Dsrna molecule that inhibits serpinc1 gene expression and use thereof

Abstract

Provided is a nucleic acid molecule that inhibits SERPINC1 gene expression via RNAi, and comprises or consists of a sense strand and an antisense strand which are complementary to each other. The nucleic acid molecule has a SERPINC1 expression inhibition rate significantly superior to that of other small nucleic acid molecules that inhibit SERPINC1 expression via RNAi.

Description

A dsRNA molecule that inhibits SERPINC1 gene expression and its application

[0001] Cross-references

[0002] This invention claims priority to the earlier application filed on March 28, 2025, with patent application number 202510383878.6 and entitled "A dsRNA molecule for inhibiting SERPINC1 gene expression and its application therein". The entire contents of that earlier application are incorporated herein by reference. Technical Field

[0003] This application relates to the field of RNAi, specifically to a dsRNA molecule that inhibits the expression of the SERPINC1 gene and its applications. Background Technology

[0004] Hemophilia is an X-linked recessive inherited bleeding disorder, classified into hemophilia A and hemophilia B based on the different clotting factors lacking. Hemophilia A (85%) lacks clotting factor VIII, while hemophilia B (15%) lacks clotting factor IX. Hemophilia A is caused by low levels or absence of clotting factor VIII, while hemophilia B is caused by a deficiency of clotting factor IX, leading to impaired blood clotting function. The SERPINC1 gene encodes antithrombin III, a plasma protease inhibitor belonging to the serine protease inhibitor superfamily. This protein inhibits thrombin and other activating serine proteases in the coagulation system, regulating the blood coagulation cascade. In hemophilia patients, inhibiting antithrombin III expression can effectively prevent bleeding.

[0005] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. dsRNA, typically 19–30 bp in length, is one of the important tools in RNAi technology. In natural organisms, longer dsRNAs, after entering cells, are specifically recognized and cleaved by the Dicer enzyme into small RNA fragments (siRNAs) of approximately 21–23 nucleotides in length. These cleaved dsRNA fragments unwind into single strands and form complexes (RISCs) with certain proteins. RISCs bind to mRNAs complementary to the antisense strand of the dsRNA within the cell and cleave them, causing their degradation and preventing protein synthesis, resulting in gene silencing. In industrial production, the chemical synthesis and modification of dsRNA are preferred to further improve the stability and efficacy of dsRNA drugs.

[0006] Small nucleic acid drugs hold promise as an effective treatment for hemophilia by lowering antithrombin III. Fitusiran, a siRNA targeting antithrombin jointly developed by Sanofi and Alnylam, binds to RNA expressing antithrombin, reducing its production and restoring the balance between clotting factors and antithrombin, thereby reducing bleeding events in hemophilia patients. This innovative therapy requires only one subcutaneous injection per month, offering significant convenience for patients in controlling bleeding risk compared to routine prophylactic clotting factor injections. However, there is still a need to develop better small nucleic acid drugs for hemophilia, offering improved efficacy, specificity, stability, targeting, and / or tolerability.

[0007] Application Overview

[0008] This application provides a nucleic acid molecule that inhibits SERPINC1 gene expression in cells via RNAi, including compositions, formulations, and uses of said nucleic acid molecule. The embodiments of this application provide ample data demonstrating that the preferred nucleic acid molecule of this application exhibits higher inhibitory efficiency against SERPINC1 mRNA. Furthermore, this application also provides compounds comprising the aforementioned nucleic acid molecule, as well as precursor molecules of the aforementioned nucleic acid molecule.

[0009] Specifically, the first aspect of this application provides a nucleic acid molecule for inhibiting SERPINC1 gene expression via RNAi, comprising or composed of complementary sense and antisense strands, wherein the length of each sense and antisense strand is independently 15-30 nt, and each nucleotide independently comprises one or more modifications selected from the following:

[0010] Modifications include locked nucleic acids, ring-opening or non-locked nucleic acid modifications, morpholinoyl modifications, tetrahydropyran modifications, 1,5-dehydrohexyl alcohol modifications, cyclohexenyl modifications, 2'-methoxyethoxy modifications (2'-MOE), 2'-O-methyl modifications (2'-OMe), 2'-O-allyl modifications, 2'-C-alkyl modifications, 2'-C-allyl modifications, 2'-fluorinated modifications (2'-F), 2'-deoxy modifications (DNA modifications), 2'-amino modifications, thiophosphate modifications, aminophosphate modifications, methylphosphonate modifications, 5'-phosphate modifications, 5'-phosphate mimic modifications, and invAb (reverse debased deoxyribonucleotide) modifications. The "5'-phosphate mimic" includes, but is not limited to: 5'-aminophosphate, 5'-methylphosphonate, 5'-thiophosphate, 5'-vinyl phosphate (5'-VP), and 5'-cyclopropyl phosphate (5'-cPrp). In some embodiments, the 5'-phosphate mimicry is modified with 5'-vinyl phosphate (5'-VP) or 5'-cyclopropyl phosphate (5'-cPrp).

[0011] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the other nucleotides in the antisense strand is substituted with methoxy or hydrogen.

[0012] It should be understood that when the 2' position of a nucleotide is replaced by a group (e.g., "fluorine", "methoxy", etc.) as described in this application, it means that the group is directly linked to the 2' carbon atom of the nucleotide's ribose. That is, when the nucleotide was originally DNA, its 2'-H becomes this group after substitution; when the nucleotide was originally RNA, its 2'-OH becomes this group after substitution. In this application, unless otherwise specified, the nucleotide in the "nucleic acid molecule that inhibits SERPINC1 gene expression by RNAi" is RNA before modification, while DNA is considered to be the nucleotide obtained by substituting the 2'-OH of the RNA nucleotide with 2'-H.

[0013] In some implementation schemes, the nucleic acid molecule of the first aspect includes:

[0014] The 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the other nucleotides in the antisense strand is substituted with methoxy groups; or

[0015] The 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen (i.e., 2' deoxygenation modification, or DNA modification). The 2' position of the other nucleotides in the antisense strand is substituted with methoxy groups.

[0016] In some embodiments, the 2' position of the 9th and 11th nucleotides of the sense strand of the nucleic acid molecule of the first aspect is substituted with fluorine from the 5' end, the 2' position of the 10th nucleotide is substituted with fluorine or hydrogen, and the 2' position of the other nucleotides is substituted with fluorine, methoxy or hydrogen.

[0017] In some embodiments, the 2' position of the 9th and 11th nucleotides of the sense strand of the nucleic acid molecule of the first aspect is substituted with fluorine from the 5' end, the 2' position of the 10th nucleotide is substituted with fluorine or hydrogen, the 2' position of the 7th and 12th nucleotides is substituted with fluorine or methoxy, and the 2' position of the other nucleotides is substituted with methoxy or hydrogen.

[0018] In some implementation schemes, the nucleic acid molecule of the first aspect includes:

[0019] The 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the other nucleotides is substituted with methoxy; or the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen, and the 2' position of the other nucleotides is substituted with methoxy; and the 2' position of the nucleotides at positions 9 and 11 of the sense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotide at position 10 is substituted with fluorine or hydrogen, and the 2' position of the other nucleotides is substituted with fluorine, methoxy, or hydrogen.

[0020] In some implementation schemes, the nucleic acid molecule of the first aspect includes:

[0021] The 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the other nucleotides is substituted with methoxy; or the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen, and the 2' position of the other nucleotides is substituted with methoxy; and the 2' position of the nucleotides at positions 9 and 11 of the sense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotide at position 10 is substituted with either fluorine or hydrogen, the 2' position of the nucleotides at positions 7 and 12 is substituted with either fluorine or methoxy, and the 2' position of the other nucleotides is substituted with either methoxy or hydrogen.

[0022] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0023] The 2' position of the 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0024] The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0025] The 2' position of nucleotides 7, 9, 10, 11, and 12, starting from the 5' end, is substituted with fluorine, while the 2' position of all other nucleotides is substituted with methoxy groups; or

[0026] The 2' position of the nucleotides at positions 7, 9, 10, and 11 starting from the 5' end is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups.

[0027] or

[0028] The 2' position of the nucleotides at positions 11 and 13 starting from the 3' end is replaced by fluorine, the 2' position of the nucleotide at position 12 starting from the 3' end is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0029] The 2' position of the nucleotides at positions 11, 13, and 15 starting from the 3' end is replaced by fluorine, the 2' position of the nucleotide at position 12 starting from the 3' end is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0030] The 2' position of the nucleotides at positions 10, 11, 12, 13, and 15, starting from the 3' end, is substituted with fluorine, while the 2' position of all other nucleotides is substituted with methoxy groups; or

[0031] The 2' position of the nucleotides at positions 11, 12, 13, and 15 starting from the 3' end is substituted with fluorine, while the 2' position of the other nucleotides is substituted with methoxy groups.

[0032] or

[0033] The 2' position of the nucleotides corresponding to the 11th and 13th positions from the 5' end of the antisense strand is replaced by fluorine, the 2' position of the nucleotide corresponding to the 12th position from the 5' end of the antisense strand is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0034] The 2' position of the nucleotides corresponding to the 11th, 13th and 15th positions from the 5' end of the antisense strand is replaced by fluorine, the 2' position of the nucleotide corresponding to the 12th position from the 5' end of the antisense strand is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0035] The 2' position of the nucleotides at positions 10, 11, 12, 13, and 15, starting from the 5' end of the antisense strand, is substituted with fluorine, while the 2' position of all other nucleotides is substituted with methoxy groups; or

[0036] The 2' position of the nucleotides at positions 11, 12, 13, and 15, starting from the 5' end of the antisense strand, is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy.

[0037] In this application, when referring to the nucleotide at position N "corresponding to the 5' end of the antisense strand" in the sense strand, this nucleotide in the sense strand refers to the sense strand nucleotide that is complementary to the Nth nucleotide at the 5' end of the antisense strand after the sense strand and antisense strand are complementary. When this nucleotide is present, it satisfies the above description of nucleotide modification. It should be understood that when the 5' end of the antisense strand is blunt, then position N "corresponding to the 5' end of the antisense strand" is position N of the sense strand starting from the 3' end; when the 5' end of the antisense strand is overhanging, for example, when the overhanging end of the antisense strand is a 2nt nucleotide, then position N "corresponding to the 5' end of the antisense strand" is position N+2 of the sense strand starting from the 3' end; when the 3' end of the sense strand is overhanging, for example, when the overhanging end of the 3' end of the sense strand is a 2nt nucleotide, then position N "corresponding to the 5' end of the antisense strand" is position N-2 of the sense strand starting from the 3' end.

[0038] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0039] The 2' position of the 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0040] The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0041] The 2' position of nucleotides 7, 9, 10, 11, and 12, starting from the 5' end, is substituted with fluorine, while the 2' position of all other nucleotides is substituted with methoxy groups; or

[0042] The 2' position of the nucleotides at positions 7, 9, 10, and 11 starting from the 5' end is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups.

[0043] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, while the 2' position of the other nucleotides is substituted with methoxy groups.

[0044] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0045] The 2' position of the 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0046] The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0047] The 2' position of nucleotides 7, 9, 10, 11, and 12, starting from the 5' end, is substituted with fluorine, while the 2' position of all other nucleotides is substituted with methoxy groups; or

[0048] The 2' position of the nucleotides at positions 7, 9, 10, and 11 starting from the 5' end is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups.

[0049] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen, and the 2' position of the other nucleotides is substituted with methoxy groups.

[0050] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0051] The 2' position of the 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0052] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen, and the 2' position of the other nucleotides is substituted with methoxy groups.

[0053] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0054] The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups.

[0055] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen, and the 2' position of the other nucleotides is substituted with methoxy groups.

[0056] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0057] The 2' position of the nucleotides at positions 7, 9, 10, 11, and 12, starting from the 5' end, is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups.

[0058] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, while the 2' position of the other nucleotides is substituted with methoxy groups.

[0059] In some implementations, the sense strand of the nucleic acid molecule of the first aspect is:

[0060] The 2' position of the nucleotides at positions 7, 9, 10, and 11 starting from the 5' end is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups.

[0061] Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, while the 2' position of the other nucleotides is substituted with methoxy groups.

[0062] In some embodiments, any of the nucleic acid molecules of the first aspect described above contains a phosphate thioester modification at at least one of the following positions:

[0063] (1) Between the first and second nucleotides starting from the 5' end of the sense strand;

[0064] (2) Between the second and third nucleotides starting from the 5' end of the sense strand;

[0065] (3) Between the first and second nucleotides starting from the 3' end of the sense strand;

[0066] (4) Between the second and third nucleotides starting from the 3' end of the sense strand;

[0067] (5) Between the first and second nucleotides starting from the 5' end of the antisense strand;

[0068] (6) Between the second and third nucleotides starting from the 5' end of the antisense strand;

[0069] (7) Between the first and second antisense chains starting at the 3' end; and

[0070] (8) Between the second and third antisense chains starting from the 3' end.

[0071] In some implementations, any of the nucleic acid molecules of the first aspect mentioned above contains phosphate thioester modification at the following positions:

[0072] (1) Between the first and second nucleotides starting from the 5' end of the sense strand;

[0073] (2) Between the second and third nucleotides starting from the 5' end of the sense strand;

[0074] (3) Between the first and second nucleotides starting from the 5' end of the antisense strand;

[0075] (4) Between the second and third nucleotides starting from the 5' end of the antisense strand;

[0076] (5) Between the first and second antisense chains starting at the 3' end; and

[0077] (6) Between the second and third antisense chains starting from the 3' end.

[0078] In some implementations, any of the nucleic acid molecules of the first aspect mentioned above contains phosphate thioester modification at the following positions:

[0079] (1) Between the first and second nucleotides starting from the 5' end of the sense strand;

[0080] (2) Between the second and third nucleotides starting from the 5' end of the sense strand;

[0081] (3) Between the first and second nucleotides starting from the 5' end of the antisense strand;

[0082] (4) Between the second and third nucleotides starting from the 5' end of the antisense strand;

[0083] (5) Between the first and second antisense chains starting at the 3' end; and

[0084] (6) Between the second and third antisense chains starting from the 3' end;

[0085] Furthermore, all other nucleotides in the nucleic acid molecule are linked by phosphate ester bonds.

[0086] In some embodiments, any of the nucleic acid molecules of the first aspect described above is linked (or conjugated) to at least one desialylate glycoprotein receptor (ASGPR) ligand. In some embodiments, the ASGPR ligand is linked (or conjugated) to the 5' end and / or 3' end of the sense strand. In some embodiments, the ASGPR ligand is linked to the 5' end of the sense strand. In some embodiments, the ASGPR ligand is linked to the 3' end of the sense strand. In some embodiments, the ASGPR ligand is a GalNAc ligand formed from one, two, three, or more N-acetylgalactosamine (GalNAc) derivatives. In this application, GalNAc polymers formed from one, two, three, or more GalNAc derivatives are referred to as monovalent GalNAc ligands, divalent GalNAc ligands, trivalent GalNAc ligands, and polyvalent GalNAc ligands, respectively. In some embodiments, the ASGPR ligand is a trivalent GalNAc ligand formed from three GalNAc derivatives.

[0087] In some embodiments, the GalNAc ligand is L96 (as shown in Formula I) or as shown in Formula II (ligand II, when X = S). - This ligand is referred to as ligand 1) or formula III (ligand 2):

[0088] Among them, in equations I and II These represent the 3' end of the sense or antisense strand of the nucleic acid molecule, respectively; preferably, they are linked to the 3' position of the 3' end nucleotide via a phosphodiester bond or a phosphothiodiester bond. In Formula II ligands, when X = S - This ligand is called ligand 1. In Formula III... This represents the connection between the sense or antisense (Oligo) 3' end of the nucleic acid molecule via a phosphate ester bond or a thiophosphate ester bond.

[0089] In some embodiments, the modifications of each nucleotide in the nucleic acid molecule are each independently selected from one or more of the following: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, thiophosphate modification, 5'-VP modification, and the nucleic acid molecule contains at least one ASGPR ligand.

[0090] In some implementations, any of the nucleic acid molecules of the first aspect mentioned above contains a modification motif selected from any of the following:

[0091] (1) Motif 1

[0092] Sense chain: NmsNmsNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmNm,

[0093] Antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNm sNm;

[0094] (2) Motif 2

[0095] Sense strand: NmsNmsNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmNm, antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0096] (3) Motif 3

[0097] Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNmNm,

[0098] Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsN msNm;

[0099] (4) Motif 4

[0100] Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm,

[0101] Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsN msNm; and

[0102] Furthermore, Nm represents a 2'-OMe modified ribonucleotide, Nf represents a 2'-F modified ribonucleotide, s represents a phosphate thioester modified ribonucleotide, Nd represents a 2' deoxyribonucleotide, and optionally when the nucleotide corresponding to Nd is U or T, Nd represents thymine deoxyribonucleotide (Td).

