Rnai formulation and use thereof

Abstract

Provided is an RNA inhibitor for inhibiting AT3 gene expression, comprising an antisense strand. The antisense strand comprises a complementary region that is complementary to at least a part of an AT3-encoding mRNA. The complementary region has a length of 17-23 nucleotides. The antisense strand comprises the following nucleotide sequence: any one of SEQ ID NOs: 1-303 and 922-1001, or a sequence differing from same by no more than 3 nucleotides.

Description

RNAi formulations and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and relates to a modified double-stranded RNAi formulation and uses thereof, in particular to a double-stranded RNAi agent for inhibiting the expression of AT3 gene and a pharmaceutical composition thereof, and uses of the double-stranded RNAi agent or the pharmaceutical composition thereof for treating diseases mediated by the expression of AT3 gene. BACKGROUND

[0002] AT3 is a member of the serpin superfamily. AT3 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 thereby regulates the coagulation cascade. The anticoagulant activity of AT3 is enhanced by the presence of heparin and other related glycosaminoglycans that catalyze the formation of the thrombin:antithrombin (TAT) complex.

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

[0004] There is a need in the art for a therapeutic method for subjects suffering from a bleeding disorder, such as hemophilia. SUMMARY

[0005] In one aspect, the present application provides an RNA inhibitor for inhibiting the expression of AT3 gene, comprising an antisense strand, the antisense strand comprising a complementary region complementary to at least a portion of mRNA encoding AT3, the complementary region having a length of 17-23 nucleotides, wherein the antisense strand comprises any one of SEQ ID NO.: 1-303, 922-1001 or a sequence differing by no more than 3 nucleotides therefrom.

[0006] In some embodiments, the RNA inhibitor for inhibiting the expression of AT3 gene further comprises a sense strand, wherein the sense strand and the antisense strand are at least 80% complementary in base.

[0007] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the sense nucleic acid strand and the antisense nucleic acid strand are present on two different nucleic acid strands.

[0008] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the sense nucleic acid segment and the antisense nucleic acid segment are present on the same nucleic acid strand, wherein the complementary region of the sense nucleic acid segment and the antisense nucleic acid segment forms a double-stranded nucleic acid structure.

[0009] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein at least one strand has a 3’ overhang of 0 to 6 nucleotides in length.

[0010] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein both strands have a 3’ overhang of 2-3 nucleotides in length, or the sense strand has a 3’ overhang of 2-3 nucleotides in length, or the antisense strand has a 3’ overhang of 2-3 nucleotides in length.

[0011] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the sense nucleic acid strand and the antisense nucleic acid strand are 16 to 35 nucleotides in length, respectively.

[0012] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein one strand of the RNA inhibitor of AT3 gene expression has at least 75% homology or complementarity to any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 643-921.

[0013] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the sense strand thereof is selected from the group consisting of any one of SEQ ID NOs: 304-642, 1002-1060, or a sequence with no more than 3 nucleotides different therefrom.

[0014] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein at least one nucleotide is a chemically modified nucleotide.

[0015] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the chemical modification is at least one of the following:

[0016] (1) modification of the phosphodiester linkage connecting the nucleotides in the nucleotide sequence of the RNA inhibitor of AT3 gene expression;

[0017] (2) modification of the ribose in the nucleotide sequence of the RNA inhibitor of AT3 gene expression;

[0018] (3) modification of bases in the nucleotide sequence of the RNA inhibitor that suppresses expression of the AT3 gene.

[0019] In some embodiments, the RNA inhibitor that suppresses expression of the AT3 gene, wherein there are at least two consecutive phosphorothioate linkages between nucleotides of the sense strand and / or the antisense strand.

[0020] In some embodiments, the RNA inhibitor that suppresses expression of the AT3 gene, wherein there are at least two consecutive phosphorothioate linkages between three consecutive nucleotides of the sense strand end and / or the antisense strand end.

[0021] In some embodiments, the RNA inhibitor that suppresses expression of the AT3 gene, wherein the modification of ribose includes fluorine substitution and / or methoxy substitution for 2’-OH.

[0022] In some embodiments, the RNA inhibitor that suppresses expression of the AT3 gene, wherein the modification of ribose includes LNA or GNA.

[0023] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the modifications of the antisense strand comprise one of: XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfmsXmsXm XmsXfsXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXuXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmXmTuXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXm PXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXdXXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfdTXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmdTXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmdXXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm PXmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXmXmsXfsXmXmXmXmXfXmXmXmXmXfXmXfXmXfXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXfXfXfXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXmXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm

[0024] XmsXfsXmXmXmXmXfXmXmXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm, wherein:

[0025] X represents a ribonucleotide, s represents a phosphorothioate linkage, Xm 2'methoxy substituted ribonucleotide, Xf represents a 2'fluoro substituted ribonucleotide, Xu represents a UNA version of a nucleotide, Xg represents a (S)-GNA version of a ribonucleotide, dT represents a 5'-methyluridine-3'-phosphate, dA represents a 2'-deoxyadenosine-3'-phosphate, P represents a phosphorylation, [Uvm] represents a 5'-(E)-vinylphosphonate-2'-Ome-U.

[0026] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the modifications of the sense strand comprise one of: XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXfXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXf XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfTgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmTgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXm XmsXmsXmXmXmXmXfImXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmImXmXmXmXmXmXmXmXm XmsXfsXmXmXmXfXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfTgXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXmsXmXmXmXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmImXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXmXmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfImXfXfXfXmImXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmImXm XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfImXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmIfXmXfXmXfXmXfXfsXmsXfXmXfXmXfImXfXfXfXmIfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmIfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmImXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmIfXmXf XfsXmsXfXmIfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmImXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmImXmXmXmXmXm XmsXmsXmXmXmXmXmXmXmXfXfXfXmXmXmXmXmXmXmXmXmXmXm

[0027] XmsXmsXmXmXmXmXfXfXfXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXm, where:

[0028] X represents ribonucleotide, I represents inosine-3'-phosphate, s represents thiophosphate linkage, Xm represents 2'-methoxy-substituted ribonucleotide, Xf represents 2'-fluorine-substituted ribonucleotide, and Xg represents the (S)-GNA form of ribonucleotide.

[0029] In some embodiments, the RNA inhibitor that inhibits AT3 gene expression further comprises a ligand, wherein the ligand is conjugated to the sense strand and / or the antisense strand.

[0030] In some embodiments, the RNA inhibitor that inhibits AT3 gene expression, wherein the ligand is conjugated to the 5' end and / or 3' end of the antisense strand.

[0031] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the ligand is conjugated to the 5' end and / or the 3' end of the sense strand.

[0032] In some embodiments, the RNA inhibitor of AT3 gene expression, or a pharmaceutically acceptable salt thereof, wherein the ligand is conjugated to the 5' end of the antisense strand, and the ligand is conjugated to the 3' end of the sense strand.

[0033] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the ligand is conjugated to the 3' end of the antisense strand, and the ligand is conjugated to the 5' end of the sense strand.

[0034] In some embodiments, the RNA inhibitor of AT3 gene expression, wherein the ligand is conjugated to the 5' end and the 3' end of the sense strand.

[0035] In some embodiments, the RNA inhibitor of AT3 gene expression, the ligand further comprises a targeting unit for enhancing uptake of the RNA inhibitor by hepatocytes.

[0036] In some embodiments, the RNA inhibitor of AT3 gene expression, the targeting unit is selected from monosaccharides and derivatives thereof.

[0037] In some embodiments, the RNA inhibitor of AT3 gene expression, the monosaccharide is selected from one or more of the following structures: mannose, galactose, D-arabinose, glucose, fructose, xylose, glucosamine, ribose.

[0038] In some embodiments, the RNA inhibitor of AT3 gene expression, the monosaccharide derivative is selected from mannose derivatives, galactose derivatives, glucose derivatives, ribose derivatives, and other derivatives.

[0039] In some embodiments, the RNA inhibitor of AT3 gene expression, the targeting unit is selected from galactose, galactosamine, N-acetylgalactosamine, and derivatives thereof.

[0040] In some embodiments, the RNA inhibitor of AT3 gene expression, the targeting unit is N-acetylgalactosamine and derivatives thereof.

[0041] In another aspect, the present application provides a pharmaceutical composition comprising the RNA inhibitor of AT3 gene expression, and further comprising a delivery vehicle, and / or a physiologically acceptable excipient and / or carrier and / or diluent.

[0042] In some embodiments, the delivery vehicle comprises a liposome.

[0043] In some embodiments, the delivery vehicle comprises a nanolipid.

[0044] In some embodiments, the use of the RNA inhibitor of AT3 gene expression and the pharmaceutical composition for the manufacture of a medicament for preventing or treating a disease or pathology or reducing the risk of a disease or pathology.

[0045] In some embodiments, the disease or pathology comprises a disease or pathology associated with elevated levels of AT3.

[0046] In some embodiments, the disease or pathology comprises hemophilia.

[0047] In another aspect, the present application provides a method of preventing or treating a disease, disorder or syndrome, the method comprising administering to a subject in need thereof an effective amount of the RNA inhibitor of AT3 gene expression, the pharmaceutically acceptable salt thereof or the pharmaceutical composition.

[0048] In some embodiments, wherein the RNA inhibitor of AT3 gene expression, the pharmaceutically acceptable salt thereof or the pharmaceutical composition is administered to the subject in a subcutaneous, intravenous, oral, rectal or intraperitoneal administration route.

[0049] In another aspect, the present application provides a method for inhibiting AT3 expression in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an effective amount of the RNA inhibitor of AT3 gene expression, the pharmaceutically acceptable salt thereof or the pharmaceutical composition.

[0050] In some embodiments, the RNA inhibitor comprises the sense strand, the antisense strand and the duplex described in Table 1-1.

[0051] In some embodiments, the RNA inhibitor comprises the sense strand, the antisense strand and the duplex described in Table 1-2.

[0052] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in conjunction with the drawings. Only the preferred embodiments of the application are shown and described in the drawings and specification. As will be realized by those skilled in the art, the application is capable of modifications in various obvious aspects, all without departing from the application as described and claimed herein. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature and explanations are made only for the purposes of assisting in understanding the application. DETAILED DESCRIPTION

[0053] The following detailed description of the application is provided as an example to enable those skilled in the art to practice the application. Other advantages in the application will be realized and appreciated by one of ordinary skill in the art.

[0054] Definitions of Terms

[0055] In the present application, the terms "iRNA," "RNAi agent," "iRNA agent," "RNA interference agent," "RNA inhibitor" are used interchangeably and generally refer to an agent comprising RNA as defined by the terms herein, and which can mediate the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). iRNAs modulate (e.g., inhibit) expression of the AT3 gene (NM_000488.4) in a cell (e.g., a cell in a subject such as a mammalian subject).

[0056] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind the endonuclease Argonaute 2 within RISC, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and are chemically modified. Design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al. (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as a single-stranded siRNA described herein or chemically modified by the methods described in Lima et al. (2012) Cell 150:883-894.

