SiRNA for inhibiting carboxypeptidase B2 gene expression, conjugate thereof, pharmaceutical composition and application

Abstract

The invention relates to siRNA (small interfering Ribonucleic Acid) for inhibiting carboxypeptidase B2 gene expression, a conjugate thereof, a pharmaceutical composition and application. The siRNA disclosed by the invention contains a positive-sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified or unmodified nucleotide. The siRNA as well as the conjugate and the pharmaceutical composition thereof disclosed by the invention can be used for effectively treating and / or preventing diseases related to CPB2 gene overexpression.

Description

Technical Field

[0001] This invention relates to siRNA that inhibits the expression of the carboxypeptidase B2 gene, siRNA conjugates, pharmaceutical compositions containing the same, methods of preparation thereof, and uses thereof. Background Technology

[0002] CPB2 (CPU, TAFI) is a carboxypeptidase, also known as a thrombin-activated fibrinolysis inhibitor, which plays an important role in the regulation of blood coagulation. It is mainly synthesized by the liver, where alkaline carboxypeptidase precursors are converted into active carboxypeptidase by thrombin (thrombomodulin) or plasmin, and then secreted into the plasma as a plasminogen (an effective fibrinolytic attenuator). When activated by the proteolytic action of the thrombin / thrombomodulin complex at the Arg92 residue, CPB2 exhibits carboxypeptidase activity. Its mechanism of action is mainly to remove the tissue plasminogen activator (tPA) and the C-terminal lysine binding site of plasminogen from fibrin, reducing its fibrinolytic capacity and thus stimulating clot formation and growth. Studies have collected peripheral blood from 45 patients with acute myocardial infarction (AMI) (25 with ST-segment elevation myocardial infarction [STEMI] and 20 with non-ST-segment elevation myocardial infarction [NSTEMI]) and 42 controls. In addition, coronary artery blood samples were collected from 11 STEMI patients during thrombus aspiration. Subsequently, proCPU and plasma CPU concentrations in all samples were measured using an activity-based assay. The results showed that AMI patients had higher levels of CPU activity than controls. Furthermore, intracoronary samples contained higher CPU levels than peripheral samples, indicating increased local CPU generation. Regarding proCPU, they found lower levels in AMI patients than in controls. These findings suggest that functional active CPU is generated in AMI patients. Furthermore, multiple studies have shown that increased CPB2 levels are associated with an increased risk of thrombosis development and formation, venous thrombosis, venous thromboembolism, deep vein thrombosis, pulmonary embolism, prosthetic valve thrombosis, post-thrombotic syndrome, atherosclerotic thrombosis, heart attack, stroke, fibrinolysis disorders, insufficient fibrinolysis, poor fibrinolysis, ischemic stroke, atrial fibrillation, myocardial infarction, peripheral artery disease, dynamic thrombosis, coronary artery disease, microvascular thrombosis, ischemic brain injury, brain edema, cerebral hemorrhage, and other diseases related to death after thromboembolic stroke.

[0003] In summary, inhibiting the expression of the CPB2 gene in patients can prevent and treat thrombosis (including venous thrombosis, deep vein thrombosis, atherosclerotic thrombosis, prosthetic valve thrombosis, microvascular thrombosis, and dynamic thrombosis), pulmonary embolism, stroke, myocardial infarction, and ischemic stroke. Currently, there are no drugs on the market that specifically target the expression of this gene; therefore, developing drugs targeting CPB2 is of significant value. Summary of the Invention

[0004] The present invention aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the CPB2 gene, thereby selectively and effectively inhibiting the expression of the CPB2 gene to achieve the purpose of disease prevention and / or treatment.

[0005] In a first aspect, the present invention provides an siRNA for inhibiting the expression of the carboxypeptidase B2 (CPB2) gene, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified, wherein the sense strand contains nucleotide sequence I, and the antisense strand contains nucleotide sequence II, wherein nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region, wherein nucleotide sequence I and nucleotide sequence II are selected from the following sequences:

[0006] (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:88;

[0007] (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:2, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:88;

[0008] (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:32, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:99;

[0009] (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:33, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:99;

[0010] (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:63, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:110;

[0011] (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:64, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:110.

[0012] In some embodiments, the sense strand and the antisense strand are complementary to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the sense strand forms a blunt end.

[0013] In some embodiments, the sense strand and the antisense strand are complementary to form the double-stranded region, and the 3' ends of both the sense strand and the antisense strand have 1-2 protruding nucleotides extending out of the double-stranded region.

[0014] In some embodiments, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; preferably, the phosphate ester group with a modifying group is a thiophosphate ester group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0015] In some implementations, the siRNA comprises a positive strand that does not contain a 3' overhang nucleotide.

[0016] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0017] In some embodiments, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs, or any combination of two or more thereof.

[0018] In some embodiments, the modified nucleotide is selected from 2'-fluoromodified nucleotides, 2'-methoxymodified nucleotides, 2'-O-CH2-CH2-O-CH3modified nucleotides, 2'-O-CH2-CH=CH2modified nucleotides, 2'-CH2-CH2-CH=CH2modified nucleotides, 2'-deoxymodified nucleotides, nucleotide analogs, or any combination of two or more thereof.

[0019] In some implementations, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluorinated nucleotide or a non-fluorinated nucleotide.

[0020] In some preferred embodiments, in the positive strand, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 in a 5' to 3' orientation, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 5, 7, 8, and 9, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 9, 10, and 11, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 8, 9, and 10, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 8, 9, and 11, with the remaining positions being non-fluorinated nucleotides; or, 2 The 2'-fluorinated nucleotides are located at positions 7 and 9, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotides are located at positions 7, 9, and 11, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotides are located at positions 7, 11, 13, and 15, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotides are located at positions 8, 9, 10, and 12, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotides are located at positions 7, 9, 11, 13, and 15, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotides are located at positions 9, 11, and 13, with the remaining positions being non-fluorinated nucleotides. The modified nucleotide; or, the 2'-fluorinated nucleotide is located at positions 7, 9, 11, and 13, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotide is located at positions 3, 7, 8, and 9, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotide is located at positions 3, 9, 11, and 13, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotide is located at positions 3, 7, 9, and 11, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotide is located at positions 7, 11, and 13, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotide is located at positions 7, 11, 13, with the remaining positions being non-fluorinated nucleotides; or, the 2'-fluorinated nucleotide is located at positions 7, 11, 13, 14, 15, 16, 17, 18, 19, 19, 10, 1 ... Positions 2 and 13, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 3, 7, 11, and 16, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 3, 7, 11, and 17, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 3, 7, 11, 16, and 17, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 9, 11, 12, and 13, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 5, 9, 10, and 11, with the remaining positions being non-fluorinated nucleotides.Alternatively, the 2'-fluorinated nucleotides are located at positions 5, 9, 11, and 13, with the remaining positions occupied by non-fluorinated nucleotides; and / or;

[0021] In the antisense strand, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16, with the remaining positions being non-fluorinated nucleotides, in the 5' to 3' direction; or, 2'-fluorinated nucleotides are located at positions 2, 6, and 14, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16, and 18, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 5' to 3', with the remaining positions being non-fluorinated nucleotides. Positions 2, 4, 6, 12, 14, 16, 18, and 20, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2 and 14, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 5, 7, and 14, with the remaining positions being non-fluorinated nucleotides.

[0022] In some embodiments, each nonfluorinated nucleotide is independently selected from a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group of the nucleotide with a nonfluorinated group, and the nucleotide analog is selected from a pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA and GNA.

[0023] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or any combination of two or more thereof.