[0103] In some implementations, any of the nucleic acid molecules of the first aspect mentioned above comprises a modification motif selected from the following and a GalNAc ligand:

[0104] (1) Motif 1-GalNAc

[0105] Sense chain: NmsNmsNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmNm-Gal NAc ligand,

[0106] Antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNm sNm;

[0107] (2) Motif 2-GalNAc

[0108] Sense chain: NmsNmsNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmNm-Ga lNAc ligand,

[0109] Antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNm sNm;

[0110] (3) Motif 3-GalNAc

[0111] Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNm-Gal NAc ligand,

[0112] Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsN msNm;

[0113] (4) Motif 4-GalNAc

[0114] Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNm-Gal NAc ligand,

[0115] Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsN msNm; and

[0116] Furthermore, Nm represents a 2'-OMe modified ribonucleotide, Nf represents a 2'-F modified ribonucleotide, s represents a thiophosphate modified ribonucleotide, and -GalNAc represents a ribonucleotide linked to the corresponding GalNAc ligand via a phosphate ester bond or a thiophosphate ester bond.

[0117] In the above-mentioned modified motifs, the GalNAc ligand can be any GalNAc ligand in the art, such as L96, the ligand shown in Formula II or Formula III. If it is to indicate that the GalNAc ligand is linked to the preceding nucleotide through a phosphate ester bond, taking motif 1 as an example, it can be represented as "motif 1-p-GalNAc". If it is to indicate that the GalNAc ligand is linked to the preceding nucleotide through a phosphate ester bond, taking motif 1 as an example, it can be represented as "motif 1-s-GalNAc". If it is to explicitly indicate that GalNAc is selected from L96, taking motif 1 as an example, the corresponding motif is further represented as "motif 1-L96". If it is to indicate that L96 is linked to the preceding nucleotide through a phosphate ester bond, it is represented as "motif 1-p-L96". If it is to indicate that L96 is linked to the preceding nucleotide through a thiophosphate ester bond, it can be represented as "motif 1-s-L96". Similarly, when GalNAc is selected from ligands of formula II or ligand 1, it can be represented as "motif 1-ligand II", "motif 1-p-ligand II", "motif 1-s-ligand II", "motif 1-ligand 1", "motif 1-p-ligand 1", and "motif 1-s-ligand 1", respectively. If it further includes VP modification, it is represented as "motif 1-ligand II-VP", "motif 1-p-ligand II-VP", "motif 1-s-ligand II-VP", "motif 1-ligand 1-VP", "motif 1-p-ligand 1-VP", and "motif 1-s-ligand 1-VP". If GalNAc is selected from ligands of formula III (i.e., ligand 2), it can be represented as "motif 1-ligand 2", "motif 1-p-ligand 2", and "motif 1-s-ligand 2", respectively. If further modified with VP, it is represented as: "motif 1-ligand 2-VP", "motif 1-p-ligand 2-VP", "motif 1-s-ligand 2-VP".

[0118] In some embodiments, any of the nucleic acid molecules of the first aspect described above comprises a modification motif selected from any of the following: motif 1-L96, motif 2-L96, motif 3-L96, motif 4-L96, motif 1-ligand II, motif 2-ligand II, motif 3-ligand 2, motif 4-ligand II; optionally, any of the nucleic acid molecules of the first aspect described above comprises a modification motif selected from any of the following: motif 1-p-L96, motif 2-p-L96, motif 3-p-L96. The motifs are: motif 4-p-L96, motif 1-s-ligand II, motif 2-s-ligand II, motif 3-s-ligand II, and motif 4-s-ligand II. Optionally, any nucleic acid molecule of the first aspect above includes a modified motif selected from any of the following: motif 1-p-L96, motif 2-p-L96, motif 3-p-L96, motif 4-p-L96, motif 1-s-ligand 1, motif 2-s-ligand 1, motif 3-s-ligand 1, and motif 4-s-ligand 1.

[0119] In some embodiments, any nucleic acid molecule of the first aspect above comprises a modified phosphate mimic motif selected from the following: phosphate mimic motif 1-p-L96, phosphate mimic motif 2-p-L96, phosphate mimic motif 3-p-L96, and phosphate mimic motif 4-p-L96. In some embodiments, any nucleic acid molecule of the first aspect above comprises a modified VP motif selected from the following: VP-motif 1-p-L96, VP-motif 2-p-L96, VP-motif 3-p-L96, and VP-motif 4-p-L96.

[0120] In some embodiments, any nucleic acid molecule of the first aspect above comprises a modified phosphate ester mimic motif selected from the following: phosphate ester mimic motif 1-s-ligand 1, phosphate ester mimic motif 2-s-ligand 1, phosphate ester mimic motif 3-s-ligand 1, and phosphate ester mimic motif 4-s-ligand 1. In some embodiments, any nucleic acid molecule of the first aspect above comprises a modified VP motif selected from the following: VP-motif 1-s-ligand 1, VP-motif 2-s-ligand 1, VP-motif 3-s-ligand 1, and VP-motif 4-s-ligand 1.

[0121] In some embodiments, any nucleic acid molecule of the first aspect comprises a modifying motif selected from the following: motif 1-ligand 2, motif 2-ligand 2, motif 3-ligand 2, and motif 4-ligand 2. In some embodiments, any nucleic acid molecule of the first aspect comprises a modifying motif selected from the following: motif 1-p-ligand 2, motif 2-p-ligand 2, motif 3-p-ligand 2, and motif 4-p-ligand 2. In some embodiments, any nucleic acid molecule of the first aspect comprises a modified phosphate mimic motif selected from the following: phosphate mimic motif 1-p-ligand 2, phosphate mimic motif 2-p-ligand 2, phosphate mimic motif 3-p-ligand 2, and phosphate mimic motif 4-p-ligand 2. In some embodiments, any of the nucleic acid molecules of the first aspect described above includes a modified VP motif selected from any of the following: VP motif 1-p-ligand 2, VP motif 2-p-ligand 2, VP motif 3-p-ligand 2 and VP motif 4-p-ligand 2.

[0122] In some embodiments, the complementary region length of the antisense strand and sense strand of the nucleic acid molecule is 15-21 bp, for example 16 bp, 17 bp, 18 bp, 19 bp or 20 bp; alternatively, the complementary region length is 16-21 bp; alternatively, the complementary region length is 17-21 bp; alternatively, the complementary region length is 18-21 bp.

[0123] In some embodiments, the antisense strand and sense strand of the nucleic acid molecule each comprise 15-21 (e.g., 16, 17, 18, 19, or 20) consecutive nucleotides of the sense or antisense strand base sequence of any dsRNA as shown in the sequence listings SER-1 to SER-100.

[0124] In some embodiments, the sense strand of the nucleic acid molecule comprises 18-21 (e.g., 18, 19, 20, or 21) consecutive nucleotides of the sense strand of any dsRNA of SER3, SER1, or SER2, or SER4 to SER-100 as shown in the sequence listing.

[0125] In some embodiments, the antisense strand of the nucleic acid molecule comprises 18-23 (e.g., 18, 19, 20, 21, 22, or 23) consecutive nucleotides of the antisense strand of any dsRNA as shown in the sequence listing SER3, SER1, SER2, SER4 to SER-100; optionally, the antisense strand of the nucleic acid molecule comprises 20-23 (e.g., 20, 21, 22, or 23) consecutive nucleotides of the antisense strand of any dsRNA as shown in the sequence listing SER3, SER1, SER2, SER4 to SER-100.

[0126] In some embodiments, the sense strand of the nucleic acid molecule comprises 18-21 (e.g., 18, 19, 20, or 21) consecutive nucleotides of the sense strand of any dsRNA of SER3, SER1, or SER2, or SER4 to SER-100 as shown in the Sequence Appendix, and the antisense strand of the nucleic acid molecule comprises 20-23 (e.g., 20, 21, 22, or 23) consecutive nucleotides of the antisense strand of any dsRNA of SER3, SER1, or SER2, or SER4 to SER-100 as shown in the Sequence Appendix.

[0127] In some embodiments, the nucleic acid molecule comprises or is any dsRNA molecule of SER3, SER1, SER2, or SER4 to SER-100 as shown in the Sequence Listing.

[0128] In some implementations, the sense chain and the antisense chain are of equal length. In some implementations, the antisense chain is 1 nt, 2 nt, or 3 nt longer than the sense chain.

[0129] In some embodiments, the sense strand length of the nucleic acid molecule of the first aspect is 18-23 nt (e.g., 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, or 23 nt), and the antisense sequence length is 20-25 nt (e.g., 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt). Alternatively, in some embodiments, the sense strand length is 21 nt, and the antisense strand length is 23 nt.

[0130] In some embodiments, the sense and / or antisense sequences of the nucleic acid molecule of the first aspect described above further include one, two, or three overhanging nucleotides in addition to the complementary region. In some embodiments, the 5' or 3' end of the sense strand has one, two, or three overhanging nucleotides. In some embodiments, the 5' or 3' end of the antisense strand has one, two, or three overhanging nucleotides. In some embodiments, the 3' end of the antisense strand has two overhanging nucleotides, and the sense sequence does not contain any overhanging nucleotides. In some embodiments, the 5' end of the antisense strand in the nucleic acid molecule forms a blunt end with the 3' end of the sense strand.

[0131] In some implementation schemes, in any of the nucleic acid molecules of the first aspect mentioned above:

[0132] (a) The sense sequence comprises 18-21 consecutive (e.g., 18, 19, 20, or 21) nucleotides of SEQ ID NO: 1.

[0133] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22, or 23) nucleotides of SEQ ID NO: 2;

[0134] (b) The sense sequence comprises 18-21 (e.g., 18, 19, 20, or 21) consecutive nucleotides of SEQ ID NO: 3.

[0135] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22, or 23) nucleotides of SEQ ID NO: 4;

[0136] (c) The sense sequence comprises 18-21 consecutive (e.g., 18, 19, 20, or 21) nucleotides of SEQ ID NO: 5.

[0137] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22, or 23) nucleotides of SEQ ID NO: 6;

[0138] (d) The sense sequence comprises 18-21 consecutive (e.g., 18, 19, 20, or 21) nucleotides of SEQ ID NO: 7.

[0139] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22, or 23) nucleotides of SEQ ID NO: 8;

[0140] (e) The sense sequence comprises 18-21 (e.g., 18, 19, 20, or 21) consecutive nucleotides of SEQ ID NO: 9.

[0141] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22, or 23) nucleotides of SEQ ID NO: 10;

[0142] (f) The sense sequence comprises 18-21 consecutive (e.g., 18, 19, 20, or 21) nucleotides of SEQ ID NO: 11.

[0143] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22, or 23) nucleotides of SEQ ID NO: 12;

[0144] (g) The sense sequence comprises 18-21 consecutive (e.g., 18, 19, 20, or 21) nucleotides of SEQ ID NO: 13.

[0145] The antisense strand base sequence comprises 20-23 consecutive (e.g., 20, 21, 22 or 23) nucleotides of SEQ ID NO: 14.

[0146] In some implementation schemes, in any of the nucleic acid molecules of the first aspect mentioned above:

[0147] (a) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 1, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 1 starting from the 3' end.

[0148] The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 2, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 2 starting from the 5' end;

[0149] (b) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 3, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 3 starting from the 3' end.

[0150] The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 4, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 4 starting from the 5' end;

[0151] (c) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 5, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 5 starting from the 3' end.

[0152] The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 6, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 6 starting from the 5' end;

[0153] (d) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 7, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 7 starting from the 3' end.

[0154] The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 8, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 8 starting from the 5' end;

[0155] (e) The sense chain sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 9, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 9 starting from the 3' end.

[0156] The antisense strand base sequence comprises at least 21 consecutive nucleotides as specified in SEQ ID NO: 10;

[0157] (f) The sense chain sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 11, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 11 starting from the 3' end.

[0158] The antisense base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 12, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 12 starting from the 5' end; or

[0159] (g) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 13, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 1 starting from the 3' end.

[0160] The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 14, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 14 starting from the 5' end.

[0161] In some implementation schemes, in any of the nucleic acid molecules of the first aspect mentioned above:

[0162] (a) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 1, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 2;

[0163] (b) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 3, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 4;

[0164] (c) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 5, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 6;

[0165] (d) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 7, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 8;

[0166] (e) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 9, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 10;

[0167] (f) The sense strand sequence comprises the base sequence shown in SEQ ID NO: 11, and the antisense strand sequence comprises the nucleotide sequence shown in SEQ ID NO: 12; or

[0168] (g) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 13, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0169] In some implementation schemes, in any of the nucleic acid molecules of the first aspect mentioned above:

[0170] (a) The sense base sequence is shown in SEQ ID NO: 1, and the antisense base sequence is shown in SEQ ID NO: 2;

[0171] (b) The sense base sequence is shown in SEQ ID NO: 3, and the antisense base sequence is shown in SEQ ID NO: 4;

[0172] (c) The sense base sequence is shown in SEQ ID NO: 5, and the antisense base sequence is shown in SEQ ID NO: 6;

[0173] (d) The sense base sequence is shown in SEQ ID NO: 7, and the antisense base sequence is shown in SEQ ID NO: 8;

[0174] (e) The sense base sequence is shown in SEQ ID NO: 9, and the antisense base sequence is shown in SEQ ID NO: 10;

[0175] (f) The sense base sequence is as shown in SEQ ID NO: 11, and the antisense base sequence is as shown in SEQ ID NO: 12; or

[0176] (g) The sense base sequence is shown in SEQ ID NO: 13, and the antisense base sequence is shown in SEQ ID NO: 14.

[0177] In some implementation schemes, in any of the nucleic acid molecules of the first aspect mentioned above:

[0178] (a) The sense base sequence is shown in SEQ ID NO: 1, and the antisense base sequence is shown in SEQ ID NO: 2;

[0179] (b) The sense base sequence is shown in SEQ ID NO: 3, and the antisense base sequence is shown in SEQ ID NO: 4;

[0180] (c) The sense base sequence is shown in SEQ ID NO: 5, and the antisense base sequence is shown in SEQ ID NO: 6;

[0181] (d) The sense base sequence is shown in SEQ ID NO: 7, and the antisense base sequence is shown in SEQ ID NO: 8;

[0182] (e) The sense base sequence is shown in SEQ ID NO: 9, and the antisense base sequence is shown in SEQ ID NO: 10;

[0183] (f) The sense base sequence is as shown in SEQ ID NO: 11, and the antisense base sequence is as shown in SEQ ID NO: 12; or

[0184] (g) The sense base sequence is shown in SEQ ID NO: 13, and the antisense base sequence is shown in SEQ ID NO: 14.

[0185] Furthermore, the modified motif is selected from motif 4, optionally from motif 4-GalNAc ligand, optionally from motif 4-L96, motif 4-ligand 1 or motif 4-ligand 2, and further optionally from motif 4-p-L96, motif 4-s-ligand 1 or motif 4-p-ligand 2;

[0186] Alternatively, the modified motif may be selected from motif 3, or optionally from motif 3-GalNAc ligand, or optionally from motif 3-L96, motif 3-ligand 1 or motif 3-ligand 2, or further optionally from motif 3-p-L96, motif 3-s-ligand 1 or motif 3-p-ligand 2.

[0187] A second aspect of this application also relates to a nucleic acid molecule that inhibits SERPINC1 gene expression via RNAi, comprising or composed of complementary sense and antisense strands, wherein the sense strand is 18-25 nt in length (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nt) and the antisense strand is 20-27 nt in length (e.g., 20, 21, 22, 23, 24, 25, 26, or 27 nt), wherein the nucleic acid molecule comprises 18-21 (e.g., 18, 19, 20, or 21) consecutive nucleotides of the sense strand sequence of any dsRNA of SER3, SER1, or SER2, or SER4 to SER-100 as shown in the Sequence Appendix, and 20-23 (e.g., 20, 21, 22, or 23) consecutive nucleotides of the antisense strand sequence of the any dsRNA, wherein each nucleotide independently comprises one or more modifications selected from: lock Nucleic acid modification, ring-opening or non-locked nucleic acid modification, morpholino modification, tetrahydropyran modification, 1,5-dehydrated hexadiol modification, cyclohexenyl modification, 2'-methoxyethoxy modification (2'-MOE), 2'-O-methyl modification (2'-OMe), 2'-O-allyl modification, 2'-C-alkyl modification, 2'-C-allyl modification, 2'-fluorinated modification (2'-F), 2'-deoxy modification (DNA modification), 2'-amino modification, thiophosphate modification, ammonia The modification includes 5'-phosphate ester modification, methylphosphonate modification, 5'-phosphate ester modification, 5'-phosphate ester mimicry modification, and invAb (reverse debased deoxyribonucleotide) modification, wherein the "5'-phosphate ester mimicry" includes, but is not limited to: 5'-aminophosphate, 5'-methylphosphonate, 5'-thiophosphate, 5'-vinyl phosphate (5'-VP), and 5'-cyclopropyl phosphate (5'-cPrp); and contains at least one desialylate glycoprotein receptor (ASGP R) ligand linked to the sense strand. It should be understood that, as used herein, when describing a nucleic acid molecule as containing "a sequence of N consecutive nucleotides" of a sense or antisense strand of a dsRNA as shown in the sequence listing, it means that the base sequence of the N consecutive nucleotides contained in the nucleic acid molecule is identical to the sequence consisting of the N consecutive bases in that base sequence.