[0057] "AT3" as used herein refers to a particular polypeptide expressed in a cell. AT3 is also known as Serpinc1. The human AT3 mRNA transcript sequence can be found, for example, in GenBank Accession No. GI:254588059 (NM_000488, e.g., NM_000488.4). The mouse Serpinc1 mRNA sequence can be found, for example, in GenBank Accession No. GI:237874216 (NM_080844, e.g., NM_080844.5).

[0058] As used herein, the term "Serpinc 1" also refers to a particular polypeptide expressed in a cell from a naturally occurring DNA sequence variant of the Serpinc 1 gene, for example, a single nucleotide polymorphism of the Serpinc 1 gene. Numerous SNPs within the Serpinc 1 gene have been identified and can be found, for example, at NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov / snp). Non-limiting examples of SNPs within the Serpinc 1 gene can be found at NCBI dbSNP Accession Nos. rs677; rs5877; rs5878; rs5879; rs941988; rs941989; rs1799876; rs19637711; rs2008946; and rs2227586.

[0059] As used herein, a "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an AT3 gene, including mRNA that is a processing product of the primary transcript. 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 the portion of the nucleotide sequence of an mRNA molecule formed during transcription of an AT3 gene.

[0060] A target sequence can be from about 9-36 nucleotides in length, for example, about 15-30 nucleotides in length. For example, a target sequence can be from 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 nucleotides in length. The ranges and ranges of length cited above are also intended to be part of the present application.

[0061] In certain embodiments, "iRNA" as used herein is double-stranded RNA and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientation with respect to a target RNA (i.e., AT3 gene). In some embodiments of the application, double-stranded RNA (dsRNA) triggers the degradation of a target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0062] The duplex structure can be of any length that allows for the specific degradation of the desired target RNA by the RISC pathway, and can range in length from about 19 to 36 base pairs, e.g., about 19-30 base pairs, e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also intended to be part of this application. In certain embodiments, the iRNA agents of the application are dsRNAs comprising 15-23 nucleotides in each strand that interact with a target RNA sequence (e.g., AT3 gene) to direct cleavage of the target RNA. In certain embodiments, the iRNA of the application is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence (e.g., AT3 target mRNA sequence) to direct cleavage of the target RNA.

[0063] In the present application, the terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, siRNA, miRNA, shRNA, RNAi agent, and primer. A polynucleotide can be modified at one or more bases, sugars and / or phosphates, or substituted with any of a variety of moieties known or described herein. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be modified after polymerization, such as by conjugation with a labeling component. The term encompasses double- and single-stranded forms of polynucleotides. Unless otherwise indicated or required by context, any embodiment of the present application as a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms, known or predicted, which can exist in equilibrium.

[0064] In the present application, the term "target nucleic acid" or "target sequence" generally refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an AT3 gene, including mRNA that is a processing product of the primary transcription product. The target portion of the sequence should be at least long enough to be a substrate for iRNA-directed cleavage at or near the position of the portion of the nucleotide sequence of an mRNA molecule formed during transcription of an AT3 gene. In one embodiment, the target sequence is within the protein coding region of AT3. The target sequence can be about 19-36 nucleotides in length, for example, preferably about 19-30 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths also are part of the present application.

[0065] In the present application, the term "nucleotide sequence" generally refers to a series or order of nucleobases, nucleotides and / or nucleosides, whether modified or unmodified, described by a series of letters using the standard nucleotide nomenclature and the symbol table for modified nucleotides described herein.

[0066] In the present application, the term "oligonucleotide" generally refers to a polymer that is composed of a plurality of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs thereof) linked by phosphodiester bonds (or related structural variants or synthetic analogs thereof). Thus, while the term "oligonucleotide" generally refers to a polymer of nucleotide residues and the linkages between them that are naturally occurring, it is to be understood that the scope of this term also includes various analogs, including but not limited to: peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methylphosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can depend on the particular application. Oligonucleotides are generally short in length, typically about 10-30 nucleotide residues, but the term can also refer to molecules of any length, although the terms "polynucleotide" or "nucleic acid" are generally used for larger oligonucleotides.

[0067] In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides. The term "modified oligonucleotide" generally means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0068] In the present application, the term "modified nucleoside" generally means a nucleoside comprising at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. A modified nucleoside comprises a modified sugar moiety and / or a modified nucleobase.

[0069] In the present application, the term "nucleobase" generally means a heterocyclic pyrimidine or purine compound that is a component of all nucleic acids and includes adenine (a), guanine (g), cytosine (c), thymine (t), and uracil (u). A nucleotide can include a modified nucleotide or nucleotide mimic, an abasic site (Ab or X), or a surrogate moiety in place of. As used herein, "nucleobase sequence" generally means the order of consecutive nucleobases independent of any sugar, linkage, or nucleobase modification. The term "unmodified nucleobase" or "naturally occurring nucleobase" generally means a naturally occurring heterocyclic nucleobase of RNA or DNA: purine bases adenine (a) and guanine (g); and pyrimidine bases thymine (t), cytosine (c) (including 5-methyl c), and uracil (u). "Modified nucleobase" generally means any nucleobase that is not a naturally occurring nucleobase.

[0070] In the present application, the term "sugar moiety" generally means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside. The term "naturally occurring sugar moiety" generally means a furanoribosyl group as found in naturally occurring RNA or a deoxyfuranoribosyl group as found in naturally occurring DNA. "Modified sugar moiety" means a substituted sugar moiety or a sugar surrogate.

[0071] In the present application, the term "internucleoside linkage" generally means a covalent linkage between adjacent nucleosides in an oligonucleotide. A "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage. A "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0072] In the present application, the term "antisense oligonucleotide" refers to a single-stranded oligonucleotide molecule having a nucleobase sequence complementary to a corresponding segment of a target nucleic acid (e.g., a genomic sequence of interest, a pre-mRNA, or an mRNA molecule). In certain embodiments, an antisense oligonucleotide is 12 to 30 nucleobases in length. In certain embodiments, an antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence complementary to a sequence of a target nucleic acid, such as an AT3 polynucleotide.

[0073] In the present application, the term "antisense strand" generally refers to a strand of an RNA inhibitor (e.g., a dsRNA) that includes a region of substantial complementarity to a target sequence. As used herein, the term "region of complementarity" generally refers to a region on an antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, mismatches can be internal or at the terminal regions of the molecule. Generally, the most tolerated mismatches are at the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' terminus and / or the 3' terminus.

[0074] In the present application, the term "sense strand" (S) generally refers to a strand of an RNA inhibitor that includes a region of substantial complementarity to a region that is the term antisense strand as defined herein. The "sense" strand is sometimes referred to as the "sense" strand, the "passenger" strand, or the "anti-guide" strand. By virtue of their sequences, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications on the sense strand or using a 5' end cap.

[0075] In the present application, the term "complementary" when used to describe a first nucleotide sequence (e.g., an RNAi agent sense strand or AT3 mRNA) with respect to a second nucleotide sequence (e.g., an RNAi agent antisense strand) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds) and form a duplex or double helix structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetics, as long as the above requirements regarding their hybridization ability are fulfilled. "Complementary" does not necessarily have nucleobase complementarity at every nucleoside. Rather, some mismatches can be tolerated.

[0076] In the present application, the term "fully complementary" generally means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence. As used herein, "partially complementary" generally means that in a pair of hybridized nucleobase sequences, at least about 70% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. As used herein, "substantially complementary" generally means that in a pair of hybridized nucleobase sequences, at least about 90% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The terms "complementary," "fully complementary," and "substantially complementary" as used herein can be used with respect to base pairing between a sense strand and an antisense strand of an RNA inhibitor or between an antisense strand of an RNA inhibitor and a sequence of an AT3 mRNA. Sequence identity or complementarity is independent of modification. For purposes of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.

[0077] In the present application, the term "homologous" or "homology" generally refers to the number of nucleotides of a subject nucleic acid sequence that have matched the same nucleotides of a reference nucleic acid sequence, typically determined by a sequence analysis program (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877), or by visual inspection. As used herein, the term "complete homology" or "completely homologous" generally refers to complete (100%) homology or "identity" between a reference sequence and a subject nucleic acid sequence. As used herein, the term "substantially homologous" or "substantial homology" generally refers to a subject sequence sharing at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the homologous nucleotides at the same nucleotide positions in a reference sequence.

[0078] In the present application, the term "ligand" generally refers to any compound or molecule that is capable of covalently or otherwise chemically binding to a biologically active substance, such as an oligonucleotide. In certain embodiments, a ligand is capable of directly or indirectly interacting with another compound, e.g., a receptor, which can be present on the surface of a cell, or alternatively can be an intracellular and / or intercellular receptor, the interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or binding.

[0079] In the present application, the terms "induce," "inhibit," "enhance," "elevate," "increase," "decrease," "reduce," and the like generally indicate a quantitative difference between two states. For example, "an amount effective to inhibit the activity or expression of AT3" means that the level of AT3 activity or expression in a treated sample will be lower than the level of AT3 activity or expression in an untreated sample. The terms are applicable, for example, to expression levels and activity levels. The terms "decrease" and "reduce" are used interchangeably and generally mean any change that is less than the original. "Decrease" and "reduce" are relative terms, requiring a comparison between before and after measurements. "Decrease" and "reduce" include complete depletion.

[0080] In certain embodiments, the term "decrease" can be an overall decrease of about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% in the expression level / amount of a gene, gene product, e.g., a protein, or a biomarker in a first sample as compared to the expression level / amount of the corresponding gene, gene product, e.g., a protein, or a biomarker in a second sample, as detected by standard methods known in the art, such as those described in the present application. In certain embodiments, the term "decrease" refers to a decrease in the expression level / amount of a gene or biomarker in a first sample, wherein the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold of the expression level / amount of the corresponding gene or biomarker in a second sample. In certain embodiments, the first sample is a sample obtained from a subject, and the second sample is a reference sample.

[0081] In the present application, the term "expression" generally means the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of a 5'-cap), and translation.

[0082] In the present application, the term "pharmaceutically acceptable" generally refers to a nontoxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such formulations can typically contain salts, excipients, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations can also typically include compatible solid or liquid fillers, diluents, or encapsulating material that are suitable for administration to humans. When used in medicine, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can conveniently be used to prepare pharmaceutically acceptable salts, and are not excluded from the scope of the present application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts derived from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0083] In the present application, the term "lipid nanoparticle" or "LNP" generally refers to a vesicle comprising a lipid layer that encapsulates a pharmacologically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described, for example, in Chinese Patent No. CN103189057B, the entire contents of which are incorporated herein by reference.

[0084] In the present application, the term "preventing and / or treating" includes not only preventing and / or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and / or alleviating one or more symptoms associated with a disease, reducing the severity and / or duration of a disease and / or any symptoms associated therewith and / or preventing further increases in the severity of a disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and generally any pharmacological effect that is beneficial to the patient being treated. The RNAi agent or pharmaceutical composition of the present application forms a viable therapeutic agent need not achieve a complete cure or eradication of any symptom or manifestation of a disease. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but need not eliminate every manifestation of a disease to be considered a useful therapeutic agent. Similarly, a therapeutic agent prophylactically administered forms a viable prophylactic agent need not be completely effective in preventing the onset of a disorder. It is sufficient to simply reduce the impact of a disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another therapy, or by producing another beneficial effect), or to reduce the likelihood of a disease occurring or worsening.

