg 19121719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1217ccgcucugga uugcucugc 19121819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1218cccuugaagu aaauggugu 19121919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1219gaacuugccu uccugguac 19122019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1220cucuucucag gcuugggca 19122119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1221aguucaguuu ggcaaagaa 19122219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1222uccuucucua ccuuggcca 19122319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1223uccagccacu cuugcagca 19122419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1224gagcgguaaa ugcacaugg 19122519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1225aucgacaagg cccaugucu

19122619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1226aagggauuug ucuccaaaa 19122719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1227cuguucugag cccucaucc 19122819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1228uuggucauag caaaagccg 19122919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1229aaguccaggg gcuggagcu 19123019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1230aagaagugga ucugaucag 19123119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1231ggcaugugga ccaccagca 19123219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1232ucuuuccucu aaaucucgc 19123319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1233cccagaagcc aaugagcag 19123419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1234ugguaggucu cauugaagg 19123519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1235ggcccuugaa guaaauggu 19123619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1236caaaaauagg aagaggugc 19123719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1237ccugggugcc uucagccac 19123819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1238aagccguccu caaugcgga 19123919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1239uuggcugugc agaugucca 19124019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1240aauaccuggg aguuuggac 19124119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1241ucugggguga guuccuucu 19124219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1242uucuucauuu accucaaga 19124319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1243ugcagauguc cacagggcu 19124419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1244uuuguugaug gccgcucug 19124519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1245uaugguguca aacuuaaau 19124619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1246cuuggccagg cucuucuca 19124719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1247aagaugagga ccaugguga 19124819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1248uuguagaaca guuccuucc 19124919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1249cuucucuuau aaaaaccag 19125019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1250ugucauuaca ggcacccag 19125119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1251gcaaucacaa cagcgguac 19125219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1252ucugccaggu gcugauaga 19125319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1253cauucuugga aucugccag 19125419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1254uuccgaggga augacaucg 19125519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1255gucacgcagu cccagaagc 19125619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1256acagggcucc cgugacagg 19125719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1257acaggcccuu gaaguaaau 19125819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1258cugauagaaa gugguagca 19125919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1259auuggccuug gacaguucc 19126019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1260acuaacuugg aggauuugu 19126119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1261ucuccgggga gcgguaaau 19126219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1262cguggagaua cucaggggu 19126319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1263ugggcaagug gagauagug 19126419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1264auggaaguag uuuguauuu 19126519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1265aguaaauggu guuaaccag 19126619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1266ucaggcuugg gcaagauga 19126719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1267gagauagugu gaucugagg 19126819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1268cagagguucc agaggacag 19126919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1269ccgcuaaucu uccacaggg 19127019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1270uagcaaaagc cguggagau 19127119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1271cuccaauuca uccagccac 