[0188] In some implementations, the nucleic acid molecule has the following properties:

[0189] (a) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 1.

[0190] The antisense strand base sequence comprises 20, 21, 22 or 23 consecutive nucleotides of SEQ ID NO: 2;

[0191] (b) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 3.

[0192] The antisense strand base sequence comprises 20, 21, 22 or 23 consecutive nucleotides of SEQ ID NO: 4;

[0193] (c) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 5.

[0194] The antisense strand base sequence comprises 20, 21, 22 or 23 consecutive nucleotides of SEQ ID NO: 6;

[0195] (d) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 7.

[0196] The antisense strand base sequence comprises 20, 21, 22 or 23 consecutive nucleotides of SEQ ID NO: 8;

[0197] (e) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 9.

[0198] The antisense strand base sequence comprises 20, 21, 22 or 23 consecutive nucleotides of SEQ ID NO: 10;

[0199] (f) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 11.

[0200] The antisense strand base sequence comprises 20, 21, 22, or 23 consecutive nucleotides of SEQ ID NO: 12; or

[0201] (g) The sense sequence comprises 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO: 13.

[0202] The antisense strand base sequence comprises 20, 21, 22 or 23 consecutive nucleotides of SEQ ID NO: 14.

[0203] In some implementations, the nucleic acid molecule has the following properties:

[0204] (a) The sense chain sequence comprises 18 consecutive nucleotides of SEQ ID NO: 1.

[0205] The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 2;

[0206] (b) The sense chain sequence comprises 18 consecutive nucleotides of SEQ ID NO: 3.

[0207] The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 4;

[0208] (c) The sense chain sequence comprises 18 consecutive nucleotides of SEQ ID NO: 5.

[0209] The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 6;

[0210] (d) The sense chain sequence comprises 18 consecutive nucleotides as specified in SEQ ID NO: 7.

[0211] The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 8;

[0212] (e) The sense chain sequence comprises 21 consecutive nucleotides of SEQ ID NO: 9.

[0213] The antisense strand base sequence comprises 23 consecutive nucleotides of SEQ ID NO: 10;

[0214] (f) The sense base sequence comprises 18 consecutive nucleotides as specified in SEQ ID NO: 11.

[0215] The antisense base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 12; or

[0216] (g) The sense base sequence comprises 18 consecutive nucleotides as defined in SEQ ID NO: 13.

[0217] The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 14.

[0218] In some embodiments, the base sequence of the nucleic acid molecule comprises the sense and antisense strand sequences of any dsRNA as shown in the Sequence Appendix Tables SER-1 to SER-100, or the sequence formed by replacing the base pair formed by the 5' end of the sense strand and the antisense strand with an AU base pair.

[0219] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 1, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 2.

[0220] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 3, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 4.

[0221] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 5, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 6.

[0222] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 7, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 8.

[0223] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 9, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 10.

[0224] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 11, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 12.

[0225] In some embodiments, the sense base sequence comprises a base sequence as shown in SEQ ID NO: 13, and the antisense base sequence comprises a base sequence as shown in SEQ ID NO: 14.

[0226] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 1, and the antisense base sequence is as shown in SEQ ID NO: 2.

[0227] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 3, and the antisense base sequence is as shown in SEQ ID NO: 4.

[0228] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 5, and the antisense base sequence is as shown in SEQ ID NO: 6.

[0229] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 7, and the antisense base sequence is as shown in SEQ ID NO: 8.

[0230] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 9, and the antisense base sequence is as shown in SEQ ID NO: 10.

[0231] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 11, and the antisense base sequence is as shown in SEQ ID NO: 12.

[0232] In some embodiments, the sense base sequence is as shown in SEQ ID NO: 13, and the antisense base sequence is as shown in SEQ ID NO: 14.

[0233] In some embodiments, each nucleotide in the nucleic acid molecule independently comprises one or more modifications selected from the following: 2'-methoxyethoxy modification (2'-MOE), 2'-O-methyl modification (2'-OMe), 2'-fluoro modification (2'-F), 2'-deoxy modification (DNA modification), thiophosphate modification, 5'-phosphate or 5'-phosphate mimic modification; and comprises at least one desialyl glycoprotein receptor (ASGPR) ligand linked to the sense strand.

[0234] In some embodiments, each nucleotide in the nucleic acid molecule independently comprises one or more modifications selected from the following: 2'-O-methyl modification, 2'-fluorine modification, 2'-deoxy modification, thiophosphate modification, 5'-vinylphosphate modification, and includes at least one desialylate glycoprotein receptor (ASGPR) ligand linked to the 3' end of the positive strand.

[0235] In some embodiments, the 2' position of each nucleotide of the nucleic acid molecule is modified. In some embodiments, the 2' position of each nucleotide of the nucleic acid molecule independently includes a modification selected from any of the following: 2'-F modification, 2'-OMe modification, and 2'-deoxy modification. It should be understood that, in this application, 2'-deoxy modification means the deoxygenation of the 2' hydroxyl group of the ribonucleotide or the substitution of the 2' hydroxyl group with hydrogen.

[0236] In some embodiments, any of the nucleic acid molecules of the second aspect described above includes a 5'-phosphate mimicry modification at the 5' end of the antisense strand, wherein the "5'-phosphate mimicry" includes, but is not limited to: 5'-aminophosphate, 5'-methylphosphonate, 5'-thiophosphate, 5'-vinyl phosphate (5'-VP), and 5'-cyclopropyl phosphate (5'-cPrp). In some embodiments, any of the nucleic acid molecules of the second aspect described above includes a 5'-vinyl phosphate modification (5'-VP) or a 5'-cyclopropyl phosphate modification (5'-cPrp) at the 5' end of the antisense strand.

[0237] In some embodiments, any of the nucleic acid molecules of the second aspect described above contains a phosphate thioester modification at at least one of the following positions: (1) between the first and second nucleotides starting from the 5' end of the sense strand; (2) between the second and third nucleotides starting from the 5' end of the sense strand; (3) between the first and second nucleotides starting from the 3' end of the sense strand; (4) between the second and third nucleotides starting from the 3' end of the sense strand; (5) between the first and second nucleotides starting from the 5' end of the antisense strand; (6) between the second and third nucleotides starting from the 5' end of the antisense strand; (7) between the first and second nucleotides starting from the 3' end of the antisense strand; and (8) between the second and third nucleotides starting from the 3' end of the antisense strand.

[0238] In some embodiments, any of the nucleic acid molecules of the second aspect described above contains a phosphate thioester modification at the following positions: (1) between the first and second nucleotides starting at the 5' end of the sense strand; (2) between the second and third nucleotides starting at the 5' end of the sense strand; (3) between the first and second nucleotides starting at the 5' end of the antisense strand; (4) between the second and third nucleotides starting at the 5' end of the antisense strand; (5) between the first and second nucleotides starting at the 3' end of the antisense strand; and (6) between the second and third nucleotides starting at the 3' end of the antisense strand.

[0239] In some embodiments, any of the nucleic acid molecules of the second aspect described above contains phosphate thioester modifications at the following positions: (1) between the first and second nucleotides starting from the 5' end of the sense strand; (2) between the second and third nucleotides starting from the 5' end of the sense strand; (3) between the first and second nucleotides starting from the 5' end of the antisense strand; (4) between the second and third nucleotides starting from the 5' end of the antisense strand; (5) between the first and second nucleotides starting from the 3' end of the antisense strand; and (6) between the second and third nucleotides starting from the 3' end of the antisense strand; and the other nucleotide linkages of the nucleic acid molecule are phosphate ester bonds.

[0240] In some embodiments, the ASGPR ligand contained in the nucleic acid molecule of the second aspect is attached (or conjugated) to the 5' end and / or 3' end of the sense strand. In some embodiments, the ASGPR ligand is attached to the 5' end of the sense strand. In some embodiments, the ASGPR ligand is attached to the 3' end of the sense strand. In some embodiments, the ASGPR ligand is a GalNAc ligand formed from one, two, three, or more N-acetylgalactosamine (GalNAc) derivatives. In this application, GalNAc polymers formed from one, two, three, or more GalNAc derivatives are referred to as monovalent GalNAc ligands, divalent GalNAc ligands, trivalent GalNAc ligands, and polyvalent GalNAc ligands, respectively. In some embodiments, the ASGPR ligand is a trivalent GalNAc ligand formed from three GalNAc derivatives.

[0241] In some embodiments, the GalNAc ligand contained in the nucleic acid molecule of the second aspect is attached (or conjugated) to the 5' end and / or 3' end of the sense strand. In some embodiments, the GalNAc ligand is attached to the 5' end of the sense strand. In some embodiments, the GalNAc ligand is attached to the 3' end of the sense strand. In some embodiments, the GalNAc ligand is L96, the ligand shown in Formula II, or ligand 1. In some embodiments, L96 is linked to the nucleotide via a phosphate ester bond. In some embodiments, the ligand shown in Formula II or ligand 1 is linked to the nucleotide via a phosphate thioester bond.

[0242] In some implementations, the nucleic acid molecules of the first and second aspects mentioned above are each independently dsRNA or siRNA with a hairpin structure (i.e., nucleic acid molecules formed by linking dsRNA through nucleotide chains or chemical bonds).

[0243] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is a dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequence is the same as the sense strand and antisense strand sequence of any dsRNA as shown in the sequence appendix SER-1 to SER-100; (2) the modifying motif is selected from motif 1, motif 2, motif 3 or motif 4.

[0244] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is a dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequence is the same as the sense strand and antisense strand sequence of any dsRNA as shown in the sequence appendix SER-1 to SER-100; (2) the modified motif is selected from motif 1-GalNAc, motif 2-GalNAc, motif 3-GalNAc or motif 4-GalNAc.

[0245] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is a dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequence is the same as the sense strand and antisense strand sequence of any dsRNA from SER-1 to SER-100 as shown in the sequence appendix; (2) the modified motif is selected from motif 1-L96, motif 2-L96, motif 3-L96 or motif 4-L96.

[0246] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is a dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequence is the same as the sense strand and antisense strand sequence of any dsRNA from SER-1 to SER-100 as shown in the sequence appendix; (2) the modified motif is selected from motif 1-p-L96, motif 2-p-L96, motif 3-p-L96 or motif 4-p-L96.

[0247] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequence is the same as the sense strand and antisense strand sequence of dsRNA of SER-3, SER-3-U, SER-33-U, SER-62, SER-97, and SER-97-U in the sequence appendix; (2) the modified motif is selected from motif 4.

[0248] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequences are the same as the sense strand and antisense strand sequences of dsRNAs SER-3, SER-3-U, SER-33-U, SER-62, SER-97, and SER-97-U in the sequence appendix; (2) the modified motif is selected from motif 4-GalNAc.

[0249] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequence is the same as the sense strand and antisense strand sequence of dsRNA of SER-3, SER-3-U, SER-33-U, SER-62, SER-97, and SER-97-U in the sequence appendix; (2) the modified motif is selected from motif 4-L96.

[0250] In some embodiments of the nucleic acid molecules in the first and second aspects mentioned above, the nucleic acid molecule is dsRNA, wherein the nucleic acid molecule has the following characteristics: (1) its sense strand and antisense strand base sequences are the same as the sense strand and antisense strand sequences of dsRNAs SER-3, SER-3-U, SER-33-U, SER-62, SER-97, and SER-97-U in the sequence appendix; (2) the modified motif is selected from motif 4-p-L96.

[0251] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 19 nt nucleotide selected from the antisense strand nucleotide sequence. In some embodiments, the sense strand of the nucleic acid molecule in any of the aforementioned aspects is 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 19 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence. In some embodiments, the sense strand of the nucleic acid molecule in any of the aforementioned aspects is 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 19 nt nucleotide selected from the antisense strand nucleotide sequence. In some implementations, the nucleic acid molecule of any of the foregoing aspects has a sense strand length of 19-25 nt, comprising a continuous 19 nt nucleotide from the 3' end of any dsRNA sense strand nucleotide sequence selected from Table A and a continuous 19 nt nucleotide from the 5' end of any antisense strand nucleotide sequence.

[0252] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the antisense strand nucleoside sequence. In some embodiments, the sense strand of the nucleic acid molecule in any of the aforementioned aspects is 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleoside sequence. In some embodiments, the sense strand of the nucleic acid molecule in any of the aforementioned aspects is 19-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleoside sequence.

[0253] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the antisense strand nucleotide sequence. In some embodiments, the sense strand of the nucleic acid molecule in any of the aforementioned aspects is 19-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence.

[0254] In some implementations, the sense strand and antisense strand of the nucleic acid molecule in any of the foregoing aspects are each 19-25 nt long independently, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 23 nt nucleotide selected from an antisense strand nucleotide sequence.

[0255] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide selected from the antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-23 nt long, comprising a continuous 19 nt nucleotide starting from the 3' end of any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence.

[0256] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence.

[0257] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 19-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 23 nt nucleotide selected from a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide selected from a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of a paired antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-25 nt long, comprising a continuous 19 nt nucleotide starting from the 3' end of any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleotide sequence.

[0258] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the foregoing aspects are each independently 21-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence.

[0259] In some implementations, the sense strand and antisense strand of the nucleic acid molecule in any of the foregoing aspects are each independently 21-25 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 23 nt nucleotide selected from an antisense strand nucleotide sequence.

[0260] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the antisense strand nucleoside sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleoside sequence. In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the aforementioned aspects are each independently 21-23 nt long, comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide starting from the 5' end of the antisense strand nucleoside sequence.

[0261] In some embodiments, the sense strand and antisense strand of the nucleic acid molecule in any of the foregoing aspects are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence.

[0262] In some implementations, the sense strand and antisense strand of the nucleic acid molecule in any of the foregoing aspects are each independently 21-23 nt long, comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 23 nt nucleotide selected from an antisense strand nucleotide sequence.

[0263] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 19 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 19 nt nucleotide selected from the 3' end of any dsRNA sense strand nucleoside sequence in Table A and a continuous 19 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some implementations, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly containing a continuous 19 nt nucleotide from the 3' end of the sense strand nucleotide sequence of any dsRNA in Table A and a continuous 19 nt nucleotide from the 5' end of the antisense strand nucleotide sequence.

[0264] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 3' end of any dsRNA sense strand nucleoside sequence in Table A. In some implementations, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 19 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide from the 3' end of the sense strand nucleotide sequence of any dsRNA in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence.

[0265] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 21 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 21 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 21 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A.

[0266] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 21 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 21 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 21 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A.

[0267] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 23 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 23 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 23 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 19 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide selected from the 5' end of any dsRNA sense strand nucleoside sequence in Table A.

[0268] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 19 nt and an antisense strand length of 21 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence.

[0269] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleoside sequence. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 21 nt and an antisense strand of 21 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleoside sequence.

[0270] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleoside sequence. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleoside sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleoside sequence.

[0271] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 23 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence. In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand length of 23 nt and an antisense strand length of 23 nt, each correspondingly comprising a continuous 21 nt nucleotide selected from any dsRNA sense strand nucleotide sequence in Table A and a continuous 21 nt nucleotide from the 5' end of the antisense strand nucleotide sequence.

[0272] In some embodiments, the nucleic acid molecule of any of the aforementioned aspects has a sense strand of 21 nt and an antisense strand of 23 nt, each correspondingly comprising a consecutive 21 nt nucleotide from a dsRNA sense strand nucleoside sequence selected from Table A and a consecutive 23 nt nucleotide from an antisense strand nucleoside sequence.