[0085] In the present application, the terms "disease" or "disorder" are used interchangeably and generally refer to any deviation from the normal state of a subject, for example, any change in the state of the body or of some of its organs, impairs or interferes with the performance of the functions, and / or causes symptoms such as discomfort, dysfunction, pain, or even death in a person affected or exposed to it. A disease or disorder can also be referred to as a distemper, an ailing, an ailment, a malady, a disorder, a sickness, an illness, a complaint.

[0086] In the present application, the term "administering" generally refers to introducing a pharmaceutical preparation of the present application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting a cell, organ or tissue with the drug can be employed. The administration can include, without limitation, intravenous, intra-arterial, intranasal, intra-abdominal, intramuscular, subcutaneous transdermal or oral. The daily dose can be divided into one, two or more doses of a suitable form for administration at one, two or more times during a certain period of time.

[0087] In the present application, the term "bleeding disorder" is a disease or condition that results in poor clotting and / or excessive bleeding. The bleeding disorder can be an inherited disorder (such as hemophilia or von Willebrand disease), or an acquired disease associated with, for example, disseminated intravascular coagulation, preeclampsia, vitamin K deficiency, autoimmune disease, inflammatory bowel disease, ulcerative colitis, a skin disorder (e.g., psoriasis, pemphigus), a respiratory disease (e.g., asthma, chronic obstructive pulmonary disease), an allergic drug reaction, for example, as a result of drug (e.g., aspirin, heparin, and warfarin) therapy, diabetes, acute hepatitis B infection, acute hepatitis C infection, a malignancy or solid tumor (e.g., prostate, lung, colon, pancreatic, gastric, biliary, head and neck, cervical, breast, melanoma, renal, and / or hematological malignancy). In one embodiment, the inherited bleeding disorder is hemophilia, e.g., hemophilia A, B, or C. In one embodiment, a subject having an inherited bleeding disorder (e.g., hemophilia) has developed an inhibitor, e.g., an alloantibody inhibitor, in place of a clotting therapy and is referred to herein as an "inhibitor subject." In one embodiment, the inhibitor subject has hemophilia A. In another embodiment, the inhibitor subject has hemophilia B. In yet another embodiment, the inhibitor subject has hemophilia C.

[0088] In the present application, the term "contacting" generally refers to bringing two or more different types of substances together in any order, in any manner, and for any length of time. The contacting can occur in vivo, ex vivo, or in vitro. In certain embodiments, it can refer to bringing the RNAi agent or composition of the present application into direct contact with a cell or tissue. In other embodiments, the term refers to bringing the RNAi agent or composition of the present application into indirect contact with a cell or tissue. For example, the methods of the present application include methods in which a subject is contacted with the RNAi agent or composition of the present application, and then the RNAi agent or composition contacts the cell or tissue by diffusion or any other active or passive transport process known in the art by which the compound circulates in the body.

[0089] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent generally refers to any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability due to the disease). A "preventive effective amount" or "preventive effective dose" of a drug generally refers to an amount of drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of disease progression or relapse, inhibits the development or relapse of the disease. The ability of a therapeutic agent or preventive agent to promote disease remission or inhibit disease progression or relapse can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, predicting efficacy in humans in animal model systems, or by measuring the activity of the agent in an in vitro assay. In some embodiments, "effective amount" refers to an amount of RNA inhibitor that produces the expected pharmacological, therapeutic, or preventive outcome.

[0090] In this application, the term "subject" generally refers to a human or non-human animal (including mammals) requiring diagnosis, prognosis, improvement, prevention, and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models.

[0091] In this application, the terms “comprising,” “including,” “having,” “may,” “containing,” and variations thereof are generally intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The term “composed of” generally indicates that no other components can exist (or similarly, features, integers, steps, etc.). Unless the context clearly specifies otherwise, the singular forms such as “a,” “an,” “the” in English, and “a,” “a,” “the,” and “the” in Chinese generally include the plural form of the things referred to.

[0092] In this application, the term "about" generally means large, roughly, or around. When the term "about" is used to refer to a range of values, a cutoff value or a specific value is used to indicate that the stated value may differ from the listed value by up to 10%. Therefore, the term "about" can be used to cover variation of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from a specific value.

[0093] It should be understood that the term "at least" preceding a number or series of numbers includes the number adjacent to the term "at least" and all subsequent numbers or integers logically included, as will be apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 of the 21 nucleotides in a nucleic acid molecule" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" precedes a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0094] It should be understood that "no more than" or "less than" as used herein refers to the value or integer adjacent to the phrase and logically lower, as will be apparent from the context. For example, a duplex having "no more than 3 nucleotides" of overhang has 3, 2, 1, or 0 nucleotides of overhang. When "no more than" precedes a series of numbers or a range, it should be understood that "no more than" can modify each number in the series or range. Ranges as used herein are inclusive of both the lower and upper limits.

[0095] DETAILED DESCRIPTION

[0096] Antisense strand and sense strand

[0097] In one aspect, the application provides an RNA inhibitor that inhibits expression of an AT3 gene.

[0098] In certain embodiments, the RNA inhibitor comprises a single-stranded oligonucleotide or a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of an AT3 gene in a cell, such as a cell of a subject (e.g., a mammal). The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of an mRNA formed during expression of the AT3 gene. The region of complementarity is about 12-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, or 12 nucleotides in length).

[0099] A dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure (region of complementarity) under conditions in which the dsRNA is used. One strand (antisense strand) of the dsRNA comprises a region of complementarity that is substantially complementary, and typically fully complementary, to a target sequence. The target sequence can be derived from a sequence of an mRNA formed during expression of an AT3 gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand such that, when combined under suitable conditions, the two strands can hybridize and form a duplex structure. Typically, the duplex structure is 12 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 12 to 30 nucleotides in length.

[0100] In certain embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 24 to about 30 nucleotides in length. Generally, the length of the dsRNA is sufficient to serve as a substrate for Dicer enzyme. For example, it is well known in the art that dsRNAs greater than about 21-23 nucleotides in length can serve as a substrate for Dicer. It is also understood by those skilled in the art that the region of RNA targeted for cleavage is typically a portion of a larger RNA molecule, often an mRNA molecule. A "portion" of a target is a contiguous nucleotide of an mRNA target that is of sufficient length to allow it to serve as a substrate for RNAi directed cleavage (i.e., cleavage via the RISC pathway).

[0101] It is also understood by those skilled in the art that the duplex region is the primary functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20 base pairs. Thus, in one embodiment, a RNA molecule or complex of RNA molecules having a duplex region in excess of 30 base pairs is a dsRNA to the extent that it is a functional duplex (e.g., 15-30 base pairs) targeted for cleavage of a desired RNA.

[0102] In certain embodiments, there is at least about 80% base complementarity between the sense and antisense strands.

[0103] In certain embodiments, the sense and antisense strands are each independently 15-30 nucleotides.

[0104] In certain embodiments, the sense and antisense strands are each independently 17-25 nucleotides.

[0105] In certain embodiments, the sense and antisense strands are each independently 19-23 nucleotides.

[0106] In some embodiments, the sense strand is selected from any one of SEQ ID NOs: 304-642, 1002-1060, or a sequence differing by no more than 3 nucleotides therefrom.

[0107] In some embodiments, the sense strand of the RNA inhibitor is selected from Table 1-1 or a sequence differing by one, two, or three nucleotides from each sequence in Table 1-2, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides.

[0108] In some embodiments, the antisense strand is selected from any one of SEQ ID NOs: 1-303, 922-1001, or at least 15 contiguous nucleotides differing by no more than 3 nucleotides therefrom, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides.

[0109] In some embodiments, the antisense strand of the RNA inhibitor is selected from Table 1-1 or at least 15 contiguous nucleotides differing by one, two, or three nucleotides from each sequence in Table 1-2, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides.

[0110] In some embodiments, both the sense and antisense strands of the RNA inhibitor have 3' overhangs of 2-3 nucleotides in length, or the sense strand has a 3' overhang of 2-3 nucleotides in length, or the antisense strand has a 3' overhang of 2-3 nucleotides in length.

[0111] In some embodiments, the RNA inhibitor has a 3' overhang of 2 nucleotides in length only on the antisense strand.

[0112] In some embodiments, the sense and antisense strands of the RNA inhibitor are selected from the sequences in Table 1-1 or at least 15 contiguous nucleotides differing by one, two, or three nucleotides from each sequence in Table 1-2, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides.

[0113] Table 1-1 Sense and Antisense Strands of RNA Inhibitors

[0114] wherein A is adenosine-3'-phosphate, C is cytidine-3'-phosphate, G is guanosine-3'- phosphate, U is uridine-3'-phosphate, I is inosine-3'-phosphate, and dT represents 2'- deoxythymidine-3'-phosphate.

[0115] Modified Nucleotides

[0116] To enhance the stability of the above-described RNA inhibitors in vivo, the sense and antisense strands of the above-described RNA inhibitors can be modified without affecting or even enhancing their activity, wherein the nucleotides can have a modifying group, and the entire strand or part of the strand can be modified. In certain embodiments, one or more nucleotides of the sense and / or antisense strand are modified to form a modified nucleotide.

[0117] All the nucleotides in the small activating nucleic acid molecules described herein can be natural or unmodified nucleotides, or at least one of the nucleotides can be a chemically modified nucleotide, which is one or a combination of the following modifications:

[0118] (1) modification of the phosphodiester bond of the nucleotides in the nucleotide sequence of the small activating nucleic acid molecules;

[0119] (2) modification of the 2’-OH of the ribose in the nucleotide sequence of the small activating nucleic acid molecules;

[0120] (3) modification of the base in the nucleotide sequence of the small activating nucleic acid molecules.

[0121] The chemical modifications are well known to those skilled in the art, and the modification of the phosphodiester bond refers to modification of the oxygen in the phosphodiester bond, including phosphorothioate modification and boranophosphonate modification. Both modifications can stabilize the siRNA structure, and maintain high specificity and high affinity of base pairing.

[0122] The modification of the ribose refers to modification of the 2’-OH in the pentose of the nucleotide, i.e., introducing certain substituents at the hydroxyl position of the ribose, such as 2’-fluoro modification, 2’-oxymethyl modification, 2’-oxaethylenemethoxyl modification, 2,4’-dinitrophenol modification, locked nucleic acid (LNA), 2’-amino modification, 2’-deoxy modification.

[0123] The modification of the base refers to modification of the base of the nucleotide, such as 5’-bromouracil modification, 5’-iodouracil modification, N-methyluracil modification, 2,6-diaminopurine modification.

[0124] In some embodiments, wherein the modification of the ribose includes fluorine substitution and / or methoxy substitution of the 2’-OH.

[0125] In some embodiments, wherein the modification of the ribose includes UNA, LNA or GNA.