[0024] In some preferred embodiments, in the positive strand, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 5, 7, 8, and 9, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 9, 10, and 11, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 8, 9, and 10, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 8, 9, and 10, with the remaining positions being 2'-methoxymodified nucleotides. 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 7 and 9, with the nucleotide at position 11 of the positive strand being a 2'-deoxy modified nucleotide, and the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 7, 9, and 11, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 7, 11, 13, and 15, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 8, 9, 10, and 12, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 7, 9, 11, 13, and 15, with the remaining positions being 2'-methoxy modified nucleotides. The nucleotides are: 5, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 9, 11, and 13, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 7, 9, 11, and 13, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 3, 7, 8, and 9, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 3, 9, 11, and 13, with the remaining positions being 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 3, 7, 9, and 11, with the remaining positions being 2'-methoxy modified nucleotides. The modified nucleotide; or, the 2'-fluoro modified nucleotide is located at positions 7, 11, and 13, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotide is located at positions 7, 11, 12, and 13, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotide is located at positions 3, 7, 11, and 16, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotide is located at positions 3, 7, 11, and 17, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotide is located at positions 3, 7, 11, 16, and 17, and the remaining positions are 2'-methoxy modified nucleotides;Alternatively, the 2'-fluoromodified nucleotide is located at positions 9, 11, 12, and 13, with the remaining positions being 2'-methoxymodified nucleotides; or, the 2'-fluoromodified nucleotide is located at positions 5, 9, 10, and 11, with the remaining positions being 2'-methoxymodified nucleotides; or, the 2'-fluoromodified nucleotide is located at positions 5, 9, 11, and 13, with the remaining positions being 2'-methoxymodified nucleotides; and / or;

[0025] In the antisense strand, 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 in the 5' to 3' direction, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 6, and 14, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16, and 18, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 4, 6, 12, 14, 16, 18, and 20, with the remaining positions being 2'-methoxymodified. The nucleotides are: 1. A 2'-fluoro modified nucleotide is located at positions 2 and 14, with the remaining positions being 2'-methoxy modified nucleotides; 2. A 2'-fluoro modified nucleotide is located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, with the remaining positions being 2'-methoxy modified nucleotides; 3. A 2'-fluoro modified nucleotide is located at positions 2, 3, 4, 5, 7, 10, and 14, with position 6 being a GNA-modified nucleotide and the remaining positions being 2'-methoxy modified nucleotides; 4. A 2'-fluoro modified nucleotide is located at positions 2, 7, 10, and 14, with the remaining positions being 2'-methoxy modified nucleotides; 5. A 2'-fluoro modified nucleotide is located at positions 2, 5, 7, and 14, with the remaining positions being 2'-methoxy modified nucleotides.

[0026] In some embodiments, neither the 5' nor 3' end of the positive strand is connected to a reverse debased deoxyribose residue. In other embodiments, only the 5' end of the positive strand is connected to a reverse debased deoxyribose residue. In still other embodiments, only the 3' end of the positive strand is connected to a reverse debased deoxyribose residue. In yet another embodiment, both the 5' and 3' ends of the positive strand are connected to a reverse debased deoxyribose residue.

[0027] In some preferred embodiments, the reverse debased deoxyribose residue is linked to the 3' terminal nucleotide and / or 5' terminal nucleotide of the positive strand via a phosphodiester bond, a thiophosphate group, or other nucleoside internucleotide bond.

[0028] In some embodiments, at least one of the following nucleotide linkages in the siRNA is a phosphate thioester linkage:

[0029] The connection between the first and second nucleotides starting at the 5' end of the positive strand;

[0030] The connection between the second and third nucleotides starting at the 5' end of the positive strand;

[0031] The connection between the first and second nucleotides starting at the 3' end of the positive strand;

[0032] The connection between the second and third nucleotides starting at the 3' end of the positive strand;

[0033] The connection between the reverse debased deoxyribose residue starting at the 3' end of the positive strand and the first nucleotide;

[0034] The link between the first and second nucleotides starting at the 5' end of the antisense strand;

[0035] The link between the second and third nucleotides starting at the 5' end of the antisense strand;

[0036] The link between the third and fourth nucleotides starting at the 5' end of the antisense strand;

[0037] The link between the first and second nucleotides starting at the 3' end of the antisense strand;

[0038] The link between the second and third nucleotides starting at the 3' end of the antisense strand.

[0039] In some preferred embodiments, the siRNA is oriented from the 5' end to the 3' end.

[0040] (1) The positive chain contains thiophosphate groups located at the positions shown below:

[0041] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0042] Between the second and third nucleotides starting at the 5' end of the positive strand; and

[0043] Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and

[0044] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0045] or,

[0046] (2) The positive chain contains thiophosphate groups located at the positions shown below:

[0047] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0048] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0049] or,

[0050] (3) The positive chain contains thiophosphate groups located at the positions shown below:

[0051] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0052] Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and

[0053] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0054] or,

[0055] (4) The positive chain contains thiophosphate groups located at the positions shown below:

[0056] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0057] or

[0058] (5) The positive chain contains thiophosphate groups located at the positions shown below:

[0059] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0060] Between the second and third nucleotides starting at the 5' end of the positive strand; and

[0061] The reverse debasing deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide and the first nucleotide.

[0062] In some preferred embodiments, the siRNA is oriented from the 5' end to the 3' end.

[0063] (i) The antisense chain contains thiophosphate groups located at the positions shown below:

[0064] Between the first nucleotide and the second nucleotide starting at the 5' end of the antisense strand; and

[0065] Between the second and third nucleotides starting at the 5' end of the antisense strand; and

[0066] Between the first nucleotide and the second nucleotide starting at the 3' end of the antisense strand; and

[0067] Between the second and third nucleotides starting at the 3' end of the antisense strand;

[0068] or

[0069] (ii) The antisense chain contains a thiophosphate group located at the position shown below:

[0070] Between the first nucleotide and the second nucleotide starting at the 5' end of the antisense strand; and

[0071] Between the second and third nucleotides starting at the 5' end of the antisense strand; and

[0072] Between the third and fourth nucleotides starting at the 5' end of the antisense strand; and

[0073] Between the first and second nucleotides starting at the 3' end of the antisense strand.

[0074] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or any combination of two or more thereof.

[0075] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0076] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0077] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0078] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0079] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0080] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0081] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 8, 9, and 10 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0082] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16, and 18 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0083] In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxylated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0084] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0085] In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 8, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0086] In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 4, 6, 12, 14, 16, 18, and 20 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0087] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2 and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0088] In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 7 and 9 of the sense strand, with the nucleotide at position 11 of the sense strand being a 2'-deoxy-modified nucleotide, and the remaining positions being 2'-methoxy-modified nucleotides; 2'-fluorinated nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18 of the antisense strand, with the remaining positions being 2'-methoxy-modified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0089] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7 and 9 of the sense strand, with the nucleotide at position 11 of the sense strand being a 2'-deoxymodified nucleotide, and the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0090] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0091] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0092] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 11, 13, and 15 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0093] In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 8, 9, 10, and 12 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxylated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0094] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 11, 13, and 15 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0095] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 11, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0096] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 11, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0097] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 11, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0098] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 8, and 9 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0099] In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 3, 7, 8, and 9 of the sense strand in a 5' to 3' orientation, with the remaining positions occupied by 2'-methoxylated nucleotides, and the 3' end is removed; 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions occupied by 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0100] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 9, 11, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0101] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 9, 11, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is removed; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0102] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0103] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is removed; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0104] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 11, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0105] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 11, 12, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0106] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 11, and 16 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0107] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 11, and 17 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0108] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 11, 16, and 17 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0109] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with position 6 being a GNA-modified nucleotide, and the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0110] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 11, 12, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0111] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 11, 12, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0112] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 9, 11, 12, and 13 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0113] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 5, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is removed; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0114] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 5, 9, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0115] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0116] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0117] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 8, and 9 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is removed; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0118] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0119] In some specific embodiments, the present invention provides siRNAs selected from Table 1; preferably, the siRNAs are selected from N-ER-FY028061M37, N-ER-FY028061M40, N-ER-FY028061M44, N-ER-FY028061M45, N-ER-FY028151M37, N-ER-FY028151M40, N-ER- FY028151M44, N-ER-FY028151M45, N-ER-FY028179M46, N-ER-FY028179M47, N-ER-FY0281 79M48, N-ER-FY028179M49, N-ER-FY028061M54, N-ER-FY028151M54, N-ER-FY028179M53.

[0120] The present invention also provides an siRNA conjugate containing the siRNA of the present invention and a conjugating group conjugated to the siRNA (as shown in the following formula, where a double helix structure represents the siRNA and the conjugating group is attached to the 3' end of the positive strand of the siRNA):

[0121]

[0122] In the above conjugate structure, X can be selected as O or S, and in some embodiments, X is O.

[0123] In some embodiments, in the siRNA conjugate, the sense strand and antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region;

[0124] or,

[0125] In the siRNA conjugate, the sense strand and antisense strand of the siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand is blunt, and the 3' end of the antisense strand is blunt.

[0126] In some embodiments, the conjugating group (wherein, exemplarily shown in some conjugating groups, a phosphate ester bond or a thiophosphate ester moiety) is selected from:

[0127]

[0128]

[0129]

[0130] In some specific embodiments, the siRNA conjugate is selected from the siRNA conjugates in Table 2; preferably, the siRNA conjugate is selected from N-ER-FY028061M37L96, N-ER-FY028061M40L96, N-ER-FY028061M44L96, N-ER-FY028061M45L96, N-ER-FY028151M37L96, N-ER-FY028151M40L96, N-ER -FY028151M44L96, N-ER-FY028151M45L96, N-ER-FY028179M46L96, N-ER-FY028179M47L96, N-ER-FY02817 9M48L96, N-ER-FY028179M49L96, N-ER-FY028061M54L96, N-ER-FY028151M54L96, N-ER-FY028179M53L96.

[0131] The present invention also provides a pharmaceutical composition comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, and a pharmaceutically acceptable carrier.