[0273] As described in this application, “Table A” is shown below:

[0274] Table A. Modified dsRNA molecules

[0275] In some embodiments of the nucleic acid molecules described in the first and second aspects above, the nucleic acid molecules comprise:

[0276] A sense chain comprising a nucleotide sequence identical to at least 19 nt of the following sequence except for the presence of 0, 1, or 2 different ribose modifications: GmCmCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm(SEQ ID NO: 215); and

[0277] The antisense strand contains a nucleotide sequence that is identical to at least 19 nt of the following sequence except for the presence of 0, 1 or 2 different ribose modifications: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmCmUm (SEQ ID NO: 216);

[0278] or

[0279] A sense chain comprising a nucleotide sequence identical to at least 19 nt of the following sequence except for the presence of 0, 1, or 2 different ribose modifications: GmCmCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm (SEQ ID NO: 217); and

[0280] The antisense strand contains a nucleotide sequence that is identical to at least 19 nt of the following sequence except for the presence of 0, 1 or 2 different ribose modifications: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmCmUm (SEQ ID NO: 216).

[0281] In some embodiments of the nucleic acid molecules described in the first and second aspects above, the nucleic acid molecules comprise (1) or (2):

[0282] (1) A sense strand containing the following nucleoside sequence: CmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUmCmUm (SEQ ID NO: 219), and

[0283] Antisense strands containing the following nucleoside sequences: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCm (SEQ ID NO: 218);

[0284] (2) A sense strand containing the following nucleoside sequence: CmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm(SEQ ID NO: 220), and

[0285] The antisense strand containing the following nucleoside sequence: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCm (SEQ ID NO: 218).

[0286] In some embodiments of the nucleic acid molecule described in the first and second aspects above, the nucleic acid molecule contains a phosphothioester bond at at least one of the following positions:

[0287] Between the first and second nucleotides starting at the 5' end of the sense strand,

[0288] Between the second and third nucleotides starting from the 5' end of the sense strand,

[0289] Between the first and second nucleotides starting at the 3' end of the sense strand,

[0290] Between the second and third nucleotides starting from the 3' end of the sense strand,

[0291] Between the first and second nucleotides starting at the 5' end of the antisense strand,

[0292] Between the second and third nucleotides starting from the 5' end of the antisense strand,

[0293] Between the first and second nucleotides starting from the 3' end of the antisense strand, and

[0294] Between the second and third nucleotides starting from the 3' end of the antisense strand;

[0295] The positions of the nucleotides are counted starting from the first nucleotide with a base at the end of each nucleotide;

[0296] Or, when the first nucleotide at the 5' end and / or the 3' end is a baseless nucleotide, the counting of nucleotide positions does not exclude baseless nucleotides.

[0297] In some embodiments of the nucleic acid molecules of the first and second aspects described above, the nucleic acid molecules comprise an ASGPR ligand, optionally the ASGPR ligand being attached to the 5' end and / or the 3' end of the sense strand; optionally, the GalNAc ligand is L96 as shown in Formula I, or a ligand shown in Formula II or Formula III.

[0298] In some embodiments of the nucleic acid molecules described in the first and second aspects above, the nucleic acid molecules may or may not contain 5' phosphate mimicry modification.

[0299] In some embodiments of the nucleic acid molecules described in the first and second aspects above, the nucleic acid molecules comprise (1) a sense strand: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-ASGPR ligand (SEQ ID NO: 221-ASGPR ligand); and

[0300] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222); or

[0301] (2) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-ASGPR ligand (SEQ ID NO: 223-ASGPR ligand); and

[0302] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222);

[0303] The -ASGPR ligand indicates that the ASGPR ligand is linked to the 3' position of the nucleotide via a phosphate ester bond or a thiophosphate ester bond.

[0304] In some embodiments of the nucleic acid molecules described in the first and second aspects above, the nucleic acid molecules comprise:

[0305] (1) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-ligand 2 (SEQ ID NO: 221-ligand 2); and

[0306] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222);

[0307] (2) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-ligand 2 (SEQ ID NO: 223-ligand 2); and

[0308] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222);

[0309] (3) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-ligand 1 (SEQ ID NO: 221-ligand 1); and

[0310] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222); or

[0311] (4) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-ligand 1 (SEQ ID NO: 223-ligand 1); and

[0312] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222);

[0313] (5) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-L96 (SEQ ID NO: 221-L96); and

[0314] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222); or

[0315] (6) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-L96 (SEQ ID NO: 223-L96); and

[0316] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCm sUm(SEQ ID NO: 222);

[0317] (7) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-ligand 2 (SEQ ID NO: 221-ligand 2); and

[0318] Antisense chain: Phosphate mimic -AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(Phosphate mimic - SEQ ID NO: 222);

[0319] (8) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-ligand 2 (SEQ ID NO: 223-ligand 2); and

[0320] Antisense chain: Phosphate mimic -AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(Phosphate mimic - SEQ ID NO: 222);

[0321] (9) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-ligand 1 (SEQ ID NO: 221-ligand 1); and

[0322] Antisense chain: Phosphate mimic -AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(Phosphate mimic - SEQ ID NO: 222); or

[0323] (10) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-ligand 1 (SEQ ID NO: 223-ligand 1); and

[0324] Antisense chain: Phosphate mimic -AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(Phosphate mimic - SEQ ID NO: 222);

[0325] (11) Sense chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-L96 (SEQ ID NO: 221-L96); and

[0326] Antisense chain: Phosphate mimic -AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(Phosphate mimic - SEQ ID NO: 222); or

[0327] (12) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-L96 (SEQ ID NO: 223-L96); and

[0328] Antisense chain: Phosphate ester mimic - AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCm GmGmCmsCmsUm (Phosphate ester mimic - SEQ ID NO: 222).

[0329] In some embodiments, the phosphate ester analogues in (7)-(12) above are vinyl phosphate esters. In some embodiments, L96 in (1)-(12) above is linked to the 3' position of the nucleotide via a phosphodiester bond. In some embodiments, ligand 1 in (1)-(12) above is linked to the 3' position of the nucleotide via a thiophosphate diester bond.

[0330] This application also provides a second nucleic acid molecule comprising the nucleic acid molecules of the first or second aspect described above. In some embodiments, the second nucleic acid molecule may comprise any two or more nucleic acid molecules from the first and second aspects, wherein the two or more nucleic acid molecules are linked to each other by sense strands and / or antisense strands. In some embodiments, the second nucleic acid molecule is a dual-targeting or multi-targeting siRNA, that is, the second nucleic acid molecule comprises at least one nucleic acid molecule from the first and second aspects described above, and the sense and / or antisense strands of the at least one nucleic acid molecule are linked to siRNA targeting other target sites of SERPINC1 mRNA or siRNA targeting other gene mRNAs.

[0331] Furthermore, this application also provides a third nucleic acid molecule that can be transcribed into a dsRNA or shRNA precursor in cells, wherein the dsRNA or shRNA is or comprises any of the nucleic acid molecules described in the first and second aspects above. In some embodiments, the third nucleic acid molecule comprises any of the nucleic acid molecules described in the first and second aspects above, and one or more additional nucleotides. In some embodiments, the third nucleic acid molecule is a circular or linear nucleic acid molecule. In some embodiments, the third nucleic acid molecule is a circular or linear plasmid. In some embodiments, the nucleic acid molecule belongs to an artificially constructed viral genome, selected from, but not limited to, lentiviral vectors or other retroviral vectors, adenovirus vectors, AAV vectors, poxvirus vectors, baculovirus vectors, and herpes simplex virus vectors. In some embodiments, the nucleic acid molecule belongs to a cellular genome, such as a nuclear genome, mitochondrial nucleic acid, or cytoplasmic free nucleic acid. In some embodiments, the third nucleic acid molecule is an RNA molecule, a DNA molecule, or a chimeric molecule of RNA and DNA.

[0332] It should be understood that the scope of this application does not exclude other compounds or compositions containing the nucleic acid molecules of this project. As long as these compounds or compositions contain the nucleic acid molecules of this project and the nucleic acid molecules still function as SERPINC1RNAi, then the compounds or compositions fall within the scope of this application.

[0333] A third aspect of this application also provides a nucleic acid delivery body comprising any one of the nucleic acid molecules or their salts described in the first and second aspects above, a precursor of the aforementioned nucleic acid molecule, the aforementioned second nucleic acid molecule, or the aforementioned third nucleic acid molecule. In some embodiments, the nucleic acid delivery body is a liposome, lipid nanoparticles or other polymers, endosomes, exosomes, or vesicles.

[0334] This application also provides viral particles comprising the aforementioned second nucleic acid molecule. In some embodiments, the viral particles are enveloped or capped viral particles. In some embodiments, the viral particles are pseudovirus particles. In some embodiments, the viral particles belong to AAV, baculovirus, poxvirus, herpesvirus, alphavirus, lentivirus, or other retroviruses.

[0335] The system also provides cells containing the nucleic acid molecules described in the first or second aspect, or a second or third nucleic acid molecule. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells, such as stem cells, liver cells, or vascular endothelial cells.

[0336] A fourth aspect of this application also provides a pharmaceutical composition comprising any one of the nucleic acid molecules or salts thereof from the first and second aspects described above, or the second or third nucleic acid molecule or salt thereof described above, or a delivery medium for the third aspect described above, or the aforementioned cells, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition is used to treat or prevent SERPINC1-mediated diseases or symptoms in a subject. In some embodiments, the SERPINC1 gene-mediated diseases or symptoms may be caused by overexpression of the SERPINC1 gene or overproduction of the SERPINC1 protein, and may be regulated by downregulating SERPINC1 gene expression. The treatment refers to the relief, reduction, or cure of SERPINC1 gene-mediated diseases or symptoms, such as a decrease in serum SERPINC1 levels. For example, a reduction in serum SERPINC1 levels or concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0337] A fifth aspect of this application also provides a method and / or combination therapy for treating a subject suffering from a condition that would benefit from inhibiting or reducing the expression of the SERPINC1 gene, such as SERPINC1-associated diseases, wherein the method or combination therapy comprises administering, alone or in combination with other therapeutic agents, the RNA molecule or biological material of this application that inhibits the expression of the SERPINC1 gene.

[0338] In some embodiments, this application provides the use of the RNA molecule of this application that inhibits SERPINC1 gene expression in the preparation of a medicament for reducing SERPINC1 levels in patients with this need.

[0339] The combination therapy of this application comprises administering to a patient suffering from a SERPINC1-associated disease an RNA molecule of this application that inhibits SERPINC1 gene expression and an additional therapeutic agent. The combination therapy of this application reduces SERPINC1 levels in the subject (e.g., by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 99%).

[0340] In some implementations, the additional therapeutic agent may be an anti-SERPINC1 antibody or its antigen-binding fragment or derivative.

[0341] A fifth aspect of this application also provides the use of the aforementioned nucleic acid molecules or salts thereof, the second nucleic acid molecule, the third nucleic acid molecule, the aforementioned nucleic acid delivery device of the fourth aspect, as well as the aforementioned viral particles, the aforementioned cells, and the aforementioned pharmaceutical compositions. In some embodiments, this application provides the use of the aforementioned nucleic acid molecules of the first or second aspect, the aforementioned second nucleic acid molecule, the third nucleic acid molecule, the aforementioned nucleic acid delivery device of the fourth aspect, as well as the aforementioned viral particles, the aforementioned cells, and the aforementioned pharmaceutical compositions to inhibit the expression of SERPINC1 mRNA or protein. In some embodiments, this application provides the use of the aforementioned nucleic acid molecules of the first or second aspect or salts thereof, the aforementioned second or third nucleic acid molecules, the aforementioned nucleic acid delivery device of the fourth aspect, as well as the aforementioned viral particles, the aforementioned cells, and the aforementioned pharmaceutical compositions to prevent or treat SERPINC1-mediated diseases or symptoms in a subject.

[0342] In some embodiments, this application provides the use of the aforementioned first or second aspect nucleic acid molecule or its salt, the aforementioned second nucleic acid molecule, the third nucleic acid molecule, the aforementioned third aspect nucleic acid delivery body, the aforementioned viral particles, the aforementioned cells, and the aforementioned pharmaceutical composition in diseases requiring control of hemorrhagic disorder levels. This application also provides the use of the aforementioned first or second aspect nucleic acid molecule or its salt, the aforementioned second nucleic acid molecule, the aforementioned third nucleic acid molecule, the aforementioned third aspect nucleic acid delivery body, the aforementioned viral particles, and the aforementioned cells in the preparation of a medicament for inhibiting or reducing the transcription of SERPINC1 mRNA or the expression of SERPINC1 protein in target cells, preventing or treating SERPINC1-mediated diseases or symptoms in a subject, wherein the disease or symptom is a hemorrhagic disorder; preferably, the disease or symptom is hemophilia; more preferably, the disease or symptom is hemophilia A or hemophilia B.

[0343] This application also provides methods for inhibiting or reducing the transcription of SERPINC1 mRNA or the expression of SERPINC1 protein in target cells, methods for preventing or treating SERPINC1-mediated diseases or symptoms in subjects, and methods in diseases requiring reduction of hemorrhagic disorders. The aforementioned methods include administering an effective amount of the aforementioned first or second nucleic acid molecule, the aforementioned second nucleic acid molecule, the aforementioned third nucleic acid molecule, or the aforementioned fourth nucleic acid delivery body, the aforementioned viral particles, the aforementioned cells, or the aforementioned pharmaceutical composition to a patient or cell in need.

[0344] In some embodiments, the SERPINC1-mediated disease or symptom is a cardiovascular disease or symptom. In some embodiments, the SERPINC1-mediated disease or symptom is a bleeding disorder; preferably, the disease or symptom is hemophilia; more preferably, the disease or symptom is hemophilia A or hemophilia B.

[0345] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. The aspects and embodiments of this application described herein include aspects and embodiments described as "comprising," "forming," and "substantially consisting of." Attached Figure Description

[0346] Figure 1 shows the effect of the modified dsRNA and the positive control Fitusiran of this application on the relative protein level of human antithrombin (ATIII protein).

[0347] Figure 2 shows the knockdown ability and persistence of the SER3-motif 3-p-ligand 2-VP sequence on ATIII protein in cynomolgus monkeys.

[0348] Figure 3 shows the knockdown ability of the sequences SER3-motif 3-p-ligand 2-VP and SER3-motif 3-p-ligand 2 on ATIII protein in humanized mice.

[0349] Application details

[0350] The present invention provides a nucleic acid molecule and its salt that can induce RNA transcripts of the SERPINC1 gene mediated by the RNA-induced silencing complex (RISC), a second nucleic acid molecule that can transcribe the nucleic acid molecule, a delivery system for the nucleic acid molecule and the second nucleic acid molecule, a virus or cell that can transcribe the nucleic acid molecule, and the use of the nucleic acid molecule and the second nucleic acid molecule.

[0351] the term

[0352] For the purposes of this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the described technology pertains. All technical and patent disclosures referenced herein are incorporated herein by reference in their entirety.

[0353] As used herein, "dsRNA" refers to double-stranded RNA. Since siRNA is a double-stranded RNA, the term "dsRNA" encompasses siRNA. dsRNA also includes double-stranded RNAs longer than siRNA, where "longer than siRNA" can mean that its sense strand is longer than siRNA, or that its antisense strand is longer than siRNA, or that both its sense and antisense strands are longer than siRNA. Typically, double-stranded RNAs longer than the siRNA sequence they contain are cleaved into siRNA by a type III endonuclease called Dicer after entering the cell. In some embodiments, the lengths of the two strands of the dsRNA are each independently 15 to 30 nt (in this application, "nt" refers to a nucleotide). When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in the RISC unwind the siRNA double helix. When it binds to a target mRNA complementary to the antisense strand of the siRNA, one or more endonucleases in the RISC cleave the target, inducing gene silencing. Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but this does not preclude the inclusion of one or more non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or two strands. In some embodiments, the dsRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, dsRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0354] As used herein, the terms "Serpin family C member 1," "SERPINC1," and "AT III" are used interchangeably and are also referred to in the art as antithrombin III. SERPINC1 can be of mammalian origin. In some embodiments, SERPINC1 is of primate origin. In some embodiments, SERPINC1 is of human origin. In some embodiments, SERPINC1 is of cynomolgus monkey origin. As used herein, "SERPINC1 gene mRNA" refers to the mRNA encoding the AT III protein, which can be transcribed from the SERPINC1 gene DNA and can be mature mRNA or pre-mRNA, and therefore may or may not contain introns. Because the SERPINC1 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the SERPINC1 gene mRNA sequence of this application is intended to include all mRNA sequences transcribed from SERPINC1 gene mutants. The human SERPINC1 gene mRNA sequence can be found, for example, in the National Geographic Library accession number GI:1848049903 (NM_000488.4). The rhesus monkey SERPINC1 mRNA sequence can be found, for example, in the National Geographic Library accession number GI:157167169 (NM_001104583.1). The cynomolgus monkey SERPINC1 mRNA sequence can be found, for example, in the National Geographic Library accession number GI:2739197436 (XM_005540037.4). The mouse SERPINC1 mRNA sequence can be found, for example, in the National Geographic Library accession number GI:1826689169 (NM_080844.5). The rat SERPINC1 mRNA sequence can be found, for example, in the National Geographic Library accession number GI:58865629 (NM_001012027.1). Other instances of the SERPINC1 gene mRNA sequence are readily available using publicly available databases, such as GenBANK. Unless otherwise specified, the human SERPINC1 gene mRNA, NM_000488.4, is used as the reference sequence, meaning that the nucleotide positions in the SERPINC1 gene mRNA are numbered according to the nucleotide numbers in the 5' to 3' directions in the reference sequence NM_000488.4. As used herein, the term "reference sequence" refers to a standard sequence used for homologous sequence alignment and can be used to define the nucleotide positions in a homologous polynucleotide or polynucleotide sequence.For example, "the site number of the base in the SERPINC1 mRNA sequence is the corresponding base number in the reference sequence NM_000488.4" means that after introducing vacancies or deleting nucleotides into the SERPINC1 gene mRNA sequence to make the SERPINC1 gene mRNA sequence and the reference sequence have the same bases at as many positions as possible, the nucleotides in the reference sequence are numbered sequentially starting from the first nucleotide at the 5' end. The SERPINC1 gene mRNA sequence and the reference sequence are defined by matching the corresponding nucleotides and defining the position of the nucleotide with the same number.