19127219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1272auuggcugau acuaacuug 19127319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1273accaugguga ugucaucac 19127419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1274aucauuguca uucuuggaa 19127519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1275aacuuaaaua ccuccauca 19127619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1276uccuaucaca uuggaauac 19127719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1277augucccgcg gcuuggcug 19127819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1278aaugcggaag cggggcaug 19127919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1279guuacaguuc cuaucacau 19128019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1280uugaagguaa gggauuugu 19128119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1281aaggacaaaa gauaaaccu 19128219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1282uugguggccu ccgggaucu 19128319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1283aggggugaca ggaaaaugu 19128419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1284auaaaaacca ggaaaggcc 19128519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1285aacauuuuac uuaacacaa 19128619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1286uugcucugca uuuuccuug 19128719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1287ugaucagaug uuuucucag 19128819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1288ccacaggccc uugaaguaa 19128919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1289aaagucaccc uguuggggu 19129019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1290cuucugcaac aauaccugg 19129119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1291aacuuugacu uccacaggc 19129219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1292aaaucucgca gagguucca 19129319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1293uccuuguguu cucagggcu 19129419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1294augcaucuga gacauagag 19129519RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1295ugauucggcc uucggucuu 19129619RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1296aaagaauaag aacauuuua 19129719RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1297aaaccuuccu uuuuccaga 19129819RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1298aaggccuguu ggccuugaa 19129919RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1299agggcugaac uuugacuuc 19130019RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1300acagugagcu cauugaugg 19130119RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1301aagauaauag uguucagag 19130219RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1302uaaauggugu uaaccagca 19130319RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1303aaaacugguu cuuuccucu 19130419RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1304ucuuccacag ggcugggca 19130521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1305ggagaagaag gcaacugagt t 21130621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1306ugaccaagcu gggugccugt t 21130721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1307gguuaacacc auuuacuuct t 21130821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1308gcugguuaac accauuuact t 21130921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1309uaaugacacc cuccagcaat t 21131021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1310cguguucagc aucuaugaut t 21131121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1311uugaggacgg cuucaguuut t 21131221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1312cggcgugucu gggaacugut t 21131321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1313uuaacaccau uuacuucaat t 21131421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1314cccugaaaag uccaaacuct t 21131521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1315cgagaugacc ucuaugucut t 21131621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1316ucuacaaggc ugauggagat t 21131721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1317agcucacugu ucuggugcut t 21131821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1318aggagcagcu gcaagacaut t 21131921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1319gccaccaacc ggcgugucut t 21132021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1320cagaacagaa gaucccggat t 21132121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1321ccuugucgau cuguucagct t 21132221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1322aggcaaguuc cguuaucggt t 21132321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1323uuuuguccuu gcugcucaut t 21132421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1324agaccuacca ggacaucagt t 21132521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1325aacugaacug ccgacucuat t 21132621DNAArtificial SequenceDescription