[0132] The present invention also provides a kit comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.

[0133] The present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for the preparation of an agent that inhibits the expression of the CPB2 gene.

[0134] The present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for the preparation of medicaments for the prevention and / or treatment of diseases related to CPB2 gene overexpression.

[0135] In some specific implementations, the disease is thrombosis, pulmonary embolism, stroke, myocardial infarction, and ischemic stroke; preferably, the thrombosis includes venous thrombosis, deep vein thrombosis, atherosclerotic thrombosis, prosthetic valve thrombosis, microvascular thrombosis, and dynamic thrombosis.

[0136] The present invention also provides a method for inhibiting CPB2 gene expression, comprising contacting or administering the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to cells expressing CPB2.

[0137] The present invention also provides a method for treating and / or preventing diseases associated with CPB2 gene overexpression, comprising administering a therapeutically effective or preventively effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to a subject in need.

[0138] In some specific implementations, the disease is thrombosis, pulmonary embolism, stroke, myocardial infarction, and ischemic stroke; preferably, the thrombosis includes venous thrombosis, deep vein thrombosis, atherosclerotic thrombosis, prosthetic valve thrombosis, microvascular thrombosis, and dynamic thrombosis.

[0139] The present invention also provides the siRNA of the present invention for use in treatment or as a medicament, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.

[0140] Beneficial effects

[0141] The siRNA, pharmaceutical composition, and siRNA conjugates provided by this invention have shown excellent CPB2 gene expression inhibitory activity in in vitro cell experiments, demonstrating good potential for treating diseases related to CPB2 gene overexpression. For example, the siRNA and its conjugates disclosed in this invention can reduce the expression of CPB2 mRNA in the liver, with low toxicity and good plasma stability, showing promising clinical application prospects.

[0142] The siRNA provided by this invention showed a good inhibitory effect on the CPB2 gene in Huh7 cells. Detailed Implementation

[0143] definition

[0144] Throughout this specification, unless otherwise specified, in this technical field, "G", "C", "A", "T" and "U" generally represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively. However, it is also generally known in the art that each of "G", "C", "A", "T" and "U" typically also represents a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of "G", "C", "A", "T", and "U" include all the above-mentioned possible cases. In this document, "nucleotide", "ribonucleic acid", and "ribonucleotide" are used interchangeably, as are "deoxyribonucleic acid" and "2'-deoxyribonucleic acid". Lowercase letters a, u, c, g: indicate nucleotides modified with 2'-methoxy groups; Af, Gf, Cf, Uf: indicate nucleotides modified with 2'-fluoride groups; lowercase letter s: indicates that the two nucleotides adjacent to s are linked by thiophosphate groups; "dG", "dC", "dA", "dT", and "dU": indicate that the ribonucleotide at the corresponding position in the motif sequence is modified with 2'-deoxy groups; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP: indicates that the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide; (Underline + Bold + Italic): indicates a GNA-modified nucleotide; Base indicates a base, such as A, U, G, C, or T; "(invAb)": indicates a reverse debasing deoxyribose residue.

[0145] In the foregoing and hereinafter, "2'-fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine. "Non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group. These nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides may be selected from one of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxy-modified nucleotides.

[0146] "Alkyl" includes straight-chain, branched, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl, and similar groups. For example, "C 1-6 The "C" in "alkyl" 1-6 "" refers to a group consisting of 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched chain or cyclic form.

[0147] "Alkoxy" herein refers to an alkyl group that is attached to the remainder of a molecule by an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, etc.

[0148] "Nucleotide analogues" refer to groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include pseudouracil (Ψ), isonucleotides, bridged nucleic acids (BNA), or acyclic nucleotides.

[0149] Pseudouracil (Ψ) refers to a natural structural analog of a uracil nucleoside, in which the ribose is not linked to the N1 of the uracil ring, but rather to the C5 of the pyrimidine ring. Its structural formula is as follows:

[0150]

[0151] BNA refers to a restricted or inaccessible nucleotide. BNA can contain a five-membered, six-membered, or seven-membered ring with a "fixed" C3'-endoglycan condensation bridging structure. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is shown in formula (1), ENA in formula (2), and cET BNA in formula (3).

[0152]

[0153]

[0154] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (4), and GNA is shown in formula (5).

[0155]

[0156] In formulas (4) and (5) above, R is selected from H, OH or alkoxy (O-alkyl).

[0157] Heteronucleotides are compounds formed by changing the position of the bases in the ribose ring of a nucleotide. For example, compounds formed by moving the bases from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (6) or (7):

[0158]

[0159] In the compounds of formulas (6)-(7) above, Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0160] In some embodiments, the nucleotide analogue is selected from one of pseudouracil, isonucleotides, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or any combination of two or more thereof. In some preferred embodiments, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, wherein, as stated above and below, the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0161] The term "2'-methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group. The term "thiophosphate group" refers to a thiophosphate group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0162] The "thiophosphate group" refers to the following formula:

[0163]

[0164] The "5'-phosphate nucleotide" refers to the structure of the following formula:

[0165]

[0166] In the context of this specification, the terms "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in the case of a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0167] Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0168] In the preceding and following text, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered a nucleotide difference at that position.

[0169] In this context, a "protruding end" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of an siRNA when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. A "flat-ended" or "knock-off" siRNA means that there are no unpaired nucleotides at that end of the siRNA, i.e., no nucleotide protrusions. A "flat-ended" siRNA is a double-stranded siRNA that is double-stranded throughout its entire length, meaning there are no nucleotide protrusions at either end of the molecule. A "double-stranded region" refers to the complementary double-stranded region of the siRNA formed by the sense and antisense strands.

[0170] In the preceding and following text, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of a pentose sugar, and it is the main type of nucleotide that exists freely in organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of a pentose sugar, and may include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-thiophosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-thiophosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methylguanosine-3'-phosphate. The terms siRNA, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-phosphate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-phosphate, and 2'-deoxythymidine-3'-phosphate are used interchangeably in the context of this disclosure. The terms "iRNA," "RNAi reagent," "iRNA agent," and "RNA interference agent" as used herein are interchangeable and refer to terms defined herein that include siRNA and mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate, for example, the expression of target genes in cells (such as the cells of a subject, like a mammalian subject).

[0171] As used herein, "2'-deoxy-modified nucleotide" or "2'-deoxy modification" refers to the replacement of ribonucleotides A, U, C, and G with their corresponding deoxyribonucleotides dA, dT, dC, and dG during the modification process. For example, following the 5' to 3' direction, the 11th nucleotide of the positive strand (SEQ ID NO:2) of N-ER-FY028061D2 ​​is replaced with a deoxyribonucleotide, which means that the 11th ribonucleotide "A" is replaced with "dA"; similarly, uracil ribonucleotide (U) is replaced with its corresponding deoxyribonucleotide (dT); cytosine ribonucleotide (C) is replaced with its corresponding cytosine deoxyribonucleotide (dC); and guanine ribonucleotide (G) is replaced with its corresponding guanine deoxyribonucleotide (dG). In this document, "2'-deoxy-modified nucleotide" or "2'-deoxy modification" is also referred to as "nucleotide replacement with deoxyribonucleotide".

[0172] Throughout this invention specification, particularly in the description of methods for preparing siRNA, pharmaceutical compositions, or siRNA conjugates, unless otherwise specified, the term "nucleoside monomer" refers to a modified or unmodified nucleoside phosphorus amide monomer used in solid-phase phosphorus amide synthesis, depending on the type and sequence of nucleotides in the desired siRNA or siRNA conjugate. Solid-phase phosphorus amide synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this invention are commercially available.

[0173] In the context of this invention, unless otherwise stated, "conjugation" refers to the covalent connection between two or more chemical parts, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical parts. Further, "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts having a specific function to siRNA. siRNA conjugated compound should be understood, depending on the context, as a collective term for multiple siRNA conjugated compounds or a siRNA conjugated compound represented by a specific chemical formula. In the context of this specification, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to siRNA to ultimately form the siRNA conjugated compound of this invention.

[0174] Various hydroxyl protecting groups can be used in this invention. Generally, protecting groups insensitize chemical functional groups to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially impairing the rest of the molecule. In some embodiments, protecting groups are stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used in this invention include monomethoxytriphenylmethyl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used in this invention include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0175] As used in this specification, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition.

[0176] The term “subject” as used in this specification refers to any animal, such as a mammal or marsupial. Subjects of this invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any kind of poultry.