[0126] The term "LNA" denotes bicyclic nucleoside analogs comprising a C2*-C4* biradical (bridge), and is referred to as "locked nucleic acid". It can mean an LNA monomer, or, when used in the context of "LNA oligonucleotide", LNA means an oligonucleotide containing one or more such bicyclic nucleotide analogs. In certain aspects, the bicyclic nucleoside analog is an LNA nucleotide, and these terms can thus be used interchangeably, and in such embodiments, both are characterized by the presence of a linking group (such as a bridge) between C2’ and C4’ of the ribose sugar ring.

[0127] UNA (unlocked nucleic acid) structures are similar to RNA but lack the C2 and C3 chemical bonds of the ribose ring, and have the following structure:

[0128] where B is a base.

[0129] GNA (glycerol nucleic acid) is a chemical substance similar to DNA or RNA, but with a different composition and does not exist in any currently known organism in nature. GNA contains a three-carbon acyclic propanediol (1,2-propanediol) backbone instead of (deoxy)ribose of DNA and RNA, constituting the simplest structure of chemically stable nucleic acids. The structure of S-(GNA) is as follows.

[0130] where B is a base.

[0131] XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXmsXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm XmsXmsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXfXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmXm XmsXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmTgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm [Xvm]sXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXuXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmXmTuXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXm PXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXdXXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfdTXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmdTXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmdXXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm PXmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXmXmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXmXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm,

[0132] wherein:

[0133] X represents a ribonucleotide, e.g. A, G, C, T, U, s represents a phosphorothioate linkage, Xm 2’-methoxy substituted ribonucleotide, Xf represents a 2’-fluoro substituted ribonucleotide, Xg represents a (S)-GNA form of a ribonucleotide, dT represents a 2’-deoxythymidine-3’-phosphate, dA represents a 2’-deoxyadenosine-3’-phosphate, P represents a phosphorylation, [Uvm] represents a 5’-(E)-vinylphosphonate-2’-Ome-U.

[0134] XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXf XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfTgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmTgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXm XmsXfsXmXmXmXfXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXmsXmXmXmXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmImXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXmXmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfImXfXfXfXmImXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmImXm XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfImXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmIfXmXfXmXfXmXf XfsXmsXfXmXfXmXfImXfXfXfXmIfXmXfXmXfXmXfXmXfXmsXmsXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXfXfXmXmXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXfXfXmXmXmXmXmXmXmXmXmXmXmXm

[0135] X represents a ribonucleotide, I represents inosine-3'-phosphate, s represents a phosphorothioate linkage, Xm 2'methoxy substituted ribonucleotide, Xf represents a 2'fluoro substituted ribonucleotide, Xg represents a (S)-GNA form of a ribonucleotide.

[0136] The modified RNA inhibitors can include as shown in Table 1-2.

[0137] Table 1-2

[0138] wherein s represents a phosphorothioate (PS) linkage. For example, a phosphorothioate linkage is used to replace the original phosphodiester linkage.

[0139] m represents a 2'-O-methyl modification of the nucleotide at the preceding position. Am represents 2'-O-methyladenosine-3'-phosphate, Cm represents 2'-O-methylcytidine-3'-phosphate, Gm represents 2'-O-methylguanosine-3'-phosphate, Um represents 2'-O-methyluridine-3'-phosphate, Im represents 2'-O-methylinosine-3'-phosphate.

[0140] f represents a 2'-fluoro modification of the nucleotide at the preceding position. Af represents 2'-fluoro adenosine-3'-phosphate, Cf represents 2'-fluoro cytosine-3'-phosphate, Gf represents 2'-fluoro guanosine-3'-phosphate, Uf represents 2'-fluoro uridine-3'-phosphate, If represents 2'-fluoro inosine-3'-phosphate.

[0141] d represents a 2'-deoxy modification of the nucleotide at the following position. dT represents 2'-deoxythymidine-3'-phosphate, dA represents 2'-deoxyadenosine-3'-phosphate, dG represents 2'-deoxyguanosine-3'-phosphate. dC represents 2'-deoxycytidine-3'-phosphate.

[0142] A represents adenosine-3'-phosphate, C represents cytidine-3'-phosphate, G represents guanosine-3'-phosphate, U represents uridine-3'-phosphate, I represents inosine-3'-phosphate, Ag represents (S)-GNA-A-phosphate, Cg represents (S)-GNA-C-phosphate, Gg represents (S)-GNA-G-phosphate, Ug represents (S)-GNA-U-phosphate, Tg represents (S)-GNA-T-phosphate, Uu represents UNA-U-phosphate, Tu represents UNA-T-phosphate, L96 represents GalNAc, VP represents 5'-(E)-vinylphosphonate, [Uvm] represents 5'-(E)-vinylphosphonate-2'-Ome-U. P represents phosphorylation or phosphate group, IB represents 3',5' inverted deoxy abasic nucleotide.

[0143] In the present invention, invAB modification refers to the linkage of an inverted abasic nucleotide at the 5' end or 3' end of a nucleotide.

[0144] Exemplary, Structure after invAB modification:

[0145] RNA inhibitors conjugated to ligands

[0146] Another aspect of the RNA inhibitors of the application relates to the manner in which the interfering nucleic acid is conjugated to a ligand to enhance the stability, activity, cellular distribution, or cellular uptake of the RNAi agent.

[0147] In certain embodiments, the distribution, targeting, or stability of the RNA inhibitor is altered by the introduction of a ligand for a receptor of the target tissue. For example, a specific ligand can provide enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cellular or organ compartment, a body tissue, an organ, or a region)) compared to the species in the absence of the ligand.

[0148] The ligand can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, 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.

[0149] The ligand can also include a targeting group, such as a cell or tissue targeting agent that binds to a specified cell type, such as a kidney cell, e.g., a lectin, a glycoprotein, a lipid, or a protein, e.g., an antibody. The targeting group can be a thyrotropin, a melanotropin, a lectin, a glycoprotein, a surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, a N-acetyl-galactosamine, a N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, a transferrin, a bisphosphate, a polyglutamic acid, a polyaspartic acid, a lipid, a cholesterol, a steroid, a bile acid, a folate, a vitamin B12, a vitamin A, a biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., a N-acetyl-galactosamine.

[0150] The sense and antisense strands of the RNA inhibitors of the application can be conveniently and routinely made by the skilled consumer using the well-known techniques of solid phase synthesis. Any other method known in the art for such synthesis, such as solution synthesis or fermentation, can additionally or alternatively be used. The use of similar techniques to prepare other oligonucleotides (such as phosphorothioate and alkylated derivatives) is known.

[0151] In certain embodiments, in addition to the standard nucleoside phosphoramidite monomers that are commercially available and routinely used in oligonucleotide synthesis, as well as non-standard nucleoside phosphoramidite monomers, the oligonucleotides or linked nucleotides of the application can be synthesized by an automated synthesizer using phosphoramidite methodology derived from ligand-nucleoside phosphoramidite monomers.

[0152] In certain embodiments, the ligand conjugation described herein is coupled to the 5' end and / or 3' end of the antisense strand, and / or the 5' end and / or 3' end of the sense strand.

[0153] For example, the ligand structure can be coupled to the 5' end and / or 3' end of the sense strand; or the ligand structure can be coupled to the 5' end of the antisense strand and the ligand structure is coupled to the 3' end of the sense strand; or the ligand structure can be coupled to the 3' end of the antisense strand and the ligand is coupled to the 5' end of the sense strand; or the ligand structure is coupled to the 5' end and 3' end of the sense strand; or the ligand structure is coupled to the 3' end of the sense strand.

[0154] In certain embodiments, the ligand described herein comprises a L96 structure, as shown in the following structural formula:

[0155] Pharmaceutical compositions

[0156] The present application also includes pharmaceutical compositions comprising the RNA inhibitors or pharmaceutically acceptable salts thereof of the present application.

[0157] In one embodiment, provided herein is a pharmaceutical composition comprising an RNA inhibitor described herein and a pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical composition comprising the RNA inhibitor can be used for the prevention and / or treatment of AT3 -associated disorders, for example, hemophilia, including hemophilia A, hemophilia B. Such pharmaceutical compositions are formulated depending on the mode of delivery. One example regimen is to formulate the composition for systemic administration by parenteral delivery, for example, subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the present application can be administered at a dose sufficient to inhibit the expression of the AT3 gene.

[0158] A pharmaceutically acceptable "excipient" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be a liquid or a solid and is selected with respect to the intended mode of administration so as to provide for the desired bulk, consistency, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition. The RNA inhibitor can be delivered in a manner that targets a particular tissue (e.g., hepatocytes).

[0159] In certain embodiments, the pharmaceutical composition further comprises a delivery vehicle (e.g., a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system).

[0160] In certain embodiments, the delivery vehicle comprises a liposome.

[0161] In certain embodiments, wherein the delivery vehicle comprises a nanolipid, which is capable of forming a liposome-nucleic acid nanoparticle with the nucleic acid molecule.

[0162] Use

[0163] In another aspect, the application provides use of the aforementioned RNA inhibitor of AT3 gene expression, or a pharmaceutically acceptable salt thereof, and the aforementioned pharmaceutical composition in the manufacture of a medicament for preventing or treating, or reducing the risk of, a disease or pathology.

[0164] In certain embodiments, wherein the disease or pathology comprises a disease or pathology associated with elevated levels of AT3.

[0165] In certain embodiments, wherein the disease or pathology comprises a bleeding disorder.

[0166] In certain embodiments, wherein the disease or pathology comprises intraoperative bleeding.

[0167] In certain embodiments, wherein the disease or pathology comprises hemophilia, including hemophilia A and / or hemophilia B.

[0168] In certain embodiments, wherein the disease or pathology comprises a disease or pathology associated with elevated levels of AT3.

[0169] In another aspect, the application provides a method of preventing or treating a disease, disorder, or syndrome, comprising administering to a subject in need thereof an effective amount of the aforementioned RNA inhibitor of AT3 gene expression, a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0170] In certain embodiments, wherein the RNA inhibitor of AT3 gene expression, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered to the subject in a subcutaneous, intravenous, oral, rectal, or intraperitoneal administration route.

[0171] In another aspect, the application provides a method for inhibiting AT3 mRNA or protein expression in a cell, tissue, or subject, comprising administering to a subject in need thereof an effective amount of the aforementioned RNA inhibitor of AT3 gene expression, a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0172] In certain embodiments, wherein the cell is a hepatocyte.

[0173] In certain embodiments, wherein the tissue is liver tissue.

[0174] In certain embodiments, wherein the cell and tissue are ex vivo.

[0175] The cells suitable for treatment using the methods of the application can be any cell that expresses an AT3 gene, for example, a liver cell, a brain cell, a gall bladder cell, a heart cell, or a kidney cell, but preferably a liver cell. The cells suitable for use in the methods of the application can be mammalian cells, and the RNAi agent inhibits expression of an AT3 gene (e.g., a human, primate, non-primate, or rat AT3 gene) by at least about 50% when contacted with the cell expressing the AT3 gene, for example, as determined by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence assays, Western blots, or flow cytometry techniques.