of Artificial Sequence Synthetic oligonucleotide 1326cauuuacuuc aagggccugt t 21132721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1327cccuggacuu caaggaaaat t 21132821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1328agcugcaagu accgcuguut t 21132921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1329acacaaggaa ggaacuguut t 21133021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1330gcaacugagg augagggcut t 21133121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1331guagccaacc cuuguguuat t 21133221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1332guuugugaac agaaguaaat t 21133321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1333gggugacuuu caaggccaat t 21133421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1334uuaucggcgc guggcugaat t 21133521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1335ccacuucuuc uuugccaaat t 21133621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1336aacaccauuu acuucaaggt t 21133721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1337gauggagagu cguguucagt t 21133821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1338caccauuuac uucaagggct t 21133921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1339uuuacuucaa gggccugugt t 21134021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1340uaagagaagu uccucugaat t 21134121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1341gcgggacauu cccaugaaut t 21134221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1342ugccccaccc uguccucugt t 21134321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1343ugguuaacac cauuuacuut t 21134421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1344cggauugccu cagaucacat t 21134521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1345ccaggacauc agugaguugt t 21134621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1346ccaguuuuca ggcggauugt t 21134721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1347caccauaucu gagaaaacat t 21134821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1348aaguaaaaau aaauacaaat t 21134921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1349gcacccaggu gcuugaguut t 21135021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1350uaacaccauu uacuucaagt t 21135121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1351ccuucaaagg ugaugacaut t 21135221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1352caaggccaau ucccgcuuut t 21135321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1353ucaguuugaa ggagcagcut t 21135421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1354gggacugcgu gaccugucat t 21135521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1355ucagccaauc gccuuuuugt t 21135621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1356ccucggaagc caucaaugat t 21135721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1357gacaaugaua acauuuucct t 21135821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1358cuuauucuuu gcaccucuut t 21135921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1359auugcuggcc guucgcuaat t 21136021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1360aaaugaagaa ggcagugaat t 21136121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1361ugaguuggua uauggagcct t 21136221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1362uccagcaacu gauggaggut t 21136321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1363aacuguaacc ucuggaaaat t 21136421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1364ucaacaaaug gguguccaat t 21136521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1365gauuagcggc cauguauuct t 21136621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1366gugcuugagu ugcccuucat t 21136721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1367ugcagaaggc cgagaugact t 21136821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1368cuauuuuugg uuugugaact t 21136921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1369cagugaagca gcugcaagut t 21137021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1370guguccaaua agaccgaagt t 21137121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1371augaauugga ggagaugaut t 21137221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1372aucuaugaug uaccaggaat t 21137321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1373ccauaaggca uuucuugagt t 21137421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1374augcauucca uaaggcauut t 21137521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1375uggugcuggu uaacaccaut t 21137621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1376aucuguucag cccugaaaat t 21137721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1377agaugaugcu ggugguccat t 21137821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1378auauggagcc aagcuccagt t 21137921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1379ccgaaucacc gaugucauut t 21138021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1380gcagagcaau ccagagcggt t 21138121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1381acaccauuua cuucaagggt t 21138221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1382guaccaggaa ggcaaguuct t 21138321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1383ugcccaagcc ugagaagagt t 21138421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1384uucuuugcca aacugaacut t 21138521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1385uggccaaggu agagaaggat t 21138621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1386ugcugcaaga guggcuggat t 21138721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1387ccaugugcau uuaccgcuct t 21138821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1388agacaugggc cuugucgaut t 21138921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1389uuuuggagac aaaucccuut t 21139021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1390ggaugagggc ucagaacagt t 21139121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1391cggcuuuugc uaugaccaat t 21139221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1392agcuccagcc ccuggacuut t 21139321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1393cugaucagau ccacuucuut t 21139421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1394ugcugguggu ccacaugcct t 21139521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1395gcgagauuua gaggaaagat t 21139621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1396cugcucauug gcuucugggt t 21139721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1397ccuucaauga gaccuaccat t 21139821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1398accauuuacu ucaagggcct t 21139921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1399gcaccucuuc cuauuuuugt t 21140021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1400guggcugaag gcacccaggt t 21140121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1401uccgcauuga ggacggcuut t 21140221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1402uggacaucug cacagccaat t 21140321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1403guccaaacuc ccagguauut t 21140421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1404agaaggaacu caccccagat t 21140521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1405ucuugaggua aaugaagaat t 21140621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1406agcccugugg acaucugcat t 21140721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1407cagagcggcc aucaacaaat t 21140821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1408auuuaaguuu gacaccauat t 21140921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1409ugagaagagc cuggccaagt t 21141021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1410ucaccauggu ccucaucuut t 21141121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1411ggaaggaacu guucuacaat t 21141221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1412cugguuuuua uaagagaagt t 21141321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1413cugggugccu guaaugacat t 21141421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1414guaccgcugu ugugauugct t 21141521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1415ucuaucagca ccuggcagat t 21141621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1416cuggcagauu ccaagaaugt t 21141721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1417cgaugucauu cccucggaat t 21141821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1418gcuucuggga cugcgugact t 21141921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1419ccugucacgg gagcccugut t 21142021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1420auuuacuuca agggccugut t 21142121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1421ugcuaccacu uucuaucagt t 21142221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1422ggaacugucc aaggccaaut t 21142321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1423acaaauccuc caaguuagut t 21142421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1424auuuaccgcu ccccggagat t 21142521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1425accccugagu aucuccacgt t 21142621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1426cacuaucucc