[0177] As used in this specification, “treatment” means a method of obtaining a beneficial or desired outcome, including but not limited to treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0178] As used in this specification, “prevention” means a method of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” siRNA, siRNA conjugates, or pharmaceutical compositions may be given to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0179] siRNA

[0180] This invention relates to an siRNA capable of inhibiting CPB2 gene expression. The siRNA of this invention contains nucleotide groups as basic structural units, as is known to those skilled in the art, wherein the nucleotide groups contain phosphate groups, ribose groups, and bases. Typically, an active, i.e., functional siRNA is approximately 12-40 nucleotides in length, and in some embodiments, approximately 15-30 nucleotides.

[0181] The siRNA of the present invention comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified, wherein the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II, wherein nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. In some embodiments, the length of the double-stranded region is 15-30 nucleotide pairs. In other embodiments, the length of the double-stranded region is 17-23 nucleotide pairs. In still other embodiments, the length of the double-stranded region is 19-21 nucleotide pairs. In yet another embodiment, the length of the double-stranded region is 19 or 21 nucleotide pairs.

[0182] The sense and antisense strands provided by this invention may have the same or different lengths. In some embodiments, the sense or antisense strand has 15-30 nucleotides. In other embodiments, the sense or antisense strand has 19-25 nucleotides. In still other embodiments, the sense or antisense strand has 19-23 nucleotides. The length ratio of the sense and antisense strands of the siRNA provided by this invention can be 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 19, 21 / 20, 21 / 21, 21 / 22, 21 / 23. Examples of possible siRNA length ratios include 22 / 19, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, 28 / 28, 29 / 29, 30 / 30, 22 / 24, 22 / 25, 22 / 26, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 19, 19 / 21, 21 / 23, 21 / 21, or 23 / 23, in which case the siRNA of this disclosure exhibits better cellular mRNA silencing activity.

[0183] Studies have found that different modification strategies can have drastically different effects on the stability, bioactivity, and cytotoxicity of siRNA. For example, CN201010106762.1 investigated various chemical modification strategies for siRNA, confirming seven effective modification methods. Compared with unmodified siRNA, one of the modification methods resulted in siRNA that improved blood stability while maintaining inhibitory activity essentially equivalent to that of unmodified siRNA.

[0184] The nucleotides in the siRNA of the present invention are each independently modified or unmodified. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides, and these modifications on the nucleotide groups do not cause the siRNA of the present invention to significantly weaken or lose its function of inhibiting CPB2 gene expression.

[0185] In some embodiments, the siRNA of the present invention contains at least one modified nucleotide. In the context of this invention, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with another group, or a nucleotide having a modified base. The modified nucleotide does not cause a significant reduction or loss of the siRNA's ability to suppress gene expression. For example, the modified nucleotide disclosed in J.K. Watts, G.G. Deleavey, and M.J. Damha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 may be selected.

[0186] In some embodiments, at least one nucleotide in the sense strand or antisense strand of the siRNA provided by the present invention is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; in other words, at least a portion of the phosphate ester group and / or ribosome in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate ester group and / or a ribosome with a modifying group. In some embodiments, the phosphate ester group with a modifying group is a thiophosphate ester group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0187] In some embodiments, the siRNA comprises a positive strand that does not contain a 3' overhanging nucleotide; that is, the positive strand of the siRNA may contain a 3' overhanging nucleotide, and excluding the 3' overhanging nucleotide of the positive strand results in a blunt end. In some embodiments, the siRNA comprises siRNA whose 3' end of the positive strand in Table 1 is modified to have a blunt end, optionally obtained by excluding the overhanging end of the positive strand in Table 1 that extends into a double-stranded region.

[0188] In some implementations, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and there is no protruding nucleotide at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as the protruding nucleotide. Then, after the nucleotide sequence formed by linking nucleotide sequence V to the 3' end of the sense strand is chemically modified, nucleotide sequence V is excluded, and correspondingly, the sense strand of the siRNA forms a blunt end.

[0189] In some implementations, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the protruding nucleotide at the 3' end of the sense strand is excluded and the nucleotide sequence of the sense strand is obtained. Accordingly, the sense strand of the siRNA forms a blunt end.

[0190] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

[0191] When the 5' terminal nucleotide of the antisense strand is attached to a 5' phosphate group or a 5' phosphate derivative, the following structure is formed:

[0192]

[0193] Formula (8) shows a structure formed by linking a 5' nucleotide to a 5' phosphate group, Formula (9) shows a structure formed by linking a 5' nucleotide to a 5' phosphate-derived group (EVP), and Formula (10) shows a structure formed by linking a 5' nucleotide to a 5' phosphate-derived group (5' methylene phosphate group). Here, Base represents a base, such as A, U, G, C, or T. R' is a hydroxyl group or is substituted by various groups known to those skilled in the art. For example, the substituted modified nucleotide can be a 2'-fluoro(2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxy modified nucleotide.

[0194] In some embodiments, the 5' terminal nucleotide of the sense or antisense strand is not linked to a 5' phosphate group, a 5' phosphate-derived group, or (invAb) (i.e., the ribonucleotide of the 5' terminal nucleotide of the sense or antisense strand is a 5' hydroxyl group), and its structure is shown below:

[0195]

[0196] Wherein, Base represents a base, such as A, U, G, C, or T. R is a hydroxyl group or hydrogen, or is substituted by various groups known to those skilled in the art, for example, R can be fluorine, alkoxy, substituted alkoxy, alkyl, substituted alkyl, amino, or substituted amino.

[0197] An exemplary modified nucleotide has the following structure:

[0198]

[0199] Wherein, Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribose group is replaced by R. These hydroxyl groups at the 2' position of the ribose group can be replaced by various groups known to those skilled in the art, for example, the substituted modified nucleotide can be a 2'-fluoro(2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxy modified nucleotide.

[0200] In some embodiments, the positive strand may contain one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capping residue.” As used herein, a “capping residue” is a nonnucleotide compound or other portion that may be incorporated at one or more ends of the nucleotide sequence of the siRNA disclosed herein. In some cases, capping residues may provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverse debased deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue appears at the 5' end, the 3' end, or both the 5' and 3' ends of the positive strand.

[0201] In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 3' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 5' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to both the 5' end and the 3' end of the positive strand. The inverted debased deoxyribose residues may be linked via phosphate ester bonds, thiophosphate ester groups, or other nucleoside internucleotide bonds. When describing modification sites in modification methods, (invAb) is not counted as the first site of the sequence. In some embodiments, inverted debased deoxyribose residues (invAb) (also referred to in the art as "inverted debasing sites") may be added. Wherein, when (invAb) is located at the 3' end of the siRNA, its structure is Formula B; when (invAb) is located at the 5' end of the siRNA, its structure is Formula C:

[0202]

[0203] siRNA conjugates

[0204] This invention relates to an siRNA conjugate containing the aforementioned siRNA and a conjugate group conjugated to the siRNA.

[0205] In this invention, the sense and antisense strands of the siRNA conjugate form a double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the siRNA conjugate. In some embodiments, the 3' end of the sense strand of the siRNA conjugate is blunt, and the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of both the sense and antisense strands of the siRNA conjugate is blunt. In this invention, "the 3' end of the sense strand is blunt" includes the case where the 3' end of the siRNA sense strand is inherently blunt, and the case where the 3' end of the siRNA sense strand has a protruding end, but the protruding end is excluded to form a blunt end.

[0206] In some preferred embodiments, the siRNA conjugate is obtained by conjugating siRNA with a conjugating group. Specifically, the sense and antisense strands of the siRNA are complementary to form a double-stranded region of the siRNA, and the 3' end of the sense strand of the siRNA forms a blunt end. The conjugating group is conjugated to the 3' end of the sense strand with the blunt end to form the siRNA conjugate.

[0207] For example, the siRNA with the sequence N-ER-FY028061M10D2 has a blunt end at the 3' end of the sense strand. The blunt end sequence at the 3' end of the sense strand is used as the nucleotide sequence for connecting the L96 conjugate group. Therefore, the sense strand of the siRNA conjugate N-ER-FY028061M10L96 is gsusucuaCfaGfAfAfucuuacuacaL96, and the antisense strand is EVPusGfsuagUfaagauucUfguagaacsusu.

[0208] In some preferred embodiments, the 3' end of the positive strand of the siRNA has a protruding nucleotide extending into a double-stranded region. The sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the positive strand is used as the nucleotide sequence for attaching the conjugate group. The conjugate group is attached to the blunt 3' end of the positive strand to form an siRNA conjugate.

[0209] In some preferred embodiments, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and there is no protruding nucleotide at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as the protruding nucleotide. The sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for attaching the conjugate group. The conjugate group is attached to the blunt 3' end of the sense strand to form an siRNA conjugate.

[0210] In some preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugate group. The conjugate group is connected to the blunt 3' end of the sense strand to form an siRNA conjugate.