[0355] As used herein, the “complementarity” of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. Percentage complementarity indicates the percentage of nucleotides in the shorter nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with the other nucleic acid molecule (e.g., approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10). “Complete complementarity” means that all consecutive residues in the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% within a region of approximately 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or to two nucleic acids hybridizing under stringent conditions. For a single base or nucleotide, according to the Watson-Crick base pairing rule, A paired with T or U, or C paired with G or I, is called complementary, paired, or matched, and vice versa; all other base pairings are called non-complementary.

[0356] As used herein, nucleic acid “hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex may include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or a combination thereof.

[0357] In this application, when referring to two polynucleotide sequences or two nucleic acid strands "hybridizing with maximum complementarity," it means hybridization in which as many nucleotides as possible in the two strands pair with each other through hydrogen bonds. In "hybridization with maximum complementarity," one or more mismatches and one or more bulges in one or more of the two strands are permissible. However, in some embodiments, the two strands "hybridizing with maximum complementarity" may hybridize without mismatches or bulges. As used herein, "complementary region" refers to all hydrogen-bonded base pairs from the 5' end to the last hydrogen-bonded base pair after hybridization. The complementary regions formed by the two hybridized strands can be continuous or spaced apart. In this application, the antisense strand and sense strand are defined relative to a third sequence complementary to one of the sequences. For example, in the phrase "nucleic acid molecule that inhibits SERPINC1 gene expression in cells via RNAi," the antisense strand and sense strand are relative to the third sequence, the SERPINC1 gene mRNA sequence. Specifically, the antisense strand refers to a sequence in the nucleic acid molecule that has a complementary region to the SERPINC1 gene mRNA sequence, and the sense strand refers to a sequence in the nucleic acid molecule that has at least 10 consecutive identical nucleotides to the SERPINC1 gene mRNA sequence. Furthermore, in this application, the 5' end of any complementary region refers to the position of the nucleotide or base pair closest to the 5' end of the sense strand or the third sequence within its complementary region. Similarly, the 3' end of any complementary region refers to the position of the nucleotide or base pair closest to the 3' end of the sense strand or the third sequence within its complementary region. In this application, the terms “close,” “near,” or “far” used to describe nucleotide positions on the same sequence or nucleoside chain refer to the number of nucleotides separating the two nucleotide positions.

[0358] The term "nucleotide" hereincludes not only naturally occurring ribonucleotide or deoxyribonucleotide monomers, but also, in this context, its related structural variants, including derivatives and analogs, which are functionally equivalent in the specific context of the use of the nucleotide, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholino oligonucleotides. In some embodiments, the "nucleotides" of this application do not contain non-natural nucleotides with modified bases. In some embodiments, the "nucleotides" of this application do not contain nucleotides with modified bases. In this application, “G”, “C”, “A”, “T” and “U” generally represent nucleotides, nucleosides or bases with guanine, cytosine, adenine, thymine and uracil as bases, respectively. Unless otherwise specified, “G”, “C”, “A”, “T” and “U” indicate nucleotides, nucleosides or bases that are not limited to those containing modifications. They can be used to represent natural nucleotides or non-natural nucleotides (or nucleosides). The non-natural nucleotides may contain ribose and / or nucleotides with modified bases, as long as the bases therein can still be complementary to their naturally paired (i.e., paired according to the Watson-Crick principle) bases through hydrogen bonds. Similarly, they can also represent natural or non-natural bases or nucleosides. To further clarify, when “G,” “C,” “A,” “T,” and “U” represent natural nucleotides, nucleosides, bases, or their derivatives, the derivatives indicate chemical modifications relative to their corresponding natural nucleotides, nucleosides, or bases, while retaining the ability to complementary pairing with their naturally paired (i.e., pairing according to the Watson-Crick principle) bases via hydrogen bonds, the affinity of this complementary pairing is greater than the affinity with other bases with which they do not naturally pair. Therefore, since T and U are derivatives of each other, unless otherwise specified, T and U are interchangeable in base sequences (e.g., the base sequence portions in the sequence appendix). However, when T or U appears as an unmodified nucleotide or nucleoside in RNA, it only represents uridine; similarly, when T appears as an unmodified nucleotide in DNA, it only represents 2'-deoxythymidine.It should be understood that in this application, whenever nucleotide sequences are mentioned, the terms "nucleotide," "nucleotide residue," and "base" are used interchangeably. The number of base pairs is measured in bp, where one bp represents one base pair. The number of nucleotides is measured in nt, where one nt represents one nucleotide.

[0359] As used in this article, "nucleotide linkage" and "nucleotide bond" are interchangeable and refer to the chemical bond (or linking group) between two adjacent nucleotides. Unless otherwise specified, the chemical bond between the two nucleotides is a phosphate ester bond or a thiophosphate ester bond. However, when a modified motif specifically indicates "s" (representing a thiophosphate ester bond), or otherwise describes the position of a nucleotide linkage other than a phosphate ester bond, then all nucleotide linkages in that modified motif except at these positions are phosphate ester bonds. For example:

[0360] (1) Sense strand: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense strand: NmNfNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

[0361] Then the linkages between the nucleotides in (1) can be either phosphate ester bonds or thiophosphate ester bonds;

[0362] However, if (2) is a sense chain: NmsNmsNmNmNmNmNmNfNmNfNfNm ...

[0363] As used herein, unless otherwise specified, the term "base sequence" refers only to the arrangement of bases in a nucleic acid molecule or nucleic acid chain. "Base" does not limit the type of base to natural or non-natural bases, and it does not limit ribose modifications in nucleotides, nor does it limit phosphate group modifications. Unless otherwise specified, the term "nucleoside sequence" refers only to the arrangement of nucleosides in a nucleic acid molecule or nucleic acid chain. It may specify that the nucleoside has a certain ribose and / or base modification, but it does not limit phosphate group modifications. However, it does not exclude the possibility of other modifications to the base or ribose in addition to the specified modifications. Unless otherwise specified, the term "nucleotide sequence" refers to the sequence of nucleotides, which may specify that the nucleotide has base, phosphate, and / or ribose modifications. Similarly, it does not exclude the possibility of other modifications to the base, ribose, and phosphate groups in addition to the specified modifications. Unless otherwise specified, nucleosides or nucleotides with clearly defined bases in their nucleotide or nucleoside sequences do not have any modifications not specified therein. It should be understood that if the nucleoside and nucleotide sequences and their context in this application do not explicitly indicate the presence of a 5'-phosphate mimic and / or targeting ligand, it means that the presence of a 5'-phosphate mimic and / or targeting ligand is not limited.

[0364] As used in this article, "ligand modification" or "targeted ligand modification" refers to the attachment of a target ligand (or "ligand") to a nucleotide. When a nucleic acid molecule or nucleotide is attached to a target ligand, it is considered to have "ligand modification" or "targeted ligand modification".

[0365] As used herein, a “targeting ligand” is a chemical group that has an affinity for a particular organ, tissue, or cell. Unless otherwise specified, it may refer to a ligand monomer or a cluster of ligands formed by concatenating ligand monomers together or by linking them via chemical bonds and / or linkers. In some embodiments, the linking of the targeting ligand to a SERPINC1 RNAi agent (e.g., the nucleic acid molecule that inhibits SERPINC1 gene expression via RNAi, as described in this application) may be performed without the use of an additional linker, for example, by designing the targeting ligand to have readily available linkers to facilitate binding with the SERPINC1 RNAi agent. In some embodiments comprising multiple SERPINC1 RNAi compositions, two or more RNAi agents may use the same linker to their respective targeting ligands. In other embodiments comprising multiple SERPINC1 RNAi compositions, two or more RNAi agents may use different linkers to their respective targeting groups. In some embodiments, a linker group is covalently concatenated to the RNAi agent. In some embodiments, the linker group is concatenated to the sense strand of the RNAi agent. In some embodiments, the linker group is conjugated to the 5' or 3' end of the sense strand of the RNAi agent. In some embodiments, the linker group is conjugated to the 5' end of the sense strand of the RNAi agent. In some embodiments, the targeting ligand, pharmacokinetic modulator, delivery polymer, and / or delivery medium for linking with the RNAi agent inherently contains a linker group, which is covalently conjugated to a portion of the targeting ligand, pharmacokinetic modulator, delivery polymer, or delivery medium other than the linker group. The linker group facilitates covalent bonding between the RNAi agent and the targeting ligand, pharmacokinetic modulator, delivery polymer, and / or delivery medium; the linker group may be conjugated to the 3' and / or 5' end of the sense or antisense strand of the RNAi agent. When the RNAi agent is linked to the targeting ligand, pharmacokinetic modulator, delivery polymer, and / or delivery medium, the linker group may be converted into a "linker" or a portion thereof in this application. Examples of linking groups include, but are not limited to: C6-SS-C6, reactive groups (such as primary amines (e.g., NH2-C6) and alkynes, alkyl groups, debased residues / nucleotides, amino acids, triyne functionalized groups, ribitols, and / or PEG groups. As used herein, a “chemical bond” is a connection between two atoms that links one chemical group (e.g., an RNAi agent) or a segment of interest to another chemical group (e.g., a targeting group, a pharmacokinetic modifier, or a delivery polymer) or a segment of interest; a chemical bond can be a stable bond (e.g., a covalent bond) or an unstable bond.The "linker" optionally includes a spacer that increases the distance between two linking atoms, the spacer further increasing the flexibility and / or length of the bond; the spacer includes, but is not limited to, alkyl, alkenyl, alkyne, aryl, aralkyl, arkenyl, and arkyne; each may contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. In some embodiments, the SERPINC1 RNAi agent is conjugated to a polyethylene glycol (PEG) moiety or to a hydrophobic group having 12 or more carbon atoms, such as cholesterol or palmitoyl. In some embodiments, the SERPINC1 RNAi agent is linked to one or more pharmacokinetic / pharmacodynamic (PK / PD) modulators. PK / PD modulators can increase the circulation time of the conjugated drug and / or increase the activity of the RNAi agent by improving cell receptor binding, improving cellular uptake, and / or other means. Various PK / PD modulators suitable for use with RNAi agents are known in the art. In some embodiments, the PK / PD modulator may be cholesterol or a cholesterol-based derivative, or in some cases, the PK / PD modulator may be composed of alkyl, alkenyl, alkynyl, aryl, aralkyl, areneyl, or arynyl groups, each of which may be linear, branched, cyclic, and / or substituted or unsubstituted. In some embodiments, these moieties are attached at the 5' or 3' end of the sense chain, at the 2' position of any given nucleotide ribosome of the sense chain, and / or at any position on the sense chain to the phosphate or thiophosphate backbone.

[0366] As used herein, "dsRNA" refers to double-stranded RNA. Since siRNA is a double-stranded RNA, the term "dsRNA" encompasses siRNA. dsRNA also includes double-stranded RNAs longer than siRNA, where "longer than siRNA" can mean that its sense strand is longer than siRNA, or that its antisense strand is longer than siRNA, or that both its sense and antisense strands are longer than siRNA. Typically, double-stranded RNAs longer than the siRNA sequence they contain are cleaved into siRNA by a type III endonuclease called Dicer after entering the cell. In some embodiments, the lengths of the two strands of the dsRNA are each independently 15 to 30 nt (in this application, "nt" refers to a nucleotide). When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in the RISC unwind the siRNA double helix. When it binds to a target mRNA complementary to the antisense strand of the siRNA, one or more endonucleases in the RISC cleave the target, inducing gene silencing. Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but this does not preclude the inclusion of one or more non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or two strands. In some embodiments, the dsRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, dsRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0367] As used herein, the "asialoglycoprotein receptor (ASGPR)" is an endocytic receptor specifically expressed by hepatocytes, primarily located on the cell membrane surface of hepatocytes facing the sinusoidal space, and exhibits specificity for sugars. The amino acid sequence of the ASGPR protein is highly conserved across species. In humans, ASGPR is mainly expressed in hepatocytes. After the nucleic acid GalNAc conjugate (also referred to as GalNAc ligand in this application) recognizes and binds to the ASGPR receptor, it is rapidly endocytosed by hepatocytes to form an endosome. Subsequently, the pH within the endosome decreases, causing the nucleic acid GalNAc conjugate to dissociate from the ASGPR receptor, and the nucleic acid molecule escapes from the endosome. During this escape process, the nucleic acid molecule also rapidly dissociates from GalNAc. In this application, any targeting ligand that can specifically bind to ASGPR is referred to as an "ASGPR ligand."

[0368] As used herein, “GalNAc” or “N-acetylgalactosamine” refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term “GalNAc” or “N-acetylgalactosamine” includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Optionally, the GalNAc compound of this application is in the β-form, namely 2-(acetylamino)-2-deoxy-β-D-galactopyranose. As used herein, “GalNAc derivative” refers to a derivative of GalNAc that retains its binding to ASGPR and induces hepatocyte endocytosis. Multiple GalNAc and / or GalNAc derivative monomers (components of the GalNAc conjugate, the units that exert ASGPR binding activity) are conjugated to form the "GalNAc conjugate" described in this application. The "GalNAc conjugate" belongs to the category of "GalNAc derivatives." Non-limiting examples of GalNAc conjugates or ASGPR ligands in this application include ligands with structures of Formula I (L96), Formula II (including ligand 1), and Formula III (ligand 2), the structures of which are shown below: Also known as L96; When X = S - It is also called ligand 1;

[0369] Also known as ligand 2;

[0370] Among them, Equations I to III These represent the 3' ends of the sense or antisense strand of the nucleic acid molecule, respectively. In some embodiments, Formulas I to III... These represent the 3' position of the nucleotide at the 3' end of the sense or antisense strand of the nucleic acid molecule, respectively, linked by a phosphodiester bond or a thiophosphate diester bond.

[0371] In this application, if the oligonucleotide molecule contains a ligand structure (such as a GalNAc ligand) and the ligand is linked to the nucleotide molecule via a phosphate ester bond or a thiophosphate bond, then "Nm-GalNAc" in the modified motif indicates that the GalNAc ligand is linked to the preceding Nm via a phosphate ester bond or a thiophosphate bond. When specifically indicating a phosphate ester bond, it can be described in words or represented as "Nm-p-GalNAc". "Nm-s-GalNAc" in the modified motif indicates that the GalNAc ligand is linked to the preceding Nm via a phosphate ester bond or a thiophosphate bond.

[0372] As used herein, the terms "protrusion," "dangling end," and "dangling sequence" are used interchangeably and refer to one or more unpaired nucleotides extending beyond the double-stranded region at the end of the strand. A nucleotide protrusion is typically formed when the 3' end of one strand extends beyond the 5' end of another strand, or when the 5' end of one strand extends beyond the 3' end of another strand. The length of a nucleotide protrusion is typically between 1 and 6 nucleotides, 1 and 5 nucleotides, 1 and 4 nucleotides, 1 and 3 nucleotides, 2 and 6 nucleotides, 2 and 5 nucleotides, or 2 and 4 nucleotides. In some embodiments, a nucleotide protrusion comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, a nucleotide protrusion comprises 1 to 4 nucleotides. In some embodiments, a nucleotide protrusion comprises 2 nucleotides. In some other embodiments, a nucleotide protrusion comprises 1 nucleotide.