acuugcccat t 21142721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1427aaauacaaac uacuuccaut t 21142821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1428cugguuaaca ccauuuacut t 21142921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1429ucaucuugcc caagccugat t 21143021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1430ccucagauca cacuaucuct t 21143121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1431cuguccucug gaaccucugt t 21143221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1432cccuguggaa gauuagcggt t 21143321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1433aucuccacgg cuuuugcuat t 21143421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1434guggcuggau gaauuggagt t 21143521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1435caaguuagua ucagccaaut t 21143621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1436gugaugacau caccauggut t 21143721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1437uuccaagaau gacaaugaut t 21143821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1438ugauggaggu auuuaaguut t 21143921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1439guauuccaau gugauaggat t 21144021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1440cagccaagcc gcgggacaut t 21144121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1441caugccccgc uuccgcauut t 21144221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1442augugauagg aacuguaact t 21144321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1443acaaaucccu uaccuucaat t 21144421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1444agguuuaucu uuuguccuut t 21144521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1445agaucccgga ggccaccaat t 21144621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1446acauuuuccu gucaccccut t 21144721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1447ggccuuuccu gguuuuuaut t 21144821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1448uuguguuaag uaaaauguut t 21144921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1449caaggaaaau gcagagcaat t 21145021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1450cugagaaaac aucugaucat t 21145121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1451uuacuucaag ggccuguggt t 21145221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1452accccaacag ggugacuuut t 21145321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1453ccagguauug uugcagaagt t 21145421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1454gccuguggaa gucaaaguut t 21145521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1455uggaaccucu gcgagauuut t 21145621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1456agcccugaga acacaaggat t 21145721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1457cucuaugucu cagaugcaut t 21145821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1458aagaccgaag gccgaaucat t 21145921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1459uaaaauguuc uuauucuuut t 21146021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1460ucuggaaaaa ggaagguuut t 21146121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1461uucaaggcca acaggccuut t 21146221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1462gaagucaaag uucagcccut t 21146321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1463ccaucaauga gcucacugut t 21146421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1464cucugaacac uauuaucuut t 21146521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1465ugcugguuaa caccauuuat t 21146621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1466agaggaaaga accaguuuut t 21146721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1467ugcccagccc uguggaagat t 21146821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1468cucaguugcc uucuucucct t 21146921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1469caggcaccca gcuuggucat t 21147021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1470gaaguaaaug guguuaacct t 21147121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1471guaaauggug uuaaccagct t 21147221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1472uugcuggagg gugucauuat t 21147321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1473aucauagaug cugaacacgt t 21147421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1474aaacugaagc cguccucaat t 21147521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1475acaguuccca gacacgccgt t 21147621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1476uugaaguaaa ugguguuaat t 21147721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1477gaguuuggac uuuucagggt t 21147821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1478agacauagag gucaucucgt t 21147921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1479ucuccaucag ccuuguagat t 21148021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1480agcaccagaa cagugagcut t 21148121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1481augucuugca gcugcuccut t 21148221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1482agacacgccg guugguggct t 21148321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1483uccgggaucu ucuguucugt t 21148421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1484gcugaacaga ucgacaaggt t 21148521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1485ccgauaacgg aacuugccut t 21148621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1486augagcagca aggacaaaat t 21148721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1487cugauguccu gguaggucut t 21148821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1488uagagucggc aguucaguut t 21148921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1489caggcccuug aaguaaaugt t 21149021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1490uuuuccuuga aguccagggt t 21149121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1491aacagcggua cuugcagcut t 21149221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1492aacaguuccu uccuugugut t 21149321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1493agcccucauc cucaguugct t 21149421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1494uaacacaagg guuggcuact t 21149521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1495uuuacuucug uucacaaact t 21149621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1496uuggccuuga aagucaccct t 21149721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1497uucagccacg cgccgauaat t 21149821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1498uuuggcaaag aagaaguggt t 21149921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1499ccuugaagua aaugguguut t 21150021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1500cugaacacga cucuccauct t 21150121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1501gcccuugaag uaaauggugt t 21150221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1502cacaggcccu ugaaguaaat t 21150321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1503uucagaggaa cuucucuuat t 21150421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1504auucauggga augucccgct t 21150521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1505cagaggacag gguggggcat t 21150621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1506aaguaaaugg uguuaaccat t 21150721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1507ugugaucuga ggcaauccgt t 21150821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1508caacucacug auguccuggt t 21150921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1509caauccgccu gaaaacuggt t 21151021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1510uguuuucuca gauauggugt t 21151121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1511uuuguauuua uuuuuacuut t 21151221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1512aacucaagca ccugggugct t 21151321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1513cuugaaguaa augguguuat t 21151421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1514augucaucac cuuugaaggt t 21151521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1515aaagcgggaa uuggccuugt t 21151621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1516agcugcuccu ucaaacugat t 21151721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1517ugacagguca cgcaguccct t 21151821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1518caaaaaggcg auuggcugat t 21151921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1519ucauugaugg cuuccgaggt t 21152021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1520ggaaaauguu aucauuguct t 21152121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1521aagaggugca aagaauaagt t 21152221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1522uuagcgaacg gccagcaaut t 21152321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1523uucacugccu ucuucauuut t 21152421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1524ggcuccauau accaacucat t 21152521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1525accuccauca guugcuggat t 21152621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1526uuuuccagag guuacaguut t