[0211] For example, the siRNA sequence N-ER-FY028061M10L96 has a protruding nucleotide extending into a double-stranded region at the 3' end of the sense strand. The blunt-ended sequence gsusucuaCfaGfAfAfucuuacuaca formed by excluding the protruding -sasc nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the L96 conjugate group. Therefore, the sequence forming the siRNA conjugate is: sense strand gsusucuaCfaGfAfAfucuuacuacaL96, antisense strand EVPusGfsuagUfaagauucUfguagaacsusu.

[0212] Generally, the conjugation group comprises at least one pharmaceutically acceptable target group, or further comprises a linker, and the siRNA, the linker, and the target group are sequentially linked. In some embodiments, there are 1-6 target groups. In some embodiments, there are 2-4 target groups. The siRNA molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the siRNA and the conjugation group can be at the 3' end or 5' end of the siRNA's sense strand, at the 5' end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3' end of the siRNA's sense strand.

[0213] In some embodiments, the conjugate group may be attached to a phosphate group, a 2'-hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugate group may also be attached to a 3'-hydroxyl group, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. When the conjugate group is attached to the end of the siRNA chain, it is usually attached to a phosphate group of the nucleotide; when the conjugate group is attached to the inner sequence of the siRNA, it is usually attached to a ribose ring or a base. Various connection methods can be found in the reference: Muthiah Manoharanet.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes. ACS Chemicalbiology, 2015, 10(5):1181-7.

[0214] In some embodiments, the siRNA and the conjugate group are linked by acid-labile or reducible chemical bonds. These bonds can degrade in the acidic environment of the endosomes, thus freeing the siRNA. For non-degradable conjugates, the conjugate group can be attached to the positive and negative strands of the siRNA to minimize the impact of the conjugate on the siRNA's activity.

[0215] In some embodiments, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA delivery, such as the various ligands described in WO2009082607A2, which are incorporated herein by reference in their entirety.

[0216] In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more ligands formed from the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0217] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to the liver surface desialylate glycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of ligand-linked siRNA to the target cells.

[0218] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the desialyl glycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently a desialyl glycoprotein, such as asialolesomucoid (ASOR) or asialofetin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0219] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0220] In some embodiments, each of the ligands may be independently selected from D-mannose, L-mannose, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannose, β-D-mannose, α-D-mannose, β-D-mannose, α-D-glucose pyranose, β-D-mannose pyranose, α-D-glucose pyranose, β-D-glucose pyranose, β-D-glucose pyranose, β-D-glucose pyranose Sugars, α-D-furanose glucose, β-D-furanose glucose, α-D-furanofructose, α-D-fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactopyranose, β-D-galactopyranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine 2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannpyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-Tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-aloxonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other options for the ligands may be found, for example, in CN201480035633.4, which is incorporated herein by reference in its entirety.

[0221] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the terms monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4, respectively, after the siRNA molecule forms a conjugate with a conjugate group containing galactose or N-acetylgalactosamine as a targeting group. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated with a conjugate group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present invention is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0222] The targeting group can be linked to the siRNA molecule via a suitable adapter. Those skilled in the art can select a suitable adapter based on the specific type of the targeting group. For details on these adapters, the types of targeting groups, and the connection methods with siRNA, please refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0223] siRNA synthesis methods

[0224] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using the conventional solid-phase phosphoramide method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and capping. When two nucleotides are linked using a phosphate ester, the linkage of the next nucleoside monomer involves these four steps. When two nucleotides are linked using a thiophosphate ester, the linkage of the next nucleoside monomer involves these four steps. The present invention selects nucleotide monomers based on the target sequence. The selected nucleotide monomers are those commonly used by those skilled in the art; for example, the nucleotide monomer for synthesizing A can be, but is not limited to, adenosine-3-phosphate. It should be understood that these monomers, when present in oligonucleotides, are linked together via 5′-3′ phosphodiester bonds or 5′-3′ thiophosphate groups. When, for example, the 3' position of the last nucleotide in the 5′ to 3′ direction is a hydroxyl group, this is achieved using conventional methods in the art.

[0225] For example, the synthesis conditions of the siRNA of the present invention can be as follows:

[0226] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% V / V). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0227] The coupling reaction conditions included: a reaction temperature of 25°C, a reaction time of 600 seconds, a coupling reagent selected from a 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, and a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65.

[0228] The oxidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the oxidizing agent being 0.05M iodine water. The molar ratio of the oxidizing agent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0229] The sulfidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 300 seconds, and the sulfidation reagent was selected from hydroflavin. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0230] The capping reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the capping reagent being a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazolium pyridine / acetonitrile solution) in a molar ratio of 1:1. The molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazolium:the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0231] After linking all nucleoside monomers, the nucleic acid sequences linked on the solid-phase support were sequentially subjected to ammonolysis, purification, and desalting to obtain the siRNA sense and antisense strands. Finally, the two strands were heated and annealed to obtain the product.

[0232] Methods for ammonolysis, purification, desalting, and annealing are well known in the art. For example, ammonolysis is performed by contacting the nucleotide sequence linked to a solid-phase support with concentrated ammonia; purification is performed by chromatography; desalting is performed by reversed-phase chromatography; and cooling is performed by gradually cooling after mixing sense and antisense strands in equimolar ratios under different stringent conditions.

[0233] The synthesized siRNAs are shown in Table 1.

[0234] siRNA conjugate synthesis method

[0235] Taking the synthesis of L96 as an example:

[0236]

[0237] The first step involves reacting DMTr-L96 with succinic anhydride to obtain compound L96-A:

[0238] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine, and diisopropylethylamine were added to dichloromethane and stirred at 25°C for 24 hours. The reaction solution was then washed with 0.5M triethylamine phosphate, and the aqueous phase was washed three times with dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. Then, column chromatography was used to purify the product to obtain pure L96-A.

[0239] The second step involves reacting L96-A with NH2-SPS to obtain L96-B:

[0240]

[0241] Preparation process: L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile. The mixture was stirred at room temperature for 5 minutes to obtain a homogeneous solution. Aminomethyl resin (NH2-SPS, 100-200 mesh) was added to the reaction solution, and the reaction was initiated at 25°C in a shaker. After 18 hours of reaction, the mixture was filtered. The filter cake was washed successively with dichloromethane and acetonitrile to obtain the filter cake. The obtained filter cake was subjected to a capping reaction with a CapA / CapB mixed solution to obtain L96-B, which is the solid-phase support containing the conjugated molecules.

[0242] The third step is to prepare siRNA conjugates.

[0243] Using L96-B as a solid-phase support, the siRNA was synthesized and ligated to the siRNA sense strand according to the siRNA synthesis method described above. The siRNA antisense strand was then synthesized using the same method, and annealed to generate the siRNA conjugate of this invention.

[0244] The synthesized siRNA conjugates are shown in Table 2.

[0245] Pharmaceutical Composition

[0246] The present invention provides a pharmaceutical composition comprising siRNA as an active ingredient and a pharmaceutically acceptable carrier as described above.

[0247] The pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA delivery, such as, but not limited to, lipid nanoparticles (LNP), magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethylethylene) phosphate. One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethyl methacrylate), PDMAEMA, and their derivatives.

[0248] The pharmaceutical composition does not have specific requirements for the content of siRNA and pharmaceutically acceptable carriers; the content of each component can be the conventional content.

[0249] In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0250] The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate buffer with a pH of 5.5-8.5.

[0251] The protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0252] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure.

[0253] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous injection, or may be administered via a spray to the lungs, or via a spray to other organs or tissues (such as the liver). In some embodiments, the pharmaceutical composition is used for intravenous injection.

[0254] In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid.

[0255] The following examples are used to further illustrate the present invention, but do not limit the present invention in any way.

[0256] Example

[0257] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0258] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0259] Example 1: Preparation of siRNA

[0260] Tianlin Biotechnology (Shanghai) Co., Ltd. synthesized siRNA molecules with the following sequence.

[0261] Table 1. siRNAs and their sequences

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] In this context, the uppercase letters “G”, “C”, “A”, and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively; the lowercase letters a, u, c, and g indicate nucleotides modified with a 2'-methoxy group; Af, Gf, Cf, and Uf indicate nucleotides modified with a 2'-fluoride group; the lowercase letter s indicates that the two nucleotides adjacent to it are linked by a phosphate thioester group; “dG”, “dC”, “dA”, “dT”, and “dU” indicate that the ribonucleotide at the corresponding position in the motif sequence is modified with a 2'-deoxy group; and EVP indicates that the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide. (Underlined + Bold + Italic): indicates GNA-modified nucleotides; "(invAb)": indicates reverse debasing deoxyribose residues.