[0373] The nucleotide at the overhang can be a ribonucleotide or a modified nucleotide as described herein. In some embodiments, the nucleotide at the overhang is a 2'-modified nucleotide (e.g., a 2'-fluorinated nucleotide, a 2'-O-methylated nucleotide), a deoxyribonucleotide, a reverse nucleotide (e.g., a reverse abase-free nucleotide, a reverse deoxyribonucleotide), or a combination thereof. For example, in one embodiment, the nucleotide at the overhang is a deoxyribonucleotide, such as deoxythymidine. In another embodiment, the nucleotide at the overhang is a 2'-O-methylated nucleotide, a 2'-fluorinated nucleotide, a 2'-methoxyethylated nucleotide, or a combination thereof. In other embodiments, the overhang comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the overhang comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dT dT-3') dinucleotide. When a nucleotide overhang is present in the antisense strand, the nucleotide in the overhang can be complementary to the target gene sequence, forming a mismatch with the target gene sequence or containing some other sequences (such as polypyrimidine or polypurine sequences, such as UU, TT, AA, GG, etc.).

[0374] Nucleotide overhangs can be located at the 5' or 3' end of one or both strands. For example, in one embodiment, the RNA molecule includes nucleotide overhangs at both the 5' and 3' ends of the antisense strand. In another embodiment, the RNA molecule includes nucleotide overhangs at both the 5' and 3' ends of the sense strand. In some embodiments, the RNA molecule includes nucleotide overhangs at the 5' end of the sense strand and / or the 5' end of the antisense strand. In other embodiments, the RNA molecule includes nucleotide overhangs at the 3' end of the sense strand and / or the 3' end of the antisense strand.

[0375] RNA molecules may include a nucleotide overhang at one end of a double-stranded RNA molecule and a blunt end at the other end. A "blunt end" means that the sense strand and antisense strand are completely base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the RNA molecule includes a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNA molecule includes a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In some embodiments, the RNA molecule includes blunt ends at both ends of the double-stranded RNA molecule. In these embodiments, the sense strand and antisense strand have the same length, and the length of the double-stranded region is the same as that of the sense strand and antisense strand (i.e., the molecule is double-stranded along its entire length).

[0376] As used herein, “GalNAc” or “N-acetylgalactosamine” refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term “GalNAc” or “N-acetylgalactosamine” includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Preferably, the GalNAc compound of this application is in the β-form, namely 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0377] As used herein, "5'-phosphate mimic," unless otherwise specified, is interchangeable with "phosphate mimic" and generally refers to a phosphate mimic linked to the 5' terminal nucleotide of a nucleic acid sequence. This mimic can be a phosphate ester, phosphonate ester, or other similar substance, linked to the 4' or 5' position of the 5' terminal nucleotide of the nucleic acid sequence. For example, it can be linked to the 4' position of the 5' terminal nucleotide of the nucleic acid sequence via a carbon-containing or carbon-free linker. It can maintain or increase the phosphorylation of the 5' terminal nucleotide of the nucleic acid sequence, reducing or preventing its degradation from 5'-3' by phosphatases and exonucleases. Non-limiting examples of the "5'-phosphate mimic" include, for example, vinylphosphonates, which are also referred to herein as "VP," "E-VP," or "5'-vp."

[0378] As used in this article, "VP modification" or "E-VP" is a phosphate ester-like modification located at the 5' end of an oligonucleotide, which forms the structure shown below by linking a vinyl phosphate ester to the 4' position of the nucleotide:

[0379] Wherein: Bx1 is a natural or modified base; T2 is a phosphate ester bond or thiophosphate bond that links the above compound to the oligonucleotide chain; and G is a substituent at the 2' position of the corresponding nucleotide, including but not limited to F, hydroxyl, H, methoxy, methoxyethoxy, etc.

[0380] In this application, the term "5'-cPrp" refers to 5'-cyclopropyl phosphate, whose chemical structure after being linked with a nucleotide is shown below:

[0381] Wherein: Bx1 is a natural or modified base; T2 is a phosphate ester bond or thiophosphate bond that links the above compound to the oligonucleotide chain; and G is a substituent at the 2' position of the corresponding nucleotide, including but not limited to F, hydroxyl, H, methoxy, methoxyethoxy, etc.

[0382] As used in this article, "3' end" specifically refers to the position of the first nucleotide or first base pair at the 3' end of a single nucleotide sequence or a double-stranded polynucleotide. "5' end" specifically refers to the position of the first nucleotide or first base pair at the 5' end of a single nucleotide sequence or a double-stranded polynucleotide.

[0383] As used in this application, the term "nucleic acid molecule" may be used to refer to any molecule having a nucleotide sequence consisting of two or more nucleotides linked by phosphate ester bonds, or modified phosphate ester bonds (e.g., thiophosphate bonds).

[0384] As used herein, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in a specific sequence. This polynucleotide chain can constitute a nucleic acid molecule or a segment of a chain within a nucleic acid molecule. Therefore, "nucleotide sequence" can be represented as a precise polynucleotide sequence composed of various nucleotides (e.g., ATCG), or as nucleotides at specific positions within a sequence, such as "nucleotides at positions 1430 to 1450 in the SERPINC1 gene mRNA sequence." In this application, unless otherwise specified, in sequences or nucleic acid molecules containing "nucleotides at specific positions within a sequence," the sequence of these nucleotides is consistent with the sequence of the aforementioned nucleotides within the sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid of RNA and DNA, and may also incorporate non-natural or artificially modified nucleotides.

[0385] In the context of RNA (e.g., mRNA, siRNA, dsRNA, or shRNA), N represents ribonucleotide, dN represents deoxyribonucleotide (DNA), Nm represents a nucleotide modified with 2'-O-Me or called a nucleotide modified with 2'-O-methyl; Nf represents a ribonucleotide modified with 2'-F or 2'-fluoro; s represents a phosphothioester modification, i.e., a 5'-thio modified phosphoester. When this modification occurs between two nucleotides or between a nucleotide and a ligand, it replaces the phosphate ester bond between the natural nucleotides and can be called a phosphothioester bond. As used herein, unless otherwise specified, "phosphothioester bond" and "phosphothiodiester bond" are used interchangeably; similarly, "phosphate ester bond" and "phosphodiester bond" are used interchangeably. Furthermore, as used herein, when describing a nucleotide whose 2' hydroxyl group is substituted with a methoxy, fluorine, or hydrogen, it is equivalent to describing the nucleotide as being modified with 2'-O-Me, 2'-F, or as a deoxyribonucleotide (2'-OH deoxy).

[0386] Furthermore, unless otherwise specified, all abbreviations or symbols appearing in this application as shown in the table below shall be interpreted as follows:

[0387] In this application, when describing the position of a nucleotide modification or the modification in the motif corresponding to the nucleotide base sequence, unless otherwise specified, the first nucleotide with a base starting from the 5' end or 3' end is referred to as the first nucleotide starting from the 5' end or 3' end of the chain. That is, when determining the position of a nucleotide modification, the nucleotides without bases at the 5' end and 3' end are not considered.

[0388] It should be understood that a nucleoside or nucleotide with "2'-OMe modification" indicates that the 2' position of the nucleoside or nucleotide is directly linked to a methoxy group; that is, when the nucleoside or nucleotide is a ribonucleoside or ribonucleotide, its 2' hydroxyl group is replaced by a methoxy group; when the nucleoside or nucleotide is a deoxyribonucleoside or deoxyribonucleotide, its 2' hydrogen is replaced by a methoxy group. Similarly, a nucleoside or nucleotide with "2'-F modification" indicates that the 2' position of the nucleoside or nucleotide is directly linked to a fluorine group. Furthermore, a nucleoside or nucleotide with "lipophilic moiety modification" indicates that the nucleoside or nucleotide is linked to a lipophilic moiety. In some embodiments, the lipophilic moiety is located in the middle of the nucleic acid chain and is linked to the 2' position of the nucleotide. In some embodiments, the lipophilic moiety is located at the end of the nucleic acid chain, for example, linked to the 3' position of the 3' terminal nucleotide of the nucleic acid chain.

[0389] Unless otherwise specified, the terms "pharmaceutically acceptable salt" or "medicinal salt" refer to a salt that, within the bounds of reasonable medical judgment, is suitable for contact with the tissues of mammals, particularly humans, without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio. Medically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salt can be prepared in situ during the final separation and purification of the compounds of this application, or solely by reacting a free base or free acid with a suitable reagent.

[0390] Unless otherwise specified, the term "isotope derivative" refers to a compound of this application that can exist in an isotopically traced or enriched form, containing one or more atoms whose atomic weights or mass numbers differ from the atomic weights or mass numbers of the most abundant atoms found in nature. Isotopes can be radioactive or non-radioactive. Commonly used isotopes for isotopic labeling include: hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O、 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2 Substitution with H can enhance metabolic stability and prolong the half-life, thereby achieving the goal of reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0391] Unless otherwise specified, the terms "solvent" or "solvent compound" refer to the physical association of the compound of this application with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvent compound" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanol compounds, methanol compounds, and isopropanol compounds. Solvation methods are well known in the art.

[0392] Unless otherwise specified, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and inhibited isomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0393] Unless otherwise specified, the term "tautomer" refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also called proton transfer tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons.

[0394] Unless otherwise indicated, the structural formulas described in this application include all isomers (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, any single stereochemical isomer of the compound of this application, or its enantiomers, diastereomers, or mixtures of geometric isomers (or conformational isomers), are within the scope of this application.

[0395] As used herein, the term "pharmaceutical composition" means a combination of at least one drug, optionally a pharmaceutically acceptable carrier or excipient, used together to achieve a particular purpose. Pharmaceutically acceptable carriers may include, for example, water, saline, glucose, buffer solutions (such as PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavorings, preservatives, or combinations thereof.

[0396] As used in this article, the term "treatment" refers to a clinical intervention aimed at altering the natural course of disease in the individual or cells receiving the treatment during the clinicopathological process. Ideal outcomes of treatment include slowing or reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0397] As used in this article, the term "prevention" refers to preventive treatment of a disease, disorder, or ailment; or delaying the onset or progression of a disease, disorder, or ailment.

[0398] In this application, the terms "effective amount" or "therapeutic effective amount" are used interchangeably and refer to an amount that has a therapeutic effect on a subject, such as: in subjects who have been given the amount, the symptoms or state of the disease are alleviated, reduced, or eliminated, or the development of the symptoms or state of the disease is delayed or suppressed compared to subjects who have not been given the amount.

[0399] As used herein, a subject, patient, or individual is considered "needed" for treatment if the subject would benefit biologically, medically, or in terms of quality of life from the treatment. In this text, the terms "subject," "patient," and "individual" are used interchangeably in certain contexts and have the same meaning.

[0400] The term “object” or “individual” as used in this article refers to mammals, such as humans, but can also refer to other animals, such as wild animals, livestock, or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0401] As used herein, the term "about" refers to the general range of error for various values ​​that is readily known to those skilled in the art. References to a value or parameter "about" herein include (and describe) an embodiment for that value or parameter itself. As used herein, when the term "about" precedes a numerical value, it indicates a range of 10% above or below that value. For example, "about 100" encompasses both 90 and 110.

[0402] As used herein, unless otherwise indicated, the singular forms “a,” “a,” and “the” include the plural forms.

[0403] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0404] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following embodiments are intended to illustrate, not limit, the scope of this application. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0405] Unless otherwise stated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These conventional techniques are described in prior art literature.

[0406] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments. Example

[0407] In the following specific embodiments of this application, each dsRNA molecule does not contain any modifications not specified in these embodiments. That is, the sequences of each dsRNA molecule used in the following specific embodiments of this application are naked sequences (i.e., sequences without modifications, only limited by bases) or only contain the modified motifs used therein and the modifications indicated in the context (e.g., indicated by Nm, Nf, Nd, s, etc.).

[0408] Example 1: Sequence Design and Synthesis of dsRNA

[0409] 1.1 dsRNA Design

[0410] Based on the human SERPINC1 gene (Gene ID, 462) mRNA sequence (NM_000488.4), multiple SERPINC1 dsRNAs were designed at different sites. All designed individual dsRNA sequences were aligned using sequence similarity software to show the lowest homology with all other non-target gene sequences. In this patent, we selected Fitusiran from Selective Enzyme Co., Ltd. as the SERPINC1 positive control sequence. The bare sequence is named ALN-AT3 in the sequence appendix, and the modified sequences are named ALN-AT3-ESC and ALN-AT3-ESC-p-L96. The bare sequences of the aforementioned dsRNA sequences and positive control sequences are shown in the sequence appendix at the end of this document.

[0411] The sequence information of Fitusiran's bare sequence (ALN-AT3) is as follows:

[0412] Sense chain: 5'-GGUUAACACCAUUUACUUCAA-3' (SEQ ID NO: 211)

[0413] Antonym: 5'-UUGAAGUAAAUGGUGUUAACCAG-3' (SEQ ID NO: 212)

[0414] The modified, unconjugated Fitusiran (ALN-AT3-ESC) sequence information is as follows:

[0415] Sense strand: 5'-GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-3' (SEQ ID NO: 213)

[0416] Antisense strand: 5'-UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsG m-3' (SEQ ID NO: 214)

[0417] The sequence information of the modified Fitusiran (ALN-AT3-ESC-p-L96) with L96 conjugation is as follows:

[0418] There is a chain of righteousness:

[0419] 5'-GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-p-L96-3' (SEQ ID NO: 213-p-L96)

[0420] Antisense strand: 5'-UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsG m-3' (SEQ ID NO: 214)

[0421] The "Af-p-L96" at the 3' end of the sense chain indicates that L96 is connected to the preceding Af via a phosphate ester bond.

[0422] The starting material for constructing GalNAc derivatives or ligands is a phosphorus amide monomer, using methods and standards commonly employed in the industry for solid-phase synthesis of phosphorus amides. During synthesis, phosphorus amides and functionalized solid supports are used. The phosphorus amides modified with GalNAc (e.g., L96, ligand 1, or ligand 2), alone or in combination with other GalNAc monomers, form polymers that can be added to any position on the oligonucleotide to form GalNAc derivatives. The GalNAc solid-phase support can be used to incorporate GalNAc modification at the 3'-terminus of the oligonucleotide.

[0423] All phosphoramidite monomers used in the preparation of dsRNA in the examples (including unmodified and phosphoramidite nucleoside monomers modified with 2'-methoxy, 2'-fluoro, 4'-PEO, and / or 5'-vinylphosphonate (5'-VP)) were commercially available or synthesized in-house; the 4'-PEO-modified phosphoramidite nucleoside monomer was referred to as compound 1H. Oligo was used. Synthesized on a CPG apparatus using a 48-type synthesizer. An acetonitrile solution of 0.6 M 5-ethimercaptotetrazole (5-HMT) was used as the activating agent. The coupling time was 300 seconds (2′OMe and 2′F). Thiophosphate or phosphate modification was introduced into the sequence via an oxidation step in the cyclic reaction. After solid-phase synthesis, the dried solid support was treated with ammonia solution at 55°C for 16 hours, then purified, desalted, and lyophilized. For 4'-PEO-modified nucleoside monomers protected with ethyl or methyl groups, after solid-phase synthesis, the solid support was treated with 7% trimethylbromosilane acetonitrile solution for 2 hours, followed by quenching of the deethylated or methylated silane group with 1 M mercaptoethanol solution for 1 hour. After this step, the dried solid support was treated with ammonia solution at 55°C for 16 hours, then purified, desalted, and lyophilized. The crude product was then purified by reversed-phase HPLC. Buffer A was 100 mM TEAA, pH... 7.5% acetonitrile and containing 5% acetonitrile, and buffer B is 100% acetonitrile. Record the UV trace at 260 nm and collect appropriate fractions. Use a microplate reader to determine the concentration using UV light, mix equimolar amounts of sense and antisense strands and add them to a new EP tube, heat at 95°C for 5 min, and slowly anneal to room temperature. Finally, use a vacuum concentrator to evaporate to dryness at room temperature to obtain the final product.

[0424] The L96 structure used in this application embodiment is shown below, prepared according to the methods of Examples 1-19 in CN104717982B:

[0425] in, This represents the 3' end of the sense or antisense strand of the dsRNA molecule linked by a phosphodiester bond.