21152721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1527uuggacaccc auuuguugat t 21152821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1528gaauacaugg ccgcuaauct t 21152921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1529ugaagggcaa cucaagcact t 21153021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1530gucaucucgg ccuucugcat t 21153121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1531guucacaaac caaaaauagt t 21153221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1532acuugcagcu gcuucacugt t 21153321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1533cuucggucuu auuggacact t 21153421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1534aucaucuccu ccaauucaut t 21153521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1535uuccugguac aucauagaut t 21153621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1536cucaagaaau gccuuauggt t 21153721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1537aaugccuuau ggaaugcaut t 21153821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1538augguguuaa ccagcaccat t 21153921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1539uuuucagggc ugaacagaut t 21154021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1540uggaccacca gcaucaucut t 21154121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1541cuggagcuug gcuccauaut t 21154221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1542aaugacaucg gugauucggt t 21154321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1543ccgcucugga uugcucugct t 21154421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1544cccuugaagu aaauggugut t 21154521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1545gaacuugccu uccugguact t 21154621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1546cucuucucag gcuugggcat t 21154721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1547aguucaguuu ggcaaagaat t 21154821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1548uccuucucua ccuuggccat t 21154921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1549uccagccacu cuugcagcat t 21155021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1550gagcgguaaa ugcacauggt t 21155121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1551aucgacaagg cccaugucut t 21155221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1552aagggauuug ucuccaaaat t 21155321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1553cuguucugag cccucaucct t 21155421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1554uuggucauag caaaagccgt t 21155521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1555aaguccaggg gcuggagcut t 21155621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1556aagaagugga ucugaucagt t 21155721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1557ggcaugugga ccaccagcat t 21155821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1558ucuuuccucu aaaucucgct t 21155921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1559cccagaagcc aaugagcagt t 21156021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1560ugguaggucu cauugaaggt t 21156121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1561ggcccuugaa guaaauggut t 21156221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1562caaaaauagg aagaggugct t 21156321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1563ccugggugcc uucagccact t 21156421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1564aagccguccu caaugcggat t 21156521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1565uuggcugugc agauguccat t 21156621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1566aauaccuggg aguuuggact t 21156721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1567ucugggguga guuccuucut t 21156821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1568uucuucauuu accucaagat t 21156921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1569ugcagauguc cacagggcut t 21157021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1570uuuguugaug gccgcucugt t 21157121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1571uaugguguca aacuuaaaut t 21157221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1572cuuggccagg cucuucucat t 21157321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1573aagaugagga ccauggugat t 21157421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1574uuguagaaca guuccuucct t 21157521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1575cuucucuuau aaaaaccagt t 21157621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1576ugucauuaca ggcacccagt t 21157721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1577gcaaucacaa cagcgguact t 21157821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1578ucugccaggu gcugauagat t 21157921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1579cauucuugga aucugccagt t 21158021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1580uuccgaggga augacaucgt t 21158121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1581gucacgcagu cccagaagct t 21158221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1582acagggcucc cgugacaggt t 21158321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1583acaggcccuu gaaguaaaut t 21158421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1584cugauagaaa gugguagcat t 21158521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1585auuggccuug gacaguucct t 21158621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1586acuaacuugg aggauuugut t 21158721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1587ucuccgggga gcgguaaaut t 21158821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1588cguggagaua cucaggggut t 21158921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1589ugggcaagug gagauagugt t 21159021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1590auggaaguag uuuguauuut t 21159121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1591aguaaauggu guuaaccagt t 21159221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1592ucaggcuugg gcaagaugat t 21159321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1593gagauagugu gaucugaggt t 21159421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1594cagagguucc agaggacagt t 21159521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1595ccgcuaaucu uccacagggt t 21159621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1596uagcaaaagc cguggagaut t 21159721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1597cuccaauuca uccagccact t 21159821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1598auuggcugau acuaacuugt t 21159921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1599accaugguga ugucaucact t 21160021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1600aucauuguca uucuuggaat t 21160121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1601aacuuaaaua ccuccaucat t 21160221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1602uccuaucaca uuggaauact t 21160321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1603augucccgcg gcuuggcugt t 21160421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1604aaugcggaag cggggcaugt t 21160521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1605guuacaguuc cuaucacaut t 21160621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1606uugaagguaa gggauuugut t 21160721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1607aaggacaaaa gauaaaccut t 21160821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1608uugguggccu ccgggaucut t 21160921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1609aggggugaca ggaaaaugut t 21161021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1610auaaaaacca ggaaaggcct t 21161121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1611aacauuuuac uuaacacaat t 21161221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1612uugcucugca uuuuccuugt t 21161321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1613ugaucagaug uuuucucagt t 21161421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1614ccacaggccc uugaaguaat t 21161521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1615aaagucaccc uguuggggut t 21161621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1616cuucugcaac aauaccuggt t 21161721DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1617aacuuugacu uccacaggct t 21161821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1618aaaucucgca gagguuccat t 21161921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1619uccuuguguu cucagggcut t 21162021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1620augcaucuga gacauagagt t 21162121DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1621ugauucggcc uucggucuut t 21162221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1622aaagaauaag aacauuuuat t 21162321DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1623aaaccuuccu uuuuccagat t 21162421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1624aaggccuguu ggccuugaat t 21162521DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1625agggcugaac uuugacuuct t 21162621DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1626acagugagcu cauugauggt t 21162721DNAArtificial SequenceDescription of Artificial Sequence