[0268] Tianlin Biotechnology (Shanghai) Co., Ltd. synthesized siRNA conjugates with the following sequences:

[0269] Table 2 siRNA conjugates and their sequences:

[0270]

[0271]

[0272]

[0273]

[0274] L96 is connected to the 3' end of the positive chain in Table 1 or the blunt end formed by the 3' end of the positive chain via a phosphodiester bond. L96 is:

[0275]

[0276] In Tables 1 and 2, if the left side of the 5' terminal nucleotide of the positive strand, the modified positive strand, and the modified positive strand with a conjugated group is not marked with P1, EVP, or (invAb), it means that the 5' terminal nucleotide is not connected to a 5' phosphate group, a 5' phosphate-derived group, or (invAb) (i.e., the ribonucleotide of the 5' terminal nucleotide is a 5' hydroxyl group), and its structure is shown in Formula X:

[0277]

[0278] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or hydrogen or is substituted by various groups known to those skilled in the art, for example, R can be fluorine, alkoxy, substituted alkoxy, alkyl, substituted alkyl, amino, substituted amino.

[0279] In Tables 1 and 2, if the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with P1 or EVP to the left, it means that the ribosome of the 5' terminal nucleotide is a 5' hydroxyl group, and its structure is as shown in Formula X.

[0280] In Tables 1 and 2, when the 3' ends of the sense strand and the modified sense strand are not connected (invAb), the 3' position of the 3' terminal nucleotides of the sense strand and the modified sense strand is a hydroxyl group; in Tables 1 and 2, the 3' terminal nucleotides of the antisense strand and the modified antisense strand are a hydroxyl group.

[0281] Example 2: siRNA and siRNA conjugates inhibit CPB2 gene expression

[0282] Experimental materials:

[0283] Huh7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-526.

[0284] RNA extraction kit, purchased from QIAGEN, catalog number QIAGEN-74106;

[0285] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0286] MEM, purchased from Gibco, item number 41090036;

[0287] Reverse transcription kit ( III 1st Strand cDNA Synthesis Kit (+gDNA wiper), purchased from Vazyme, catalog number R312-02;

[0288] TaqManTM Gene expression premix, purchased from Applied Biosystems, catalog number 4369016;

[0289] Opti-medium, purchased from Gibco, item number 31985070;

[0290] TaqMan probe primers (CPB2), purchased from Thermo, Assay ID: Hs00255533_m1;

[0291] TaqMan probe primers (GAPDH), purchased from Thermo, Assay ID: Hs99999905_m1.

[0292] Experimental methods:

[0293] 1. Resuspend Huh7 cells in PS-free (penicillin-streptomycin mixture) MEM medium to a density of 2×10⁻⁶ cells / mL. 5 A 100 μL / well cell suspension was spread into each well of a 96-well plate, resulting in 20,000 cells per well.

[0294] 2. The dry powder of the siRNA to be tested and the siRNA conjugate (for ease of description, it is collectively referred to as siRNA in the experimental process description of this embodiment) is centrifuged at low temperature and high speed, and then dissolved in enzyme-free sterile water to prepare a 10μM siRNA stock solution.

[0295] 3. Prepare 0.1 nM siRNA transfection dilution buffer and 0.01 nM siRNA transfection dilution buffer.

[0296] (1) Preparation of 0.01 μM siRNA stock solution:

[0297] a. Take 2 μL of the above 10 μM siRNA stock solution and add 18 μL of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 1 μM.

[0298] b. Take 2 μL of the 1 μM siRNA dilution solution prepared in step a), add 18 μL of ultrapure distilled water to obtain a stock solution with a final concentration of 0.1 μM siRNA.

[0299] c. Take 2 μL of the 0.1 μM siRNA stock solution prepared in step b) and add 18 μL of ultrapure distilled water to obtain a final concentration of 0.01 μM siRNA stock solution.

[0300] (2) Take 4.8 μL of the prepared 0.01 μM siRNA stock solution and add 35.2 μL of Opti-medium to obtain a 1.2 nM siRNA dilution solution;

[0301] (3) 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent was mixed with the above 1.2 nM siRNA diluent at a volume ratio of 1:1 and allowed to stand for 5 minutes to obtain 0.6 nM siRNA containing the transfection reagent diluent.

[0302] (4) Add 20 μL of 0.6 nM siRNA containing transfection reagent dilution to a 96-well plate to transfect the cultured Huh7 cells. The final volume is 120 μL (the concentration of siRNA in this system is 0.1 nM).

[0303] The 0.01 nM siRNA transfection dilution can be prepared using a similar procedure as described above.

[0304] 4. After transfection, culture for 24 hours, and set up 3 replicates for each concentration.

[0305] 5. Extract total RNA according to the RNA extraction kit instructions:

[0306] 6. Reverse transcription of total RNA to obtain cDNA, following these steps:

[0307] a) Remove gDNA using gDNAase according to the table below;

[0308] Volume / μL 5×gDNABuffer 2 Sample (RNA) 8

[0309] Run the program at 42℃ for 2 minutes;

[0310] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription:

[0311] Volume / uL The mixture in step a) 10 10×RT Mix 2 HiScript Ⅲ Enzyme Mix 2 <![CDATA[Oligo(dT) 20 VN]]> 1 Random hexamers 1 <![CDATA[RNase-free ddH2O]]> 4

[0312] 50℃, 15min; 85℃, 5s.

[0313] c) Store the reverse transcription product obtained in step b) at 4°C for real-time PCR analysis.

[0314] 7. Perform real-time PCR analysis

[0315] a) Prepare the qPCR reaction mixture as shown in the table below. Throughout the entire process, all reagents should be kept on ice.

[0316] Volume / μL <![CDATA[TaqMan TM Gene expression premix 5 20×GAPDH TaqMan probe / primer 0.5 20×target special gene TaqMan probe / primer(CPB2) 0.5 cDNA template obtained in step 6 4

[0317] b) Perform the qPCR procedure as described below.

[0318] 50℃, 2 minutes; 95℃, 10 minutes;

[0319] 95℃, 15 seconds, 60℃, 1 minute (this operation is repeated 40 times).

[0320] 8. Results Analysis

[0321] a) Use Quant Studio 6Flex software with default settings to automatically calculate the Ct value;

[0322] b) Calculate the relative expression level of the gene using the following formula:

[0323] ΔCt = Ct(CPB2 gene) – Ct(GAPDH)

[0324] ΔCt = ΔCt(test sample group) - ΔCt(Mock group), where the Mock group represents the group without siRNA compared to the test sample group;

[0325] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0326] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0327] 9. Experimental Results

[0328] The siRNA concentrations of 0.1 nM and 0.01 nM were selected for testing, and the results are shown in Table 3 and Table 3-1.

[0329] Table 3 Inhibition rate of the siRNA of the present invention

[0330]

[0331]

[0332] Table 3-1 Inhibition rate of the siRNA conjugate of the present invention

[0333]

[0334]

[0335] Note: The result of "--" is not shown.

[0336] As can be seen from Tables 3 and 3-1, the siRNA and its conjugates provided by this invention exhibit good inhibitory effects on the CPB2 gene in Huh7 cells.

[0337] Example 3: In vitro stability test of rat liver homogenate

[0338] 1. Experimental reagents and consumables

[0339] Reagent Name brand Item number / CAS number <![CDATA[1MMgCl2]]> Beyotime ST269 ammonium bicarbonate Tianjin Guangfu Fine Chemical GB663-78 Sodium dihydrogen phosphate Tianjin Guangfu Fine Chemical GB / T1267-1999 disodium hydrogen phosphate Tianjin Guangfu Fine Chemical 10039-32-4 Acetonitrile Honeywell AH015-4HC methanol Honeywell AH230-4HC 0.5MEDTA Beyotime ST066

[0340] 2. Experimental Procedure

[0341] 2.1 Preparation of liver homogenate

[0342] 2.1.1 Preparation of grinding fluid

[0343] Reagent Name Preparation method 5mM EDTA solution Pour 1 mL of 0.5 M EDTA into a 100 mL volumetric flask, dilute to the mark with purified water, and mix well. 100mM magnesium chloride solution Take 1 mL of 1M magnesium chloride into a 10 mL volumetric flask, dilute to the mark with purified water, and mix well. Grinding fluid Adjust the pH to 6.0 by adding 1 mL of EDTA solution, 1 mL of magnesium chloride solution, and 98 mL of purified water.

[0344] 2.1.2 Tissue Homogenization

[0345] Rat liver tissue (collected from SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was mixed with homogenizing solution at a ratio of 100mg:5mL to prepare liver homogenate (concentration of 20mg / mL). After preparation, the homogenate was added to a homogenizer and the homogenization parameters were set as follows.

[0346] Running speed 60Hz Runtime 30s Pause time 15s Number of runs 4 times Operating temperature -20℃

[0347] 2.2 Sample Preparation

[0348] The siRNA conjugate sample was prepared into a 1 mg / mL solution using enzyme-free water and set aside for use. The internal standard sample was prepared into a 0.125 mg / mL solution using enzyme-free water.