[0176] The term "inhibit" as used herein can be used interchangeably with "reduce," "decrease," "silence," "down-regulate," "suppress," and other similar terms, and includes any level of inhibition. Expression of an AT3 gene can be evaluated in terms of the level or change in level of any variable associated with AT3 gene expression, for example, AT3 mRNA levels or AT3 protein levels. This level can be analyzed in a single cell or in a population of cells, including, for example, a sample derived from a subject. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more variables associated with AT3 expression as compared to a control level. The control level can be any type of control level employed in the art, for example, a pre-dose baseline level or a level measured from a similar subject, cell, or sample that has never been treated or treated with a control, such as, for example, a buffer control or an inactive agent control.

[0177] Inhibition of AT3 gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or population of cells in which the AT3 gene is transcribed and which has been treated (e.g., by contacting one or more cells with an RNAi agent of the application, or by administering an RNAi agent of the application to a subject in which the cells are present) such that AT3 gene expression is inhibited, as compared to a second cell or population of cells that is essentially identical to the first cell or population of cells but has not been so treated (a control cell that has not been treated with an RNAi agent or has not been treated with an RNAi agent targeting the gene of interest). In preferred embodiments, inhibition is evaluated in a cell line that expresses high levels of AT3 using the methods provided in the Examples using appropriate concentrations of siRNA, and the level of mRNA in the intervened cell is expressed as a percentage of the level of mRNA in the non-intervened control cell.

[0178] In other embodiments, inhibition of AT3 gene expression can be evaluated by a decrease in a parameter that is functionally associated with AT3 gene expression, for example, AT3 protein levels in the blood or serum of a subject. AT3 gene silencing can be determined in any cell that expresses AT3 (endogenous or exogenous from an expression construct) and by any assay known in the art.

[0179] Inhibition of AT3 protein expression can be represented by a decrease in the level of AT3 protein expressed in cells or cell populations or in subject samples (e.g., protein levels in blood samples derived from the subject). As described above, for the evaluation of mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or a change in protein levels in subject samples (e.g., blood or serum derived from it).

[0180] Control cells, cell populations, or subject samples that can be used to evaluate AT3 gene inhibition include cells, cell populations, or subject samples that have not been exposed to the RNAi agent of this application. For example, control cells, cell populations, or subject samples may be derived from a single subject (e.g., a human or animal subject) prior to treatment with the RNAi agent or from an appropriately matched population of controls.

[0181] The level of AT3 mRNA expressed in cells or cell populations can be determined using any method known in the art for evaluating mRNA expression. For example, qRT-PCR can be used to evaluate a decrease in gene expression. A decrease in protein production can be evaluated using any method known in the art, such as ELISA. In some embodiments, a liver biopsy sample is used as tissue material to monitor decreased AT3 gene or protein expression. In other embodiments, a blood sample is used as a subject sample to monitor decreased AT3 protein expression.

[0182] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0183] Example

[0184] Example 1 Sequence Synthesis

[0185] The sense and antisense strand sequences of siRNA were synthesized using a mature solid-phase synthesis method based on phosphoramide chemistry on a Dr. Oligo 48 / Syn-HCY-12P oligonucleotide synthesizer. Oligonucleotide chain growth was achieved through a four-step cycle: deprotection, condensation, capping, and oxidation or sulfidation steps for adding each nucleotide. Synthesis was performed on a controllable porous glass (CPG) system. ) on solid supports made. The phosphoramidite monomers used were all purchased from commercial sources. For siRNAs used in in vitro screening, synthesis was performed on a Dr. Oligo 48 oligonucleotide synthesizer at a 1 pmol scale; for siRNAs used in in vivo testing, synthesis was performed on a Syn-HCY-12P oligonucleotide synthesizer at a 5 pmol or greater scale. In the case of GalNAc ligand attachment at the 3'-end of the sense strand, CPG solid supports with GalNAc ligand attached were used. Trichloroacetic acid (TCA) in 3% dichloromethane was used for deprotection of the 4,4'-dimethoxytrityl protecting group (DMT). 5-ethylthio-1H-tetrazole was used as the activator. I2 in THF / Py / H2O and 3-amino-1,2,4-dithiazole-5-thione (XH) in pyridine were used for oxidation and sulfurization reactions, respectively. After the final solid phase synthesis step, the solid support-bound oligomer was cleaved and the protecting groups were removed by treatment with 28% ammonium hydroxide solution. The crude single-stranded product was further purified by ion pair reverse phase HPLC (IP-RP-HPLC).

[0186] For sequences used in in vitro screening, the purified single-stranded oligonucleotide product from IP-RP-HPLC can be used directly for annealing.

[0187] For siRNAs used in in vivo testing, the purified single-stranded oligonucleotide product from IP-RP-HPLC was dissolved in 1.0 M NaOAc and precipitated by the addition of ice-cold EtOH, which was converted to the sodium salt. Annealing of the sense and antisense oligonucleotides to form the double-stranded siRNA product was performed by equimolar complementation in water, which was lyophilized to provide a fluffy white solid.

[0188] Example 2 In vitro screening - dual luciferase assay

[0189] The principle of dual luciferase assay is to clone the regulatory elements of the gene of interest upstream of the Firefly luciferase gene to construct a luciferase reporter plasmid. Then the cells are transfected, lysed after appropriate stimulation or treatment, and the luciferase activity is measured. The effect of stimulation before and after or different stimulations on the regulatory elements of interest is judged by the level of luciferase activity. At the same time, in order to reduce the influence of internal changes on the accuracy of the experiment, the plasmid with Renilla luciferase gene is used as a control plasmid to co-transfect the cells with the reporter gene plasmid, providing an internal control for transcriptional activity, so that the test results are not disturbed by changes in experimental conditions.

[0190] The specific experimental method is as follows:

[0191] Day 1: Cell plating

[0192] Discard the supernatant PBS and wash once for adherent cells, trypsinize and terminate digestion with complete medium (suspension cells do not need this step), slowly blow and transfer to centrifuge tube. Centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend with medium and count. Configure cells to the corresponding concentration, blow evenly, take 100 μL and add to the 96-well enzyme-labeled plate, and incubate in a 37°C, 5% CO2 incubator for 24 h.

[0193] Day 2: Transfection

[0194] After 24 h of cell plating, discard the original medium in the 96-well enzyme-labeled plate for adherent cells, add 150 μL of fresh medium to each well (for suspension cells, centrifuge and discard the original medium and replace it with fresh medium).

[0195] Configure the transfection system (preheat OPTI-MEM in a 37°C water bath in advance):

[0196] A: psiCHECK2 plasmid / siRNA solution: 100 ng of psiCHECK2 plasmid / well and different concentrations of siRNA (set 4 concentrations, 10 nM starting, 10-fold dilution), dissolved in OPTI-MEM, total volume 25 μL, mix well;

[0197] B: Lip 2000 solution: 0.4 μL of Lip 2000 reagent / well, dissolved in OPTI-MEM, total volume 25 μL, mix well and incubate at room temperature for 5 min;

[0198] Mix A (psiCHECK2 plasmid / siRNA solution) and B (Lip 2000 solution), set up negative control wells (only transfect psiCHECK2 plasmid) and background control wells (wells containing cells) at the same time, incubate at room temperature for 18 min, then add 50 μL of mixed solution to each well of the 96-well enzyme-labeled plate, and incubate in a 37°C, 5% CO2 incubator for 24 h.

[0199] Day 3: Dual luciferase activity detection

[0200] After 24 h of transfection, aspirate the cell culture medium for adherent cells (for suspension cells, centrifuge and discard the supernatant), add 100 μL of lysis buffer (thawed in advance) to each well and place on ice, shake at room temperature for 10 min, and fully lyse the cells;

[0201] Configure the firefly luciferase reaction working solution and the sea renilla luciferase reaction solution, i.e. the firefly luciferase substrate (50x) and the sea renilla luciferase substrate (50x) are diluted with the corresponding buffer to 1x working solution (incubate to room temperature).

[0202] Add 100 μL luciferase reaction solution to each well, shake plate to mix, detect the activity of luciferase, and try to complete the detection within 30 min.

[0203] Add 100 μL luciferase reaction solution to each well, shake plate to mix, detect the activity of luciferase, and try to complete the detection within 30 min.

[0204] Data processing (calculation formula):

[0205] (1-((experimental group R-background R) x (negative control group F-background F)) / ((experimental group F-background F) x (negative control group R-background R))) x 100

[0206] Background F: untransfected cells + luciferase detection reagent;

[0207] Background R: untransfected cells + luciferase detection reagent + sea anemone luciferase detection reagent;

[0208] Experimental group F: transfected cells (treated with psiCHECK2 plasmid and different concentrations of siRNA) + luciferase detection reagent;

[0209] Experimental group R: transfected cells (treated with psiCHECK2 plasmid and different concentrations of siRNA) + luciferase detection reagent + sea anemone luciferase detection reagent;

[0210] Negative control group F: transfected cells (treated with psiCHECK2 plasmid) + luciferase detection reagent;

[0211] Negative control group R: transfected cells (treated with psiCHECK2 plasmid) + luciferase detection reagent + sea anemone luciferase detection reagent.

[0212] The instrument consumables are shown in Table 2-1.

[0213] Table 2-1

[0214] The in vitro screening results of the naked sequence and the results of the modified sequence are shown in Table 2-2 and Table 2-3, respectively

[0215] Table 2-2

[0216] Table 2-3

[0217] Table 2-3 (multiple data for one concentration represent multiple experiments)

[0218] Table 2-4 (multiple data for one concentration represent multiple experiments)

[0219] Example 3 In vivo testing - batch 1

[0220] The purpose of this experiment was to investigate the procoagulant effect of the test drug on F8-KO mice by administering the test drug subcutaneously once a week for 3 consecutive weeks.

[0221] 36 male F8-KO mice (genotype: (F8-KO) ko / Y, level: SPF, source: Jiangsu Jizu Yaoke Biotechnology Co., Ltd., animal age at the beginning of the experiment: 9 weeks old) were divided into 9 groups according to body weight, 4 in each group: Control group, Fitusiran 1 mg / kg group, Fitusiran 3 mg / kg group, M-ACON-siRNA-136-30-L96 1 mg / kg, 3 mg / kg group, M-ACON-siRNA-140-34-L96 1 mg / kg, 3 mg / kg group, M-ACON-siRNA-326-5-L96 1 mg / kg, 3 mg / kg group. Each group of animals was given subcutaneous injection of test substances once a week for 3 consecutive weeks, and 24 hours after the last administration, the blood was collected from the eye socket to detect the clotting time (CT). Then the blood was collected from the heart, centrifuged to separate the plasma, and the four blood coagulation items (PT, APTT, Fbg, TT) and antithrombin (AT) were detected.

[0222] The specific operation is as follows:

[0223] 3.1 Test substances:

[0224] The information related to the test substances and reagents is as follows:

[0225] Test substance 1: M-ACON-siRNA-136-30-L96; property: dry powder; purity: 98.05%; storage condition: -80℃.