[0426] The structure of ligand 1 used in this embodiment is shown below:

[0427] In the embodiments of this application, 3'Oligo5' represents a nucleotide linked to the 3' position via a thiophosphate bond;

[0428] The structure of ligand 2 used in this embodiment is shown below:

[0429] Synthesis of ligand 1

[0430] The molecule of ligand 1 is a trivalent GalNAc conjugate prepared from two compounds 6 and one compound 8 using conventional raw materials and synthetic methods in the art. The synthetic methods for compounds 6 and 8 are shown below:

[0431] 1. Synthesis of Compound 1

[0432] ① Synthesis of compound 16:

[0433] 10 g of compound 15 (CAS #: 2140-79-6, 2'-methoxyadenosine, 2'-O-Methyladenosine) and 100 mL of pyridine were added to a flask. After stirring to dissolve, the mixture was cooled in an ice bath. 20 g of B2Cl was carefully added dropwise, and the mixture was stirred in an ice bath for 10 min under nitrogen protection, then stirred for 16 hours at room temperature. Saturated sodium bicarbonate aqueous solution was slowly added to the reaction mixture in an ice bath, and after stirring for 1 hour, the mixture was concentrated to remove pyridine. DCM was added to the residue, and after stirring for 15 min, the mixture was allowed to stand and separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to give 18 g of compound 16 as a white solid powder, with a yield of 85.3%. ESI+MS: m / z 594.3 [M+H] + .

[0434] ② Synthesis of compound 17:

[0435] 10 g of compound 16 and HOAc / Ac2O (50 mL / 50 mL) were added to a flask. After stirring to dissolve, the mixture was heated to 100 °C and stirred at 100 °C for 2 hours. The reaction mixture was cooled to room temperature and carefully poured into 200 mL of ice water. DCM was then added, and the mixture was stirred for 15 min. After standing, the liquid was separated, and the organic phase was washed successively with water, 5% sodium bicarbonate aqueous solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to give 5 g of compound 17 as a white solid powder, with a yield of 71.4%. ESI+MS: m / z 415.2 [M+H] + .

[0436] ③ Synthesis of compound 19:

[0437] Add 5 g of compound 17 and 40 mL of DCM to a three-necked flask. After stirring to dissolve, cool to -30 °C and carefully add 4.02 g of TMSOTf dropwise under nitrogen protection, stirring at -30 °C for 30 min. Then carefully add 3.17 g of a DCM solution of compound 18 (CAS#: 24697-70-9), 2-(4-hydroxybutyl)isoindoline-1,3-dione, and (2-(4-Hydroxybutyl)-1H-isoindole-1,3(2H)-dione). After the addition is complete, continue stirring at -30 °C for 1 hour. Heat the reaction solution to 0-5 °C and carefully add 5% sodium bicarbonate aqueous solution dropwise. After stirring for 15 min, allow to stand and separate the liquid. Separate the organic phase, wash successively with 5% sodium bicarbonate aqueous solution and saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate. The crude product was prepared by column chromatography and reverse phase chromatography to obtain 2.8 g of compound 19 white solid powder, with a yield of 40.5% and ESI+MS: m / z 574.3 [M+H]+.

[0438] ④ Synthesis of Compound 1:

[0439] 2.5 g of compound 19 and 25 mL of MeOH were added to a flask. The mixture was stirred until homogeneous, and 320 mg of MeONa was carefully added dropwise under nitrogen protection. The mixture was stirred at room temperature for 6 hours, followed by concentration. The crude product was then converted to a white solid powder of compound 1 by reverse phase reaction, yielding 1 g of compound 1 in 62.9% yield. ESI-MS: m / z 424.4 [M+OAc-]-.

[0440] 2. Synthesis of Compound 6

[0441] ① Synthesis of compound 2:

[0442] 1.2 g of compound 1 and 12 mL of pyridine were added to a three-necked flask. After stirring to dissolve, 1.2 g of DMTrCl was added, and the mixture was stirred at room temperature for 1 hour under nitrogen protection. Excess DMTrCl was quenched by slowly adding methanol (0.6 mL) to the reaction mixture, and after stirring at room temperature for 15 min, 303.5 mg of NaHCO3 was added. The mixture was concentrated to obtain a crude product, which was dissolved by adding DCM / H2O and washing with water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product of compound 2, which was used directly in the next reaction step.

[0443] ② Synthesis of compound 3:

[0444] Ethanol (24 mL) was added to the crude compound 2, and the temperature was slowly raised to 50 °C. After stirring to dissolve, hydrazine hydrate (0.75 mL) was added, and the mixture was stirred overnight at 50 °C. Then, the temperature was lowered to room temperature, and stirring was continued at room temperature for half an hour. At this point, a large amount of white solid gradually precipitated. The mixture was filtered, and the residue was washed with ethanol. The filtrate was concentrated under reduced pressure, then redissolved in DCM / H2O. The liquid was separated, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain the crude compound 3, which was directly used in the next reaction.

[0445] ③ Synthesis of compound 5:

[0446] 0.9 g of compound 4 (CAS#:1159408-54-4, 5-[(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-BD-galacturonic acid)oxy]pentanoic acid, 5-[(3,4,6-Tri-O-Acetyl-2-Acetylamido-2-Deoxy-BD-Galactopyranosyl)Oxy]Pentanoic Acid), DCM (24 mL), 0.84 mL of Et3N, and 2.3 g of HBTU) were added sequentially to the crude compound 3. The mixture was stirred at room temperature for 2 hours under nitrogen protection. The reaction was quenched with 9 mL of water, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by reverse-phase preparation (75% ACN-H2O) to give 1.4 g of compound 5, with a three-step yield of 44.2%. ESI-MS: m / z 1025.6 [M+OAc-]-.

[0447] ④ Synthesis of compound 6:

[0448] 1.4 g of compound 5 was dissolved in 14 mL of DCM and cooled at 0 ± 2 °C. 524.7 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 81.3 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 2 hours. The reaction mixture was cooled at 0 ± 2 °C, quenched with 14 mL of 5% NaHCO3, and the organic phase was separated. The organic phase was washed with saturated brine (1 x 14 mL) at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude product was redissolved in DCM / MTBE and carefully added dropwise to a vigorously stirred heptane solution. A large amount of viscous oil gradually precipitated from the walls and bottom of the flask. After standing for 10 min, the supernatant was decanted. The crude product was chromatographically analyzed with EA:hept. = 10:1-2:1 as eluent. After concentration and drying, 560 mg of white solid powder was obtained, with a yield of 32% and a phosphorus spectrum purity of 98.19%. ESI-MS: m / z 1225.4 [M+OAc-]-.

[0449] 3. Synthesis of Compound 8

[0450] ① Synthesis of compound 7:

[0451] 2 g of compound 5 was dissolved in 20 mL of DCM, and 248.4 mg of succinic anhydride and 0.69 mL of Et3N were added sequentially. Finally, 25.3 mg of DMAP was added, and the mixture was stirred at room temperature for 16 hours. HPLC monitoring showed a significant amount of reactant remaining, so 310.4 mg of succinic anhydride and 0.69 mL of Et3N were added. Finally, 25.3 mg of DMAP was added, and the mixture was stirred at room temperature for another 36 hours. The reaction solution was cooled at 0 ± 2 °C, and ice water was added to the reaction mixture, followed by DCM. Finally, 40 mL of 1% HOAc aqueous solution was added, and the mixture was stirred for 10 min. The organic phase was separated and washed sequentially with 1% HOAc aqueous solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product. The crude product was chromatographically analyzed using DCM:MeOH = 70:1-20:1 eluent. After concentration and drying, 0.9 g of compound 7 was obtained as a white solid powder, with a yield of 40.7%. No succinic acid residue was detected, and the succinic anhydride residue was 0.26%. ESI-MS: m / z 1065.7 [MH]-.

[0452] ② Synthesis of compound 8:

[0453] 300 mg of compound 7, 72.7 mg of DIPEA, 106 mg of HBTU, and 10 mL of acetonitrile were added to a three-necked flask. The mixture was stirred at 25 °C for 10 minutes, followed by the addition of 650 mg of solid support PS. Stirring continued at 25 °C for 24 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed with acetonitrile. The collected filter cake was concentrated under vacuum to remove the solvent, yielding 850 mg of solid. 700 mg of this solid was added to a three-necked flask, along with 1.74 g of acetic anhydride, 4.15 mg of DMAP, 103 mg of triethylamine, and 10 mL of pyridine. The mixture was stirred at 25 °C for 4 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed successively with acetonitrile, methanol, and acetonitrile. The collected filter cake was concentrated under vacuum to remove the solvent, yielding 750 mg of compound 8. The loading was determined to be 254.82 μmol / g.

[0454] Synthesis of ligand 2

[0455] The molecule of ligand 2 is a trivalent GalNAc conjugate prepared from two compounds 44 and one compound 46 using conventional raw materials and synthetic methods in the art. The synthetic methods for compounds 44 and 46 are shown below:

[0456] 1. Synthesis of Compound 44

[0457] Synthesis of compound 36:

[0458] 60 g of compound 35 (commercially purchased, CAS No.: 26287-72-9, Chinese name: 1-acetoxy-2,3,5-tribenzoyloxy-L-ribose, English name: L-Ribofuranose, 1-acetate 2,3,5-tribenzoate) was dissolved in DCM (480 mL), and 24.66 g of 4-azido-1-butanol was added. The mixture was cooled to 0-10 °C. Under nitrogen protection, 8.43 g of boron trifluoride-diethyl ether solution was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. The reaction solution was cooled to 0-10 °C, and saturated sodium bicarbonate aqueous solution (480 mL) was added dropwise. After stirring for 30 min, the organic phase was separated, washed with saturated brine (480 mL), dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum. The crude product was subjected to column chromatography, concentrated, and dried to give 48.5 g of compound 36, a white solid, with a yield of 72.9%. ESI+MS: m / z 560.2 [M+H]+.

[0459] Synthesis of compound 37:

[0460] 50 g of compound 36 was dissolved in 250 mL of MeOH and cooled to 0–5 °C in an ice bath. Under nitrogen protection, 16.09 g of a MeOH solution of MeONa (approximately 30% by mass) was carefully added dropwise. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was then cooled to 0–5 °C, and 15 mL of 10% NH4Cl aqueous solution was carefully added. After stirring for 15 min, 15 mL of 5% sodium bicarbonate aqueous solution was added, and stirring was continued for another 15 min. The mixture was then concentrated to dryness, and residual water was removed using acetonitrile (50 mL x 2). The crude product was subjected to column chromatography, concentrated, and dried to give 20 g of compound 37 as a colorless oil, with a yield of 90.9%. ESI+MS: m / z 270.1 [M+Na]+.

[0461] Synthesis of compound 38:

[0462] 20.3 g of compound 37 was dissolved in pyridine (203 mL) and cooled at -10 °C. 39.78 g of di-tert-butylsilylbis(trifluoromethanesulfonic acid) was added, followed by 95.9 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 2 hours. The reaction mixture was concentrated to dryness under reduced pressure, and DCM (203 mL) was added. The mixture was washed successively with 5% NaHCO3 aqueous solution (203 mL) and saturated brine (203 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum. The crude product was subjected to chromatography, concentration, and drying to give 26.5 g of compound 38 as a colorless oil, with a yield of 83.3%. ESI+MS: m / z 433.2 [M+Na]+.

[0463] Synthesis of compound 39:

[0464] 10 g of compound 38 was dissolved in DMF (200 mL), and 7.32 g of iodomethane was added. The mixture was then cooled at -5 to -5 °C. Subsequently, 16.81 g of cesium carbonate was added. The reaction mixture was stirred at -5 to -5 °C for 15 min, then brought to room temperature and stirred for another 19 hours. Water (200 mL) and DCM (400 mL) were added to the reaction mixture, and after stirring for 10 min, the organic phase was separated. The organic phase was washed successively with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude compound 39 was used directly in the next step. ESI+MS: m / z 424.2 [M+Na]+.

[0465] Synthesis of compound 40:

[0466] The crude compound 39 was dissolved in 100 mL of THF and cooled at -5°C. Then, 1.55 g of pyridine hydrogen fluoride (approximately 70% by mass) was carefully added dropwise. After the addition was complete, the mixture was stirred at -5°C for 4 hours. EA (100 mL) was then added to the reaction mixture, followed by washing with saturated brine (200 mL x 6). The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum. The crude product was subjected to column chromatography, concentrated, and dried to give 4.6 g of compound 40 as a colorless oil, with a two-step yield of 68.2%. ESI+MS: m / z 284.1 [M+Na]+.

[0467] Synthesis of compound 41:

[0468] 10.7 g of compound 40 was dissolved in pyridine (107 mL). Under ice bath and nitrogen protection, 14.57 g of DMTrCl was added, and after the addition was complete, the mixture was stirred at room temperature for 40 min. Methanol (10.7 mL) was slowly added to the reaction mixture to quench the reaction, and the mixture was stirred at room temperature for 15 min. Then, 3.78 g of solid NaHCO3 was added. The mixture was stirred for another 15 min, concentrated, and the crude product was obtained. DCM / H2O (107 mL / 53.5 mL) was added, stirred to dissolve, and washed with saturated brine (53.5 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography, concentrated, and dried to give 17 g of compound 41 as a colorless oil, with a yield of 73.7%. ESI-MS: m / z 622.6 [M+OAc-]-.

[0469] Synthesis of compound 42:

[0470] 17 g of compound 41 was dissolved in a THF / H2O mixture (153 mL / 17 mL). 11.87 g of PPh3 was added in three portions of 3.96 g each, with a 5-minute interval between each addition. The mixture was heated to 5°C and stirred for 12 hours. The solution was concentrated to dryness under reduced pressure. The crude product was subjected to column chromatography, concentrated, and dried to give 14.5 g of a colorless oil, with a yield of 89.4%. ESI-MS: m / z 596.4 [M+OAc-]-.

[0471] Synthesis of compound 43:

[0472] To a 70 mL solution of 12 g of compound 4 (CAS#:1159408-54-4,5-[(3,4,6-tri-O-acetyl-2-acetamido-2-Deoxy-BD-Galactopyranosyl)Oxy]pentanoic acid, 6.17 g of EDCI·HCl, 4.35 g of HOBt, and 23.81 mL of Et3N were added sequentially. The mixture was stirred at room temperature under nitrogen protection for 0.5 hours. Then, compound 42 was added, and the mixture was stirred at room temperature under nitrogen protection for another 3 hours. The reaction was quenched with 70 mL of saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude product was subjected to column chromatography, concentrated, and dried to give 12 g of compound 43 as a white solid, with a yield of 46.3%. ESI-MS: m / z 1025.4 [M+OAc-]-.

[0473] Synthesis of compound 44:

[0474] 5 g of compound 43 was dissolved in 50 mL of DCM and cooled at 0 ± 2 °C. 1.87 g of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 290 mg of 1H-tetrazolium. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 2 hours. The reaction mixture was cooled at 0 ± 2 °C, quenched with 50 mL of 5% NaHCO3, and the organic phase was separated. The organic phase was washed with 50 mL of saturated brine at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum. The crude product was prepared by reverse phase reversal, concentrated, and dried to obtain 2.3 g of compound 44 as a white solid powder, with a yield of 38.1% and a phosphine purity of 99.8%. ESI-MS: m / z 1225.6 [M+OAc-]-.

[0475] 2. Synthesis of Compound 46

[0476] Synthesis of compound 45:

[0477] 4 g of compound 43 was dissolved in DCM (40 mL), and 827.8 mg of succinic anhydride and 4 mL of pyridine were added sequentially. Finally, 505.3 mg of DMAP was added, the temperature was slowly raised to 40 °C, and the mixture was stirred at 40 °C for 16 hours. The reaction solution was cooled at 0 ± 2 °C and washed sequentially with ice water (20 mL), 1% HOAc aqueous solution (20 mL x 3), and water (20 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product. The crude product was subjected to column chromatography, concentrated, and dried to obtain 3.1 g of compound 45 as a white solid powder, with a yield of 70.3%. ESI-MS: m / z 1065.5 [MH]-.

[0478] Synthesis of compound 46:

[0479] 1.5 g of compound 45, 323.9 mg of DIPEA, 640 mg of HBTU, and 60 mL of acetonitrile were added to a three-necked flask. The mixture was stirred at 25 °C for 10 minutes, followed by the addition of 4.37 g of solid support PS. Stirring continued at 25 °C for 16 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed with acetonitrile (30 mL x 3). The collected filter cake was concentrated under vacuum to remove the solvent, yielding a solid. This solid was added to another three-necked flask, along with 7.18 g of acetic anhydride, 17.17 mg of DMAP, 0.43 g of triethylamine, and 30 mL of pyridine. The mixture was stirred at 25 °C for 4 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed successively with acetonitrile (30 mL x 3), methanol (30 mL x 3), and acetonitrile (30 mL x 3). The collected filter cake was concentrated under vacuum to remove the solvent, yielding 5 g of compound 46. The loading was determined to be 239 μmol / g.