[0349] 2.3 Sample incubation

[0350] (1) Add 250 μL of the prepared liver homogenate to a 2 mL enzyme-free tube.

[0351] (2) Add 50 μL of nucleic acid sample to the sample from step 1;

[0352] (3) The system consists of 300 μL of biological sample. Vortex and let stand for 5 min.

[0353] (4) Divide into 2 tubes, each containing 100 μL;

[0354] (5) The system was incubated at 37°C for 48 hours.

[0355] 2.4 Biological Sample Processing

[0356] Vortex each 100 μL biological sample system, mix well, add 300 μL of Clarity OTX lysis buffer (Clarity OTX Lysis-loading Buffer, purchased from Agilent-FinnoMed, catalog number AL0-8579), vortex, let stand for 30 min, add 100 μL of internal standard solution, vortex, let stand for 5 min, centrifuge for 1 min, and set aside for use (total sample volume approximately 500 μL).

[0357] 2.5 Solid-phase extraction:

[0358] (1) Preparation of solid phase extraction reagent

[0359] Activator: Add 200 mL of methanol to the mobile phase bottle and label it as activator;

[0360] Equilibrium buffer: Prepare 1M phosphate buffer solution [877mL sodium dihydrogen phosphate (1.56g / L) + 123mL disodium hydrogen phosphate (3.58g / L)], dilute 100 times, adjust pH to 5.5 with phosphate, and label as equilibrium buffer;

[0361] Rinse solution: Take 500 mL of equilibrium solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH to 5.5 with phosphoric acid, mix well, and label as rinsing solution;

[0362] Eluent: Weigh 7.9 g of ammonium bicarbonate into a 1 L mobile phase bottle, add 1 L of water, take 500 mL of ammonium bicarbonate solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH to 9 with sodium hydroxide, mix well, and label as eluent;

[0363] (2) The extraction steps are as follows:

[0364] step process activation 1 mL of the activator from the above steps, 10 min balance 2 × 1 mL of the equilibration solution from the above steps, for the first 10 min, and the second 10 min. Sample 400 μL (4 / 5 of the total volume) rinse 2×1mL of the rinsing solution from the above steps, first time for 20 minutes, second time for 20 minutes. Washout 2 × 0.75 mL of the eluent from the above steps, first time for 20 min, second time for 20 min.

[0365] 2.6 Post-processing

[0366] The eluent (600 mL twice, totaling 1200 mL) was placed in a 2 mL EP tube and concentrated under vacuum for 6 hours at 1800 rpm. The concentrated sample was then reconstituted with 100 μL of mobile phase (initial ratio), centrifuged at low speed for 2 min, and 10 μL of the supernatant was injected into a high-resolution mass spectrometer. The antisense strand ratio of the siRNA conjugate was semi-quantitatively detected using LC-MS / MS, calculated as: Residual percentage = Target molecular weight response intensity / Sum of all molecular weight response intensities × 100%. The metabolic results after in vitro incubation in rat liver homogenate for 48 hours are shown in Table 4 below.

[0367] Table 4. Remaining antisense strand percentage of siRNA conjugates

[0368] siRNAID AS% N-ER-FY028061M8L96 81.12 N-ER-FY028061M54L96 91.46 N-ER-FY028151M8L96 87.04 N-ER-FY028151M54L96 97.04 N-ER-FY028179M8L96 70.42 N-ER-FY028179M53L96 91.99

[0369] The sequences of N-ER-FY028061M8L96, N-ER-FY028151M8L96, and N-ER-FY028179M8L96 are shown in Table 5.

[0370] Table 5

[0371]

[0372] In Table 4, AS represents the antisense strand of siRNA. The larger the remaining AS, the better the drug stability and the longer the drug's duration of action. As can be seen from Table 4, the siRNA conjugate disclosed in this invention exhibits excellent in vitro stability in rat liver homogenate.

[0373] Example 4: Silencing effect of siRNA conjugate in mice expressing the human CPB2 (hCPB2) gene

[0374] 4.1 Constructing a mouse model overexpressing the hCPB2 gene using AAV

[0375] Six- to eight-week-old C57BL / 6 mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were introduced into the facility. After acclimatization feeding for 3-5 days, a single intravenous injection of adeno-associated virus (AAV) containing the hCPB2 gene (pAAV[Exp]-CBh{CPB2CDS(ns)}:T2A:SEAP, virus provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was administered via tail vein to induce target gene overexpression. The administration volume was 100 μL (3 x 10⁻⁶). 11 vg) / animal, then fed with regular feed.

[0376] 4.2 In vivo efficacy study of siRNA silencing in hCPB2 mouse model

[0377] Fourteen days after AAV virus injection, mice were divided into groups of five. Each group received a subcutaneous dose of a single 3 mpk dose of N-ER-FY028061M54L96, N-ER-FY028151M54L96, or N-ER-FY028179M53L96. SEAP protein expression was measured every 7 days after administration to determine the inhibitory rate of the siRNA conjugate on hCPB2 protein expression.

[0378] The experiment shows that the siRNA conjugate disclosed herein has high inhibitory activity against hCPB2 gene expression in vivo and can reduce hCPB2 protein levels for a long time. The inhibition rate on day 7 of the experiment was as high as 90%, indicating that the designed compound can effectively inhibit the production of hCPB2 protein. It also has a high inhibition rate of over 80% on days 70-91 of the experiment, demonstrating long-lasting inhibitory effect in vivo.

Claims

1. An siRNA for inhibiting the expression of a carboxypeptidase B2 gene, comprising a sense strand and an antisense strand, wherein each of the nucleotides in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences: (1) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 88; (2) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 2, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 88; (3) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 32, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 99; (4) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 33, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 99; (5) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 63, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 110; (6) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 64, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:

110.

2. The siRNA of claim 1, wherein, the sense strand and the antisense strand are complementary to form the double-stranded region, and the 3’ end of the antisense strand has 1-2 overhanging nucleotides extending out of the double-stranded region, and the 3’ end of the sense strand forms a blunt end; or, the sense strand and the antisense strand are complementary to form the double-stranded region, and the 3’ end of both the sense strand and the antisense strand has 1-2 overhanging nucleotides extending out of the double-stranded region. at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modification group; preferably, the modified nucleotide is selected from a 2’-fluoro-modified nucleotide, a 2’-alkoxy-modified nucleotide, a 2’-substituted alkoxy-modified nucleotide, a 2’-alkyl-modified nucleotide, a 2’-substituted alkyl-modified nucleotide, a 2’-deoxy-modified nucleotide, a 2’-amino-modified nucleotide, a 2’-substituted amino-modified nucleotide, a nucleotide analogue, or a combination of any two or more thereof; preferably, the phosphate group with a modification group is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom; and / or the siRNA comprises a sense strand that does not comprise 3’ overhanging nucleotides; and / or the siRNA comprises an antisense strand that does not comprise 5’ overhanging nucleotides. ​ ​ ​ ​ ​ ​ ​ 3. The siRNA of claim 1 or 2, wherein, ​ ​ the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

4. The siRNA according to any one of claims 1-3, wherein, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide; Preferably, in the sense strand, in the 5' to 3' direction, 2'-fluoro modified nucleotides are at positions 7, 9, 10 and 11, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 5, 7, 8 and 9, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 9, 10 and 11, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 8, 9 and 10, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 8, 9, 10 and 11, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7 and 9, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9 and 11, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 11, 13 and 15, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 8, 9, 10 and 12, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9, 11, 13 and 15, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 9, 11 and 13, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9, 11 and 13, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 8 and 9, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 9, 11 and 13, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 9 and 11, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 11 and 13, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 11, 12 and 13, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 11 and 16, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 11 and 17, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 11, 16 and 17, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 9, 11, 12 and 13, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 5, 9, 10 and 11, and the remaining positions are non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 5, 9, 11 and 13, and the remaining positions are non-fluoro modified nucleotides; and / or In the antisense strand, in the direction from 5' to 3', 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 6 and 14, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 4, 6, 12, 14, 16, 18 and 20, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2 and 14, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14, and the rest positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7 and 14, and the rest positions are non-fluoro-modified nucleotides.

5. The siRNA of claim 4, wherein, Each non-fluoro-modified nucleotide is independently selected from one of the nucleotides or nucleotide analogues in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluoro group, the nucleotide analogue being selected from one of pseudouracil, anisouracil, LNA, ENA, cET BNA, UNA and GNA.