[0226] Test substance 2: M-ACON-siRNA-140-34-L96-B01; property: dry powder; purity: 97.20%; storage condition: -80℃.

[0227] Test substance 3: M-ACON-siRNA-326-5-L96-B01; property: dry powder; purity: 95.99%; storage condition: -80℃.

[0228] 3.2 Positive control:

[0229] The information related to the positive control and reagents is as follows:

[0230] Name: Fitusiran; property: dry powder; purity: 99.76%; storage condition: -80℃.

[0231] 3.3 Experimental method

[0232] The solvent, positive control drug or test drug was administered subcutaneously once a week for 3 consecutive weeks according to the grouping of Table 3. 24 hours after the last administration, blood was collected from the retro-orbital venous plexus of the glass capillary. A small section of the capillary tube was cut off and gently stretched to the left and right until visible blood clots appeared, which was recorded as the clotting time (CT). Subsequently, blood was collected from the heart, and plasma was separated by centrifugation to detect the levels of coagulation four items (PT, APTT, Fbg, TT) and antithrombin (AT).

[0233] Table 3 - Experimental grouping

[0234] wherein sc. represents subcutaneous injection, and qw. represents once a week.

[0235] 3.4 Experimental results

[0236] 3.4.1 Clinical observation: All experimental animals had no obvious abnormalities visible to the naked eye during the entire administration period.

[0237] 3.4.2 Effect of test substances on the CT of experimental animals:

[0238] Effect of test substances on the clotting time (CT) of experimental animals: Compared with the Control group, the clotting time of mice in all administration groups was shortened to varying degrees. Among them, the clotting time of the Fitusiran 1 mg / kg and 3 mg / kg groups, the M-ACON-siRNA-140-34-L96 3 mg / kg group, and the M-ACON-siRNA-326-5-L96 3 mg / kg group showed statistically significant differences compared with the Control group (P < 0.01, P < 0.001), and the M-ACON-siRNA-326-5-L96 had the best efficacy. The shortening effect of each test substance on the clotting time of F8-KO mice showed a positive correlation with the dose, as shown in Table 4.

[0239] Table 4. Effect of subcutaneous injection of test substances once a week for 3 consecutive weeks on the clotting time of F8-KO mice (n = 4, ) ** P < 0.01, *** P < 0.001 vs. Control; ## P < 0.01 vs. Fitusiran 1 mg / kg

[0240] 3.4.3 Effect of test substances on the coagulation four items of experimental animals:

[0241] The PT of mice in the Fitusiran 1 mg / kg, 3 mg / kg groups, the M-ACON-siRNA-140-34-L96 3 mg / kg, 1 mg / kg groups, and the M-ACON-siRNA-326-5-L96 3 mg / kg group were all slightly shorter than that of the Control group.

[0242] The APTT of mice in all the dosing groups were all reduced to different degrees compared with the Control group, except for the M-ACON-siRNA-136-30-L96 1 mg / kg group. The APTT of mice in the Fitusiran 3 mg / kg and M-ACON-siRNA-140-34-L96 3 mg / kg groups were statistically significantly different from that of the Control group (P < 0.05, P < 0.001). The effect of M-ACON-siRNA-326-34-L96 on APTT was similar. The shortening of thrombin time reflects the acceleration of the coagulation process.

[0243] The Fbg formation time (s) of mice in all the dosing groups were all reduced to different degrees compared with the Control group, although the mean Fbg formation time (s) of some dosing groups was significantly reduced, but no statistically significant difference was observed, which was related to the small sample size or large standard deviation of each group of experimental animals. The Fbg content (g / L) of mice in all the dosing groups was all increased to different degrees compared with the Control group, although the mean Fbg content (g / L) of some dosing groups was significantly increased, but no statistically significant difference was observed compared with the Control group due to the small sample size or large standard deviation of each group of experimental animals. The effect of Fbg formation time shortening and content increasing was best with M-ACON-siRNA-326-34-L96. The shortening of Fbg formation time and the increase of content reflect that the measured test substances all improved the procoagulant effect of F8-KO mice to different degrees.

[0244] The TT of mice in all the dosing groups were all reduced to different degrees compared with the Control group, in which the TT of mice in the Fitusiran 3 mg / kg and M-ACON-siRNA-136-30-L96 3 mg / kg groups were statistically significantly different from that of the Control group (P < 0.05, P < 0.01), and the TT of mice in the M-ACON-siRNA-326-30-L96 group was apparently shorter. As shown in Table 5.

[0245] Table 5. Effect of the test substance on the four coagulation parameters of F8-KO mice given subcutaneously once a week for 3 consecutive weeks (n = 4, ) * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; # P < 0.05 vs. Fitusiran 1 mg / kg; + P < 0.05, ++ P < 0.01 vs. Fitusiran 3 mg / kg

[0246] In the procoagulant test of F8-KO mice, the positive control drug Fitusiran, the test substances M-ACON-siRNA-136-30-L96, M-ACON-siRNA-140-34-L96 and M-ACON-siRNA-326-5-L96 all showed certain procoagulant effects. M-ACON-siRNA-326-5-L96 was slightly better than M-ACON-siRNA-136-30-L96 and M-ACON-siRNA-140-34-L96 in terms of procoagulant effects, and was better than the positive control drug Fitusiran in terms of the formation time (s) of fibrinogen (Fbg), the content (g / L) of fibrinogen (Fbg) and thrombin time (TT).

[0247] Example 4 In vivo test - Batch 2

[0248] The purpose of this experiment was to study the procoagulant effect of the test drug on B6-F8-KO mice by subcutaneously administering the test substance once a week for 3 consecutive weeks.

[0249] 64 male B6-F8-KO mice (genotype: (F8-KO) ko / Y, level: SPF, source: Jiangsu Jizhu Yaoke Biotechnology Co., Ltd., animal age at the beginning of the experiment: 9 weeks) were evenly divided into 16 groups according to body weight, 4 in each group: Control group, Fitusiran 1 mg / kg group, M-ACON-siRNA-136-15 1 mg.kg, 3 mg / kg group, M-ACON-siRNA-136-23 1 mg.kg, 3 mg / kg group, M-ACON-siRNA-136-25 1 mg.kg, 3 mg / kg group, M-ACON-siRNA-136-43 1 mg.kg, 3 mg / kg group, M-ACON-siRNA-136-53 1 mg.kg, 3 mg / kg group, M-ACON-siRNA-140-32 1 mg.kg, 3 mg / kg group, M-ACON-siRNA-329-6 1 mg.kg, 3 mg / kg group. Each group of animals was given a test substance subcutaneously, once a week, for 3 consecutive weeks. 24 hours after the last administration, blood was collected from the orbita to detect the clotting time (CT). Subsequently, blood was collected from the heart, centrifuged to separate plasma, and the four clotting tests (PT, APTT, Fbg, TT) and antithrombin (AT) were detected.

[0250] 4.1 Test substance:

[0251] The test substance and reagent related information is as follows:

[0252] Test substance 1: M-ACON-siRNA-136-15 Property: dry powder Purity: 99.06% Storage condition: -80℃

[0253] Test substance 2: M-ACON-siRNA-136-23 Property: dry powder Purity: 99.14% Storage condition: -80℃

[0254] Test substance 3: M-ACON-siRNA-136-25 Property: dry powder Purity: 98.18% Storage condition: -80℃

[0255] Test substance 4: M-ACON-siRNA-136-43 Property: dry powder Purity: 97.86% Storage condition: -80℃

[0256] Test substance 5: M-ACON-siRNA-136-53 Property: dry powder Purity: 98.94% Storage condition: -80℃

[0257] Test substance 6: M-ACON-siRNA-140-32 Property: dry powder Purity: 98.4% Storage condition: -80℃

[0258] Test article 7: M-ACON-siRNA-329-6 Character: dry powder Purity: 96.89% Storage condition: -80°C

[0259] 4.2 Positive control:

[0260] The positive control and reagent related information are as follows:

[0261] Name: Fitusiran; Character: dry powder; Purity: 99.76%; Storage condition: -80°C.

[0262] 4.3 Experimental method

[0263] According to the grouping of Table 6, the solvent, positive control drug or test drug was administered subcutaneously once a week for 3 consecutive weeks. 24 hours after the last administration, the blood was collected by the method of glass capillary orbit retro-orbital plexus, a small section of capillary tube was cut off every 15 seconds and gently stretched to the left and right until visible blood clots appeared, which was recorded as the clotting time (CT). Then the heart blood was collected, centrifuged to separate the plasma, and the levels of four blood coagulation items (PT, APTT, Fbg, TT) and antithrombin (AT) were detected.

[0264] Table 6

[0265] Wherein sc. represents subcutaneous injection, and qw. represents once a week.

[0266] 4.4 Experimental results

[0267] 4.4.1 Clinical observation: All experimental animals had no obvious abnormalities visible to the naked eye during the entire administration period.

[0268] 4.4.2 Effect of test article on CT of experimental animals:

[0269] Effect of test article on the clotting time (CT) of experimental animals:

[0270] Compared with the Control group, the coagulation time of mice in all administration groups was shortened to varying degrees, among which the coagulation time of the Fitusiran 1 mg / kg group, the M-ACON-siRNA-136-23 1, 3 mg / kg group, the M-ACON-siRNA-136-25 3 mg / kg group, the M-ACON-siRNA-136-43 3 mg / kg group, and the M-ACON-siRNA-329-6 1, 3 mg / kg group showed statistically significant differences (P < 0.05, P < 0.01) compared with the Control group. The coagulation time of the M-ACON-siRNA-136-25 1 mg / kg group was significantly longer than that of the Fitusiran 1 mg / kg group (P < 0.05), and there was no statistically significant difference between the other test substance groups with the same administration dose (1 mg / kg) and the Fitusiran 1 mg / kg group (Table 7).

[0271] Table 7 Effect of subcutaneous injection of test substances once a week for 3 consecutive weeks on the coagulation time of B6-F8-KO mice (n = 4, )

[0272] 4.4.3 Effect of test substances on the four items of coagulation of experimental animals:

[0273] The prothrombin time (PT) of mice in the administration groups was shortened to varying degrees compared with the Control group, among which the Fitusiran 1 mg / kg group, the M-ACON-siRNA-136-23 3 mg / kg group, the M-ACON-siRNA-136-43 3 mg / kg group, and the M-ACON-siRNA-329-6 1, 3 mg / kg group showed statistically significant differences (P < 0.05, P < 0.01) compared with the Control group.

[0274] The activated partial thromboplastin time (APTT) of mice in each administration group was shortened to varying degrees compared with the Control group, among which the M-ACON-siRNA-136-23 3 mg / kg group, the M-ACON-siRNA-136-43 3 mg / kg group, the M-ACON-siRNA-140-32 3 mg / kg group, and the M-ACON-siRNA-329-6 3 mg / kg group showed statistically significant differences (P < 0.05, P < 0.01, P < 0.001) compared with the Control group. The shortening of thromboplastin time reflects the acceleration of the coagulation process.