Synthetic oligonucleotide 1627aagauaauag uguucagagt t 21162821DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1628uaaauggugu uaaccagcat t 21162921DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1629aaaacugguu cuuuccucut t 21163021DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1630ucuuccacag ggcugggcat t 21

Claims

1. A method of treating a subject having a hemophilia, comprising administering to the subject a therapeutically effective amount of a double stranded ribonucleic acid (dsRNAi) agent, wherein the dsRNAi agent comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby treating the subject having a hemophilia.

2. A method of treating a subject having a hemophilia, comprising administering to the subject a therapeutically effective amount of a double stranded ribonucleic acid (dsRNAi) agent, wherein the dsRNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby treating the subject having a hemophilia.

3. A method of preventing at least one symptom in a subject having a hemophilia, comprising administering to the subject a therapeutically effective amount of a double stranded ribonucleic acid (dsRNAi) agent, wherein the dsRNAi agent comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby preventing at least one symptom in the subject having a hemophilia.

4. A method of preventing at least one symptom in a subject having a hemophilia, comprising administering to the subject a therapeutically effective amount of a double stranded ribonucleic acid (dsRNAi) agent, wherein the dsRNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby preventing at least one symptom in the subject having a hemophilia.

5. The method of any one of claims 1-4, wherein the nucleotide sequence of the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence of any one of the antisense nucleotide sequences provided in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

6. The method of any one of claims 1-4, wherein the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand comprise the sense strand nucleotide sequence and the antisense strand nucleotide sequence of a dsRNAi agent selected from the group consisting of AD-50487.1, AD-50477.1, AD-50483.1, AD-50475.1, AD-50495.1, AD-50476.1, AD-50499.1, AD-50478.1, AD-50489.1, AD-50501.1, AD-50507.1, AD-50484.1, AD-50515.1, AD-50540.1, AD-50528.1, AD-50549.1, AD-50539.1, AD-50534.1, AD-50527.1, AD-50514.1, AD-50509.1, AD-50529.1, AD-54944.

7. The method of claim 1, wherein at least one of the nucleotides of the dsRNAi agent is a modified nucleotide.

8. The method of claim 7, wherein the modified nucleotide is selected from the group consisting of a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

9. The method of claim 8, wherein the modified nucleotide is selected from the group consisting of a 2'-O-methyl (2'-OMe) modified nucleotide and 2'-fluoro modified nucleotide.