6. The siRNA of any one of claims 1-5, wherein, Each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or a combination of any two or more thereof; Preferably, in the sense strand, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 7, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 5, 7, 8 and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9 and 10, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7 and 9, and the nucleotide at position 11 of the sense strand is a 2'-deoxy-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 13 and 15, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10 and 12, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13 and 15, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8 and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11 and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11 and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16 and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 11, 12 and 13, and the remaining positions are 2'-methoxy-modified nucleotides.or 2'-fluoro modified nucleotides at positions 5, 9, 10, and 11, and 2'-methoxy modified nucleotides at the remaining positions; or 2'-fluoro modified nucleotides at positions 5, 9, 11, and 13, and 2'-methoxy modified nucleotides at the remaining positions; and / or; In the antisense strand, in the direction from 5' to 3', 2'-fluoride-modified nucleotides are located at positions 2, 6, 14 and 16, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 6 and 14, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 4, 6, 12, 14, 16, 18 and 20, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2 and 14, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and position 6 is a GNA-modified nucleotide, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 7, 10 and 14, and the rest are 2'-methoxy-modified nucleotides; or, 2'-fluoride-modified nucleotides are located at positions 2, 5, 7 and 14, and the rest are 2'-methoxy-modified nucleotides. More preferably, neither the 5' end nor the 3' end of the sense strand is connected to a reverse abasic deoxyribose residue, or only the 5' end of the sense strand is connected to one reverse abasic deoxyribose residue, or only the 3' end of the sense strand is connected to one reverse abasic deoxyribose residue, or the 5' end and the 3' end of the sense strand are respectively connected to one reverse abasic deoxyribose residue. Further preferably, the reverse abasic deoxyribose residue is connected to the 3' end nucleotide and / or the 5' end nucleotide of the sense strand by a phosphodiester bond, a phosphorothioate group or other internucleoside bond.

7. The siRNA of any one of claims 1-6, wherein, In the direction from the 5' end to the 3' end, at least one of the connections between the following nucleotides in the siRNA is a phosphorothioate group connection: The connection between the 1st and 2nd nucleotides starting from the 5' end of the sense strand; The connection between the 2nd and 3rd nucleotides starting from the 5' end of the sense strand; The connection between the 1st and 2nd nucleotides starting from the 3' end of the sense strand; The connection between the 2nd and 3rd nucleotides starting from the 3' end of the sense strand; The connection between the reverse abasic deoxyribose residue and the 1st nucleotide starting from the 3' end of the sense strand; The connection between the 1st and 2nd nucleotides starting from the 5' end of the antisense strand; The connection between the 2nd and 3rd nucleotides starting from the 5' end of the antisense strand; the linkage between the 3rd and 4th nucleotides from the 5' terminus of the antisense strand; the linkage between the 1st and 2nd nucleotides from the 3' terminus of the antisense strand; the linkage between the 2nd and 3rd nucleotides from the 3' terminus of the antisense strand; Preferably, in the 5' to 3' direction, (1) the sense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; and between the 1st and 2nd nucleotides from the 3' terminus of the sense strand; and between the 2nd and 3rd nucleotides from the 3' terminus of the sense strand; or, (2) the sense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; or, (3) the sense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; and between the 1st and 2nd nucleotides from the 3' terminus of the sense strand; and between the 2nd and 3rd nucleotides from the 3' terminus of the sense strand; or, (4) the sense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; or (5) the sense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; and between the reverse abasic deoxyribose residue and the 1st nucleotide from the 3' terminus of the sense strand; Preferably, in the 5' to 3' direction, (i) the antisense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the antisense strand; and between the 2nd and 3rd nucleotides from the 5' terminus of the antisense strand; and between the 1st and 2nd nucleotides from the 3' terminus of the antisense strand; and between the 2nd and 3rd nucleotides from the 3' terminus of the antisense strand; or (ii) the antisense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' terminus of the antisense strand; and between the 2nd and 3rd nucleotides from the 5' terminus of the antisense strand; and between the 3rd and 4th nucleotides from the 5' terminus of the antisense strand; and between the 1st and 2nd nucleotides from the 3' terminus of the antisense strand.

8. The siRNA of any one of claims 1-7, wherein, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, a GNA modified nucleotide, a 2'-deoxy modified nucleotide, or a combination of any two or more thereof; preferably, in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 3' terminal end removed of overhang; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 3' terminal end removed of overhang; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 3' terminal end removed of overhang; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, with the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, 2'-fluoro modified nucleotides at positions 8, 9, and 10 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 3, 5, 8, 10, 14, 16, and 18 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 3' terminal overhang removed; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 4, 6, 12, 14, 16, 18, and 20 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2 and 14 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7 and 9 of the sense strand, with the nucleotide at position 11 of the sense strand being a 2'-deoxy modified nucleotide, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7 and 9 of the sense strand, with the nucleotide at position 11 of the sense strand being a 2'-deoxy modified nucleotide, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 3' terminal overhang removed; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining locations, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 8, 9, 10, and 12 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 9, 11, and 13 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 9, 11, and 13 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with a 3' overhang removed; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, and 13 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 8, and 9 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 8, and 9 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 3' terminal nucleotide of the sense strand lacking a 3' terminal cove; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 9, 11, and 13 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 9, 11, and 13 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 3' terminal nucleotide of the sense strand lacking a 3' terminal cove; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 3' terminal nucleotide of the sense strand lacking a 3' terminal cove; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11 from the 5' end to the 3' end of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end to the 3' end of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, and 16 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, and 17 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, 16, and 17 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, with the 6 position being a GNA modified nucleotide, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, 2'-fluoro modified nucleotides at positions 9, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 3' terminal overhang removed, and 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14 of the anti sense strand, and 2'-methoxy modified nucleotides in the remaining positions, with the 5' terminal nucleotide of the anti sense strand linked to a 5 '-trans vinyl phosphonate ester group, in the 5' to 3' direction; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 3, 7, 8, and 9 of the sense strand, 2'-methoxy-modified nucleotides at the remaining positions, and a 5'-trans- vinylphosphonate group attached to the 5' terminal nucleotide of the antisense strand; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 3, 7, 9, and 11 of the sense strand, 2'-methoxy-modified nucleotides at the remaining positions, and a 5'-trans- vinylphosphonate group attached to the 5' terminal nucleotide of the antisense strand. or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 3, 7, 8, and 9 of the sense strand, 2'-methoxy-modified nucleotides at the remaining positions, and a 5'-trans- vinylphosphonate group attached to the 5' terminal nucleotide of the antisense strand; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 3, 7, 9, and 11 of the sense strand, 2'-methoxy-modified nucleotides at the remaining positions, and a 5'-trans- vinylphosphonate group attached to the 5' terminal nucleotide of the antisense strand.

9. The siRNA according to any one of claims 1 to 8, wherein, the siRNA is selected from the siRNAs of Table 1; preferably, the siRNA is selected from N-ER-FY028061M37, N-ER-FY028061M40, N-ER-FY028061M44, N-ER-FY028061M45, N-ER-FY028151M40, N-ER-FY028151M44, N-ER-FY028151M45, N-ER-FY028179M46, N-ER-FY028179M47, N-ER-FY028179M48, N-ER-FY028179M49, N-ER-FY028061M54, N-ER-FY028151M54, N-ER-FY028179M53.

10. An siRNA conjugate comprising the siRNA of any one of claims 1-9 and a conjugate group conjugated to the siRNA.

11. The siRNA conjugate of claim 10, wherein, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, the 3' end of the antisense strand has 1-3 overhanging nucleotides extending out of the double-stranded region; or, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, the 3' end of the antisense strand forms a blunt end.

12. The siRNA conjugate of claim 10 or 11, wherein, the conjugate group is selected from:

13. The siRNA conjugate of any one of claims 10-12, wherein, The siRNA conjugate is selected from the siRNA conjugates of Table 2; preferably, the siRNA conjugate is selected from N-ER-FY028061M37L96, N-ER-FY028061M40L96, N-ER-FY028061M44L96, N-ER-FY028061M45L96, N-ER-FY028151M37L96, N-ER-FY028151M40L96, N-ER-FY028151M44L96, N-ER-FY028151M45L96, N-ER-FY028179M46L96, N-ER-FY028179M47L96, N-ER-FY028179M48L96, N-ER-FY028179M49L96, N-ER-FY028061M54L96, N-ER-FY028151M54L96, N-ER-FY028179M53L96.

14. A pharmaceutical composition comprising the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, and a pharmaceutically acceptable carrier.

15. A kit comprising the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, or the pharmaceutical composition of claim 14.

16. Use of the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, or the pharmaceutical composition of claim 14 in (a) or (b): (a) for the preparation of a medicament for inhibiting the expression of a CPB2 gene; (b) for the preparation of a medicament for preventing and / or treating a disease associated with overexpression of a CPB2 gene; Preferably, the disease is thrombosis, pulmonary embolism, stroke, myocardial infarction, and ischemic stroke; more preferably, the thrombosis includes venous thrombosis, deep vein thrombosis, atherosclerotic thrombosis, prosthetic valve thrombosis, microvascular thrombosis, and dynamic thrombosis.

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