[0275] The thrombin time (TT) of all the mice in the administration groups was shortened to varying degrees compared with the Control group, among which the M-ACON-siRNA-136-23 1, 3 mg / kg, M-ACON-siRNA-136-25 1 mg / kg, M-ACON-siRNA-136-43 3 mg / kg, M-ACON-siRNA-136-53 1 mg / kg, M-ACON-siRNA-140-32 1, 3 mg / kg and M-ACON-siRNA-329-6 3 mg / kg groups showed statistically significant differences (P < 0.05, P < 0.01, P < 0.001) compared with the Control group, and the shortening of the thrombin time reflected the enhanced procoagulant effect (Table 8).

[0276] Table 8. Effect of subcutaneous injection of test substances once a week for 3 consecutive weeks on the four coagulation indicators of F8-KO mice (n = 4, ) * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; # P < 0.05 vs. Fitusiran 1 mg / kg

[0277] In the procoagulant experiment of B6-F8-KO mice, the positive control drug Fitusiran 1 mg / kg, all the 1 mg / kg and 3 mg / kg of the test substances showed certain procoagulant effects, which were mainly reflected in CT, PT, APTT and TT. In combination with the indicators, M-ACON-siRNA-136-23, M-ACON-siRNA-136-43 and M-ACON-siRNA-329-6 showed better procoagulant effects, and no animal death occurred, but the efficacy at the same dose was not better than that of the positive control drug Fitusiran.

[0278] Example 5 In vivo test - batch 3

[0279] The purpose of this experiment was to study the procoagulant effect of the test drugs on B6-F8-KO mice by subcutaneous injection of the test substances once a week for 3 consecutive weeks.

[0280] 30 male F8-KO mice were divided into 7 groups according to body weight, 4 or 6 in each group: Control group, ACON-SiRNA-007 3 mg / kg (1 mg / kg for the 2nd and 3rd administration) group, ACON-SiRNA-007 10 mg / kg (3 mg / kg for the 2nd and 3rd administration) group, ACON-SiRNA-247 3 mg / kg (1 mg / kg for the 2nd and 3rd administration) group, ACON-SiRNA-247 10 mg / kg (3 mg / kg for the 2nd and 3rd administration) group, M-ACON-siRNA-206-1-L96 3 mg / kg (1 mg / kg for the 2nd and 3rd administration) group, M-ACON-siRNA-206-1-L96 10 mg / kg (3 mg / kg for the 2nd and 3rd administration) group. The animals in each group were subcutaneously injected with the test substance once a week for 3 weeks, and 24 hours after the last administration, blood was collected from the orbit to detect the clotting time (CT). Subsequently, blood was collected from the heart, and plasma was separated by centrifugation to detect the four blood coagulation items (PT, APTT, Fbg, TT) and antithrombin (AT).

[0281] 5.1 Test substance:

[0282] The test substance and reagent related information is as follows:

[0283] Test substance 1: ACON-SiRNA-247 Property: dry powder Purity: 95.20% Storage condition: -80℃

[0284] Test substance 2: M-ACON-siRNA-206-1-L96 Property: dry powder Purity: 96.01% Storage condition: -80℃

[0285] 5.2 Positive control:

[0286] The positive control and reagent related information is as follows:

[0287] Name: ACON-SiRNA-007; Property: dry powder; Purity: 95.25%; Storage condition: -80℃.

[0288] 5.3 Experimental method

[0289] According to the grouping of Table 9, the solvent, positive control drug or test drug was subcutaneously injected once a week for 3 weeks, and 24 hours after the last administration, blood was collected from the glass capillary orbit posterior venous plexus, a small section of capillary tube was cut off every 15 seconds and gently stretched to the left and right until visible blood clots appeared, which was recorded as the clotting time (CT). Subsequently, blood was collected from the heart, and plasma was separated by centrifugation to detect the four blood coagulation items (PT, APTT, Fbg, TT) and antithrombin (AT) levels.

[0290] Table 9

[0291] wherein sc. represents subcutaneous injection, and qw. represents once a week.

[0292] 5.4 Experimental results

[0293] 5.4.1 Effect of the test substances on the CT of the experimental animals:

[0294] Effect of the test substances on the clotting time (CT) of the experimental animals:

[0295] Compared with the Control group, the clotting time of the mice in all the test drug groups was shortened to varying degrees, and the clotting time of the animals in the ACON-SiRNA-247 3 mg / kg group showed statistically significant differences compared with the Control group and the positive control ACON-SiRNA-007 3 mg / kg group of the same dose (P < 0.05, P < 0.01). The shortening of the clotting time is a key indicator reflecting the clotting process, and its shortening is of great significance.

[0296] Table 10 Effect of subcutaneous injection of test substances once a week for 3 consecutive weeks on the clotting time of F8-KO mice (n = 4 or 6, ) ** P < 0.01 vs. Control; + P < 0.05 vs. ACON-SiRNA-007 3 mg / kg

[0297] 5.4.2 Effect of the test substances on the four clotting items of the experimental animals:

[0298] The prothrombin time (PT) of the mice in all the test drug groups showed no significant difference compared with the Control group.

[0299] The activated partial thromboplastin time (APTT) of the mice in the ACON-SiRNA-007 1 mg / kg, 3 mg / kg groups and the ACON-SiRNA-247 3 mg / kg group was significantly lower than that of the Control group, but no statistically significant difference was observed, which was related to the small number of experimental animals in each group. The APTT of the mice in the ACON-SiRNA-247 1 mg / kg group and the ACON-siRNA-206-1-L96 1 mg / kg, 3 mg / kg groups showed no significant change compared with the Control group. The shortening of the thromboplastin time reflects the acceleration of the clotting process.

[0300] The fibrinogen (Fbg) formation time (s) in all tested drug groups was shortened to varying degrees compared with the control group. Specifically, the Fbg(s) in the ACON-SiRNA-007 3 mg / kg group, the ACON-SiRNA-247 1 mg / kg and 3 mg / kg groups, and the M-ACON-siRNA-206-1-L96 3 mg / kg groups showed statistically significant differences compared with the control group (P<0.01, P<0.001). The fibrinogen (Fbg) content (g / L) in all tested drug groups was increased to varying degrees compared with the control group. Although the mean Fbg content (g / L) in the ACON-SiRNA-247 1 mg / kg, M-ACON-siRNA-206-1-L96 1 mg / kg and 3 mg / kg groups increased significantly, due to the large standard deviation, no statistically significant difference was observed compared with the control group. The ACON-SiRNA-007 3 mg / kg and ACON-SiRNA-247 3 mg / kg groups showed statistically significant differences compared to the untreated Control group of F8-KO mice. The shortened fibrinogen formation time and increased fibrinogen content reflect that the tested substances improved the procoagulant effect in F8-KO mice to varying degrees.

[0301] The thrombin time (TT) of mice in the ACON-siRNA-007 3 mg / kg group and the M-ACON-siRNA-206-1-L96 3 mg / kg group was significantly shorter than that in the Control group (P<0.05, P<0.01), reflecting its enhanced procoagulant effect. There were no significant differences between the other treatment groups and the Control group (Table 8).

[0302] Table 11. Effects of subcutaneous injection of the test substance once a week for 3 consecutive weeks on coagulation parameters in F8-KO mice (n = 4 or 6, ) * P<0.05, ** P<0.01, *** P<0.001 vs. Control; # P<0.05 vs. Fitusiran 1mg / kg

[0303] 5.4.3 Effects of the test substance on the four coagulation parameters in experimental animals:

[0304] There were no significant differences in plasma AT levels in the ACON-SiRNA-007 1 mg / kg, 3 mg / kg groups, the ACON-SiRNA-247 1 mg / kg, 3 mg / kg groups, and the M-ACON-siRNA-206-1-L96 1 mg / kg, 3 mg / kg groups compared to the Control group 12.

[0305] Table 12. Effect of once weekly subcutaneous injection for 3 weeks on plasma AT levels in F8-KO mice (n = 4 or 6, )

[0306] Method for in vivo mouse PK

[0307] To evaluate the in vivo pharmacokinetic behavior of siRNA, C57BL / 6J mice (SPF level, half male and half female, 6-8 weeks, 18-25 g) were selected for this study, 4 animals (2 males and 2 females) per time point, 40 animals per dose group. A single subcutaneous injection of 3 mg / kg of the test product was administered. Food status: free water diet. Plasma and liver were collected at different time points.

[0308] Collection and processing of plasma samples: 10 post-dose time points were collected within 28 days, pre-dose, 0.5 h, 1 h, 2 h, 4 h, 24 h, 72 h, 168 h, 336 h, 504 h, 672 h after administration. About 0.4 mL of blood was collected from the submandibular vein or other veins each time, and the blood collection tube contained K2-EDTA anticoagulant, which was placed on wet ice after collection. The collected whole blood was placed in a labeled collection tube, transferred to the sample centrifugation laboratory through a wet ice box, and plasma separation was completed. The whole blood was centrifuged within 1 hour after collection, under the conditions of 6800 g, 2-8°C, 6 minutes. The plasma sample was stored in an ultra-low temperature -80°C refrigerator.

[0309] Collection and processing of liver samples: after euthanasia of the animals, liver tissue was collected, rinsed with normal saline to avoid cross contamination, absorbed with filter paper, and then placed in labeled tubes / self-sealing bags (one tissue per tube / self-sealing bag). The liver samples were stored in a -80°C refrigerator.

[0310] LC-MS / MS was used to detect the concentration of siRNA in plasma and liver samples. Based on the drug concentration data of each group at each time point, the pharmacokinetic parameters AUC0-t, AUC0-∞, MRT0-∞, Cmax, Tmax, and T1 / 2, and the male / female ratio of AUC(0-t) were calculated using the pharmacokinetic calculation software Phoenix Winnolin R7.0 non-compartment model.

[0311] The plasma results are shown in the following table:

[0312] Liver results are shown in the table below:

Claims

1. An RNA inhibitor of AT3 gene expression comprising an antisense strand comprising a complementary region complementary to at least a portion of a mRNA encoding AT3, the complementary region being 17-23 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence of any one of SEQ ID NO.: 1-303, 922-1001 or a sequence differing by no more than 3 nucleotides therefrom.

2. The RNA inhibitor of claim 1, wherein the RNA inhibitor inhibits expression of an AT3 gene. It further comprises a sense strand, wherein there is at least 80% base complementarity between the sense strand and the antisense strand.

3. The RNA inhibitor of AT3 gene expression according to any one of claims 1-2, wherein the sense nucleic acid strand and the antisense nucleic acid strand are present on two different nucleic acid strands.

4. The RNA inhibitor of AT3 gene expression according to any one of claims 1-2, wherein the sense nucleic acid segment and the antisense nucleic acid segment are present on the same nucleic acid strand, wherein the complementary region of the sense nucleic acid segment and the antisense nucleic acid segment forms a double-stranded nucleic acid structure.

5. The RNA inhibitor for inhibiting AT3 gene expression according to any one of claims 1-4, characterized in that... At least one strand has a 3' overhang of 0 to 6 nucleotides in length.

6. The RNA inhibitor of claim 1-5, wherein the RNA inhibitor inhibits the expression of an AT3 gene. Both strands have a 3' overhang of 2-3 nucleotides in length, or the sense strand has a 3' overhang of 2-3 nucleotides in length, or the antisense strand has a 3' overhang of 2-3 nucleotides in length.