10. The method of claim 1, wherein the antisense strand further comprises at least one phosphorothioate internucleoside linkage.

11. The method of claim 1, wherein the antisense strand further comprises at least two phosphorothioate internucleoside linkages.

12. The method of claim 1, wherein the antisense strand further comprises at least three phosphorothioate internucleoside linkages.

13. The method of claim 1, wherein the antisense strand further comprises at least four phosphorothioate internucleoside linkages.

14. The method of any one of claims 1-4, wherein the sense strand further comprises at least one phosphorothioate internucleoside linkage.

15. The method of claim 1, wherein the sense strand further comprises at least two phosphorothioate internucleoside linkages.

16. The method of claim 1, wherein the antisense strand further comprises a 3' overhang of at least 1 nucleotide.

17. The method of claim 1, wherein the antisense strand further comprises a 3' overhang of at least 2 nucleotides.

18. The method of claim 1, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

19. The method of claim 2, wherein the region of complementarity is at least 17 nucleotides in length.

20. The method of claim 2, wherein the region of complementarity is between 19 and 21 nucleotides in length.

21. The method of claim 20, wherein the region of complementarity is 19 nucleotides in length.

22. The method of claim 1, wherein each strand is no more than 30 nucleotides in length.

23. The method of claim 1, wherein the dsRNAi agent further comprises a ligand.

24. The method of claim 23, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNAi agent.

25. The method of claim 23, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

26. The method of claim 23, wherein the ligand is ##STR00021##

27. The method of claim 23, wherein the dsRNAi agent is conjugated to the ligand as shown in the following schematic ##STR00022## and, wherein X is O or S.

28. The method of claim 27, wherein the X is O.

29. The method of claim 2 or 4, wherein the region of complementarity comprises the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID N0:562)

30. The method of any one of claims 1-4, wherein the sense strand comprises the nucleotide sequence of 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO:294), and the antisense strand comprises the nucleotide sequence of 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO:562).

31. The method of claim 30, wherein the sense strand comprises 5'-GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID N0:956) and the antisense strand comprises 5'-uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg-3' (SEQ ID NO:975), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; and s is a phosphorothioate linkage.

32. The method of claim 30, wherein the sense strand comprises 5'-GfsgsUfuAfaCfaCfCfAfuUfuAfcUfuCfaAf-3' (SEQ ID NO:941) and the antisense strand comprises 5'-usUfsgAfaGfuAfaAfuggUfgUfuAfaCfcsasg-3' (SEQ ID NO:960), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; and s is a phosphorothioate linkage.

33. The method of claim 1, wherein the Serpinc1 expression in the subject is inhibited by at least about 30%.

34. The method of claim 1, wherein the administration of the dsRNAi agent to the subject causes an increase in blood clotting and/or a decrease in Serpinc1 protein accumulation.

35. The method of claim 1, wherein the subject is a human.

36. The method of claim 1, wherein the hemophilia is hemophilia A.

37. The method of claim 1, wherein the hemophilia is hemophilia B.

38. The method of claim 1, wherein the hemophilia is hemophilia C.

39. The method of claim 1, wherein the hemophilia is hemophilia A and the subject is an inhibitor subject.

40. The method of claim 1, wherein the hemophilia is hemophilia B and the subject is an inhibitor subject.

41. The method of claim 1, wherein the hemophilia is hemophilia C and the subject is an inhibitor subject.

42. The method of claim 1, wherein the dsRNAi agent is administered to the subject at a dose of about 10 mg/kg to about 30 mg/kg.

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

44. The method of claim 1, wherein the dsRNAi agent is administered to the subject once a week.

45. The method of claim 1, wherein the dsRNAi agent is administered to the subject twice a month.

46. The method of claim 1, wherein the dsRNAi agent is administered to the subject subcutaneously at a cumulative weekly dose of about 0.5 mg/kg to about 5 mg/kg.

47. The method of claim 1, further comprising administering to the subject an additional therapeutic treatment.

48. The method of any one of claims 1-4, further comprising measuring thrombin levels in the subject.

Images