7. The RNA inhibitor of claim 1-6, wherein the RNA inhibitor inhibits the expression of an AT3 gene. The sense nucleic acid strand and the antisense nucleic acid strand are 16 to 35 nucleotides in length, respectively.

8. The RNA inhibitor of claim 1-7, wherein the RNA inhibitor inhibits the expression of an AT3 gene. One strand of the RNA inhibitor of AT3 gene expression has at least 75% homology or complementarity to a nucleotide sequence selected from any one of SEQ ID NO: 643-921.

9. The RNA inhibitor of claim 1-8, wherein the RNA inhibitor inhibits the expression of an AT3 gene. The sense strand thereof is selected from any one of SEQ ID NO: 304-642, 1002-1060 or a sequence differing by no more than 3 nucleotides therefrom.

10. The RNA inhibitor of AT3 gene expression according to any one of claims 1-9, wherein at least one nucleotide is a chemically modified nucleotide.

11. The RNA inhibitor of AT3 gene expression according to any one of claims 1-10, wherein the chemical modification is at least one of the following: (1) a modification to the phosphodiester linkage connecting the nucleotides in the nucleotide sequence of the RNA inhibitor of AT3 gene expression; (2) a modification to the ribose in the nucleotide sequence of the RNA inhibitor of AT3 gene expression; (3) a modification to the base in the nucleotide sequence of the RNA inhibitor of AT3 gene expression.

12. The RNA inhibitor of AT3 gene expression according to any one of claims 1-11, wherein there are at least two consecutive phosphorothioate linkages between the nucleotides of the sense strand and / or the antisense strand.

13. The RNA inhibitor of AT3 gene expression according to any one of claims 1-12, wherein there are at least two consecutive phosphorothioate linkages between the three consecutive nucleotides at the end of the sense strand and / or the end of the antisense strand.

14. The RNA inhibitor of expression of AT3 gene according to any one of claims 1-13, wherein the modification of ribose comprises a fluoro substitution and / or a methoxy substitution for 2'-OH.

15. The RNA inhibitor of expression of AT3 gene according to any one of claims 1-13, wherein the modification of ribose comprises UNA, LNA or GNA.

16. The RNA inhibiting agent which inhibits the expression of AT3 gene according to any one of claims 1 to 15, wherein the modification of the antisense strand comprises one of the following: XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXfXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XmsXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmTgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm [Xvm]sXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXuXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmXmTuXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXmXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXm PXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXdXXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfdTXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmdTXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmdXXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm PXmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXmXmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXmXmXfXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm PXmsXfsXmXmXmXfXfXfXfXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXmXmXmXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXmXfXmXmXmXmXfXmXfXmXfXmXmXmXmXmsXmsXm, wherein: X represents a ribonucleotide, s represents a phosphorothioate linkage, Xm a 2'methoxy substituted ribonucleotide, Xf represents a 2'fluoro substituted ribonucleotide, Xu represents a UNA version of a nucleotide, Xg represents a (S)-GNA version of a ribonucleotide, dT represents a 2'-deoxythymidine-3'-phosphate, dA represents a 2'-deoxyadenosine-3'-phosphate, P represents a phosphorylation, [Uvm] represents a 5'-(E)-vinylphosphonate-2'-Ome-U.

17. The RNA inhibitor of AT3 gene expression according to any one of claims 1-15, wherein the modifications of the sense strand comprise one of: XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXmXfXmXfXmXfXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXfXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXf XmsXfsXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfXmXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXmXmXfTgXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXmXmsXfsXmXmXmTgXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXm XmsXmsXmXmXmXmXfImXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmImXmXmXmXmXmXmXmXm XmsXfsXmXmXmXfXgXmXmXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXmXmXmXfTgXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXgXmXmXmXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmXmsXmsXm XmsXfsXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXfXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXmXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXfXfXfXmXfXmXfXmXfXmXfXmXfXmXfXmXmXmsXmsXm XmsXfsXmXmXmXfXfXfXfXmXmXmXmXfXmXfXmXmXmXmXmsXmsXm XmsXfsXfXfXmXfXmXfXmXfXmXmXmXfXmXfXmXfXmXfXmsXmsXm XmsXmsXmXmXmXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmImXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmImXmXmXmXmXmXmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmImXmXmXmXmXmXm XmsXmsXmXmXmXmXfImXfXfXfXmImXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmImXm XmsXmsXmXmXmXmXfXfXfXfXfXmXmXmXmXmXmXmXmXmXm XmsXmsXmXmXmXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmXfXmXfXmXfXmXf XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm XfsXmsXfXmXfXmXfImXfXfXfXmXfXmXfXmXfXmXfXmXf XfsXmsXfXmXfXmXfXmXfXfXfXmXfXmIfXmXfXmXfXmXfXfs Xms Xf Xm Xf Xm Xf Im Xf Xf Xf Xm If Xm Xf Xf Xf Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm Xfs Xms Xf Xm Xf Xm If Xm Xf Xf Xm Xf Xm Xf Xm Xf Xm Xf Xm Xf Xfs Xms Xf Xm Xf Xm Xf Xm Xf Xf Xf If Xm Xf Xm Xf Xm Xf Xm Xf Xfs Xms Xf Xm Xf Xm Xf Xm Xf Xf Xf Xm If Xm Xf Xm Xf Xm Xf Xm Xf Xfs Xms Xf Xm Xf Xm Xf Xm Xf Xf Xf Xm Im Xm Xf Xm Xf Xm Xf Xfs Xms Xf Xm Xf Xm Xf Xm Xf Xf Xf Xm Xf Xm Xf Xm If Xm Xf Xm Xf Xm Xf Xf Xf Xm Xf Xm Xf Xm Im Xm Xf Xfs Xms Xf Xm If Xm Xf Xm Xf Xf Xf Xm Xf Xm Xf Xm Xf Xm Xf Xfs Xms Xf Xm Im Xm Xf Xm Xf Xf Xf Xm Xf Xm Xf Xm Xf Xm Xf Xms Xms Xm Xm Xm Xm Xf Xf Xf Xf Xm Xm Xm Im Xm Xm Xm Xm Xm Xm Xms Xms Xm Xm Xm Xm Xf Xf Xf Xf Xm Xm Xm Xm Im Xm Xm Xm Xm Xm Xms Xms Xm Xm Xm Xm Xf Xf Xf Xf Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm Xms Xms Xm Xm Xm Xm Xm Xm Xf Xf Xm Xm Xm Xm Xm Xm Xm Xm XmsXmsXmXmXmXmXfXfXfXmXmXmXmXmXmXmXmXmXmXmXm, wherein: X represents a ribonucleotide, I represents an inosine-3'-phosphate, s represents a phosphorothioate linkage, Xm a 2'methoxy substituted ribonucleotide, Xf represents a 2'fluoro substituted ribonucleotide, Xg represents a (S)-GNA version of a ribonucleotide.

18. The RNA inhibitor of expression of AT3 gene according to any one of claims 1-17, further comprising a ligand, wherein the ligand is conjugated to the sense strand and / or the antisense strand.

19. The RNA inhibitor of expression of AT3 gene according to claim 18, wherein the ligand is conjugated to the 5' end and / or the 3' end of the antisense strand.

20. The RNA inhibitor of expression of AT3 gene according to any one of claims 18-19, wherein the ligand is conjugated to the 5' end and / or the 3' end of the sense strand.

21. The RNA inhibitor of expression of AT3 gene according to any one of claims 18-20, or a pharmaceutically acceptable salt thereof, wherein the ligand is conjugated to the 5' end of the antisense strand and the ligand is conjugated to the 3' end of the sense strand.

22. The RNA inhibitor of expression of AT3 gene according to any one of claims 18-20, wherein the ligand is conjugated to the 3' end of the antisense strand and the ligand is conjugated to the 5' end of the sense strand.

23. The RNA inhibitor of expression of AT3 gene according to any one of claims 18-20, wherein the ligand is conjugated to the 5' end and the 3' end of the sense strand.

24. The RNA inhibitor of expression of AT3 gene according to any one of claims 18-23, the ligand further comprising a targeting unit, a structure for enhancing uptake of the RNA inhibitor by hepatocytes.

25. The RNA inhibitor of expression of AT3 gene according to claim 24, the targeting unit selected from the group consisting of monosaccharides and derivatives thereof.

26. The RNA inhibitor of expression of AT3 gene according to any one of claims 24-25, said monosaccharide being selected from one or more of the following structures: mannose, galactose, D-arabinose, glucose, fructose, xylose, glucosamine, ribose.

27. The RNA inhibitor of expression of AT3 gene according to any one of claims 24-26, said monosaccharide derivative being selected from a mannose derivative, a galactose derivative, a glucose derivative, a ribose derivative and other derivatives.

28. The RNA inhibitor of expression of AT3 gene according to any one of claims 24-27, said targeting unit being selected from galactose, galactosamine, N-acetylgalactosamine and derivatives thereof.

29. The RNA inhibitor of expression of AT3 gene according to any one of claims 24-28, said targeting unit being N-acetylgalactosamine and derivatives thereof.

30. A pharmaceutical composition comprising the RNA inhibitor of expression of AT3 gene according to any one of claims 1-29, and further comprising a delivery vehicle, and / or a physiologically acceptable excipient and / or carrier and / or diluent.

31. The pharmaceutical composition according to claim 30, wherein said delivery vehicle comprises a liposome.

32. The pharmaceutical composition according to claim 31, wherein said delivery vehicle comprises a nanolipid.

33. Use of the RNA inhibitor of expression of AT3 gene according to any one of claims 1-29 and of the pharmaceutical composition according to any one of claims 30-32 for the manufacture of a medicament for the prevention or treatment of, or reduction of the risk of, a disease or pathology.

34. The use according to claim 33, wherein said disease or pathology comprises a disease or pathology associated with elevated levels of AT3.

35. The use according to any one of claims 33-34, wherein said disease or pathology comprises a hemophilia.

36. A method of preventing or treating a disease, disorder or syndrome, said method comprising administering to a subject in need thereof an effective amount of the RNA inhibitor of expression of AT3 gene according to any one of claims 1-29, a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to any one of claims 30-32.

37. The method according to claim 36, wherein said RNA inhibitor of expression of AT3 gene, a pharmaceutically acceptable salt thereof or said pharmaceutical composition is administered to the subject in a subcutaneous, intravenous, oral, rectal or intraperitoneal administration route.

38. A method for inhibiting the expression of AT3 in a cell, tissue or subject, comprising administering to the cell, tissue or subject an effective amount of the RNA inhibitor of expression of AT3 gene according to any one of claims 1-29, a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to any one of claims 30-32.

39. The RNA inhibitor according to claims 1-29, comprising the duplexes described in Table 1-1.

40. The RNA inhibitor of claims 1-29, comprising a duplex described in Table 1-2.

41. The RNA inhibitor of claims 1-29, comprising a duplex described in Table 2-1, 2-2, 2-3, or 2-4.

Images