[0480] Preparation of oligonucleotide sequences with ligands linked to the 3' end of oligonucleotides

[0481] Synthesizing nucleic acid chains from the 3' to 5' end: One ligand carrier (e.g., compound 8 or compound 46) is sequentially reacted with two ligand monomers (e.g., compound 6 or compound 44); or, starting with a universal carrier (e.g., a UnyLinker-type carrier), synthesizing nucleic acid chains from the 3' to 5' end: Three ligand monomers (e.g., compound 6 or compound 44) ​​are reacted sequentially. Through conventional solid-phase synthesis cycles (deprotection, coupling, oxidation, and capping), the precursor (or intermediate) of the ligand linked to the solid-phase carrier is obtained. Further solid-phase synthesis cycles are then performed using the corresponding 2'-modified monomer. After the reaction is complete, the synthesized molecule is dissociated from the solid-phase carrier by ammonolysis, yielding the ligand-linked oligonucleotide sequence.

[0482] Preparation of oligonucleotide sequences with ligands attached to the inside or 5' of the oligonucleotide.

[0483] Nucleic acid chains are synthesized from the 3' to the 5' end. A solid-phase synthesis cycle is performed using the corresponding 2'-modified monomer. Then, the ligand monomer (such as compound 6 or compound 44) ​​or other monomers acting as linkers are reacted sequentially through conventional solid-phase synthesis cycle steps (deprotection, coupling, oxidation, and capping) to obtain an intermediate oligonucleotide containing a ligand at the 5' end, linked to a solid-phase support. (If the 5' end is further extended using a solid-phase synthesis cycle with the corresponding 2'-modified monomer, an intermediate intermediate with a ligand internally linked to the oligonucleotide is obtained; that is, two nucleic acid chains linked by a ligand.) After the reaction is complete, the entire synthesized molecule is dissociated from the solid-phase support by ammonolysis, yielding an oligonucleotide sequence containing a ligand internally or at the 5' end.

[0484] Example 2: High-throughput screening and detection of SERPINC1 gene-dsRNA in vitro activity

[0485] 2.1 SERPINC1 gene dsRNA transfection of HepG2 cells (naked sequence)

[0486] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. After cell digestion, the cell density was adjusted to 4 × 10⁶ cells / year. 5 Cells were seeded at a rate of 1 ml / well in 12-well plates. To prepare the transfection complex (100 μL): 45 μL of Opti-MEM and 5 μL of dsRNA (naked nucleic acid, unmodified, sequence as shown in the sequence appendix at the end of this article) at different concentrations were mixed; 48 μL of Opti-MEM and 2 μL of RNAiMax transfection reagent were mixed and allowed to stand for 5 min. Then, the two mixtures were combined and allowed to stand for 20 min to form the transfection complex. The transfection complex was added to the 12-well plates, with replicates for each sample, and the plates were incubated at 37°C with 5% CO2 for 24 h. Cells were harvested 24 h post-transfection, and RNA was extracted using the TRIZOL assay.

[0487] 2.2 Real-time quantitative PCR analysis

[0488] Cells were lysed 24 hours after transfection, and total RNA was extracted using the Novizan FastPure Cell / Tissue Total RNA Isolation Kit V2 (refer to the Novizan RC112-01 instruction manual). cDNA was then reverse transcribed using Takara PrimeScript RT Master Mix RR036Q. qPCR primer sequences are shown in the primer section of the sequence appendix at the end of this article. Human GAPDH was used as an internal reference gene, and PCR reactions were performed using a Bio-Rad CFX96 real-time PCR instrument. The Mock group was used as a control for normalization, ensuring that the SERPINC1 gene mRNA expression level in the Mock group was 1.

[0489] 2.3 Data Analysis

[0490] After the PCR reaction, relative quantification was performed using the reference gene as a standard and CFX96 software, followed by statistical analysis using GarphPad software. Table 1 and Figure 1 show the high-throughput screening validation results of dsRNA (naked nucleic acid, unmodified) molecules in HepG2 cells.

[0491] Table 1: Results of single-dose 1 nM high-throughput screening in HepG2 cells (unmodified)

[0492] Example 3: In vitro activity detection of modified SERPINC1 gene-dsRNA

[0493] High-throughput screening of SERPINC1 gene dsRNA in HepG2 cells yielded 19 preferred sequences (sequences are shown in the sequence appendix at the end of the article), which were then modified. The modified motif is motif 4 as shown below:

[0494] Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm,

[0495] Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm

[0496] Meanwhile, we selected the modified but unconjugated Fitusiran (ALN-AT3-ESC) as a positive control sequence for comparison with SERPINC1. The specific modification method is described in section 1.1 of Example 1.

[0497] Table 2 shows the screening results of candidate sequences and positive control sequences in HepG2 cells at a single dose (0.1 nM).

[0498] Table 2: Single-dose screening of modified dsRNA in HepG2 cells

[0499] The results show that multiple candidate sequences in this embodiment exhibit superior or equivalent effects to Fitusiran.

[0500] Example 4: Optimization of Serpinc1-dsRNA

[0501] 4.1 Inhibitory Activity Detection

[0502] To further confirm the preferred dsRNA molecules, we replaced the above preferred sequences with U of the antisense strand (that is, replaced the base pair containing the 5' end nucleotide of the antisense strand of the dsRNA with the AU base pair, where the 5' end nucleotide of the antisense strand is U and the 3' end nucleotide of the sense strand is A) to form SER33-U, SER35-U, SER36-U, SER97-U and SER3-U, respectively. The modification methods of the dsRNA and Fitusiran in this application are the same as those in Example 3.

[0503] Table 3: Single-dose (1 nM) screening of modified dsRNA in HepG2 cells

[0504] As can be seen, the multiple candidate sequences in this embodiment still exhibit results equivalent to or superior to those of Fitusiran. This result further demonstrates that the preferred sequences obtained through the screening in the above embodiments have the potential to efficiently reduce SERPINC1 nucleic acid levels.

[0505] Example 5: In vitro efficacy detection of modified dsRNA

[0506] This embodiment further validated the activity of the modified candidate complex in HepG2 cells. The modification methods of dsRNA and Fitusiran in this application are the same as in Example 3.

[0507] 5.1 Detection of mRNA expression levels in HepG2 cells

[0508] The detection steps were the same as in Example 2, with the human GAPDH gene as the internal reference gene. The detection primers used are shown in the primer section of the sequence appendix at the end of the article. Table 4 shows the average mRNA level in HepG2 cells treated with modified dsRNA relative to the target gene expression level in the untreated group (the relative mRNA expression level in the untreated group was set to 1).

[0509] Table 4. IC50 data of modified dsRNA in HepG2 cells.

[0510] The results once again demonstrate the highly efficient inhibitory activity of the candidate sequence against the target gene.

[0511] Example 6: In vivo efficacy test

[0512] The experiment used 6-8 week old SPF-grade humanized SERPINC1 homozygous mice (Nanmo Biological Experimental Animal Co., Ltd.), randomly divided into groups of 8 mice each (half male and half female). Each group received a single subcutaneous injection of either modified dsRNA conjugated with L96 or Fitusiran, while the Blank group received saline. Specific dosages are shown in Table 5. Blood samples were collected every 7 days after administration, and plasma was collected via phorectomy for SERPINC1 protein content detection.

[0513] In this embodiment, the dsRNA is modified with the motif 4-p-L96 as shown below:

[0514] Sense strand: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNm-p-L96, antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm

[0515] The modified Fitusiran (ALN-AT3-ESC-p-L96) with L96 is described in section 1.1 of Example 1.

[0516] Table 5 3mpk Single Dosage Regimen

[0517] 6.1 SERPINC1 protein ELISA detection

[0518] Plasma was diluted 1:200 or 1:1000 and the residual SERPINC1 content in each group was detected using the Abcam ab108801 ELISA kit. The specific operation steps were referred to the ab108834 ELISA test instructions. The absorbance value was detected at a wavelength of 450 nm using an ELISA reader and analyzed using GraphPad software. The inhibition rate percentage is a relative result relative to the untreated group. The results are shown in Figure 1 (the naming of each group refers to the naked sequence number shown in Table 5).

[0519] The results showed that, in a single 3mpk dose, the plasma SERPINC1 levels in mice treated with the candidate dsRNA molecules were significantly lower than those in the control group. On day 7, the inhibition rates of SER97, SER62, and SER3 were all above 75%, and after day 21, the candidate sequences showed superior inhibition of the target gene compared to *Syngonium oxyphylla*. These results demonstrate that our designed multiple dsRNA molecules targeting SERPINC1 can effectively inhibit the expression and secretion of SERPINC1 protein in the liver, representing a potential therapeutic agent targeting SERPINC1.

[0520] Example 7: In vitro efficacy detection of dsRNA after different modifications of SER3

[0521] To further confirm the modifications of the preferred dsRNA molecules, we tested the in vitro activity of SER3 sequences with different modifications.

[0522] 7.1 Detection of mRNA expression levels in HepG2 cells

[0523] The detection procedure was the same as in Example 2, using the human GAPDH gene as the internal reference gene. The detection primers used are shown in the primer section of the sequence appendix at the end of the article. Table 6 shows the average mRNA level in HepG2 cells treated with modified dsRNA relative to the target gene expression level in the untreated group (the relative mRNA expression level in the untreated group was set to 1). The results showed that the activity of the SER3 sequence modified with motif 3 (i.e., SER3-motif 3) was significantly higher than that of other modifications.

[0524] Table 6: Single-dose (0.1 nM) inhibitory activity of modified dsRNA in HepG2 cells

[0525] The dsRNA modification method in this embodiment is as follows:

[0526] SER3-Modal 4

[0527] Significant chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm(SEQ ID NO: 223)

[0528] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222)

[0529] SER3-E19

[0530] Significant chain: GmsCmsCmGmAmGmAmUmGfAmCfCmUfCmUmAmUmGmUmCmUm(SEQ ID NO: 224)

[0531] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222)

[0532] SER3-Modality 3

[0533] Significant chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm(SEQ ID NO: 221)

[0534] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222)

[0535] SER3-E23

[0536] Sense chain: GmsCmsCmGmAmGmAfUmGfAmCfCmUmCmUmAmUmGmUmCmUm(SEQ ID NO: 225)

[0537] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222)

[0538] Example 8: Detection of the in vitro efficacy of dsRNA with different SER3 modifications in primary monkey liver cells.

[0539] In this embodiment, the sequence of SER3-motif 3-p-ligand 2-VP is:

[0540] sense chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-p-ligand 2 (SEQ ID NO: 221-p-ligand 2);

[0541] Antonym chain: VP-AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(VP-SEQ ID NO: 222).

[0542] 8.1 Detection of mRNA expression levels in PMH (primary monkey hepatocytes)

[0543] Primary monkey hepatocytes were seeded at a density of 300,000 cells per well into pre-coated 24-well plates. After adhesion, different dsRNAs were transfected into the primary monkey hepatocytes using RNAiMax. Twenty-four hours after transfection, total RNA was extracted from the cells using Trizol. The detection procedure was the same as in Example 2, using the monkey-GAPDH gene as an internal control gene (primers are shown in the monkey-GAPDH sequence appendix at the end of the article), and the detection primers used are shown in the primer section of the sequence appendix at the end of the article.

[0544] The results are shown in Table 7. SER3-motif 3-p-ligand 2-VP significantly inhibited Serpinc1 in primary monkey hepatocytes compared to Fitusiran (as shown in Example 1, ALN-AT3-ESC-p-L96).

[0545] Table 7: Single-dose (0.05 nM) inhibitory activity of modified dsRNA in primary monkey hepatocytes

[0546] Example 9: In vivo efficacy test in cynomolgus monkeys

[0547] Two male cynomolgus macaques were used in the experiment, divided into two groups of one macaque per group. On Day 0, each group received a subcutaneous injection of the ginseng drug Fitusiran and SER3-motif 3-p-ligand 2-VP, respectively. The molecular structures of both drugs were identical to those in Example 8, and the dosage was 1 MPK. Blood samples were collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each group on Days 7, 3, 2, 7, 14, 21, 28, 35, 42, 49, 56, and 63. ATIII activity was detected using a colorimetric method (using the SIEMES Antithrombin III Assay Kit (chromogenic substrate method), and the procedure was performed according to the kit's instructions). For data processing, the average ATIII activity of Day 7 and Day 3 was used as the normalized value for Day 0.

[0548] As shown in Figure 2, at a dose of 1 MPK, SER3-motif 3-p-ligand 2-VP showed a more sustained knockdown and activity inhibition of ATIII protein expression compared to Yangshen.

[0549] Example 10: In vivo efficacy test in humanized mice

[0550] The experiment used 6-8 week old SPF-grade humanized SERPINC1 heterozygous mice (Nanmo Biological Experimental Animal Co., Ltd.), randomly divided into groups of 6 mice each (half male and half female). The mice received a single subcutaneous injection of 1 MPK. Each group received a different modified dsRNA conjugated to liver delivery, while the Blank group received saline. Specific dosages are shown in Table 8. Blood was collected every 7 days after administration, and plasma was collected via phorectomy for SERPINC1 protein content detection (using ELISA).

[0551] Table 81 mpk Single Dosage Regimen

[0552] In this embodiment, the sequence of SER3-motif 3-p-ligand 2 is as follows:

[0553] Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-p-ligand 2 (SEQ ID NO: 221-p-ligand 2),

[0554] Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222);

[0555] The SER3-motif 3-p-ligand 2-VP sequence is shown in Example 8.

[0556] As shown in Figure 3, at a dose of 1 MPK, the sequences SER3-motif 3-p-ligand 2-VP and SER3-motif 3-p-ligand 2 had comparable knockdown and activity inhibition capabilities against ATIII protein in humanized mice.

[0557] Example 11: In vitro activity assay of dsRNA

[0558] In this embodiment, the activity levels of multiple dsRNAs were further investigated in HepG2 cells in vitro.

[0559] The specific steps are as follows:

[0560] (1) dsRNA transfection of HepG2 cells

[0561] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. After cell digestion, the cell density was adjusted to 2.5 × 10⁻⁶ cells / year. 5Cells were seeded at a rate of 0.5 mL per well in a 24-well plate. To prepare the transfection complex: dsRNA was diluted with Opti-MEM to obtain 50 μL of dsRNA-Opti-MEM mixtures of different concentrations. Simultaneously, 49 μL of Opti-MEM and 1 μL of RNAiMax transfection reagent were mixed and incubated separately for 5 min. Then, the two mixtures were combined and incubated for 20 min to form the transfection complex. The RNAiMax-only treatment group served as a blank control. The transfection complex was added to the 24-well plates and incubated for 48 h in a 5% CO2, 37°C incubator.

[0562] (2) Real-time quantitative PCR analysis

[0563] Cells were lysed 48 h after transfection, and total RNA was extracted using the Novizan FastPure Cell / Tissue Total RNA Isolation Kit V2 (refer to the Novizan RC112-01 instruction manual), and then reverse transcribed into cDNA. The qPCR primer sequences are shown in Table 1. Human GAPDH-2 gene was used as an internal reference gene (primers are shown in the sequence listing at the end of the article), and PCR reactions were performed using a Bio-Rad CFX96 real-time PCR instrument.

[0564] (3) Data Analysis

[0565] After the PCR reaction, the 2–ΔΔCt method was used to normalize the expression of SERPINC1 mRNA in the blank control group (using human GAPDH as an internal reference and transfection reagent as a control) to a level of 1. Statistical analysis was then performed using GraphPad software. The results are shown in Tables 9 and 10.

[0566] Table 9: In vitro activity of naked dsRNA sequences, where T represents thymine deoxyribonucleoside and U represents uracil deoxyribonucleoside in each dsRNA sequence.

[0567] As shown in Table 9, SER3-1, SER3-3 and SER3 all have better inhibitory effects on SERPINC mRNA at the cellular level, especially SER3, which is more active than SER3-1 and SER3-3.

[0568] Table 10. In vitro activity of modified dsRNA

[0569] The SER3-motif 3-p-L96 sequences tested in Table 10 are shown below, where L96 is linked to the 3' position of the 3' terminal nucleotide of the sense strand via a phosphodiester bond. The Fituriran sequence is shown as ALN-AT3-ESC-p-L96 in Example 1:

[0570] SER3-motif 3-p-L96

[0571] Significant chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-p-L96 (SEQ ID NO: 221-p-L96),

[0572] Antonym chain: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222).

[0573] As shown in Table 10, SER3-motif 3-p-L96 at concentrations of 0.01 nM and 0.1 nM was superior to Fitusiran in knocking down the SERPINC1 target gene.

[0574] Example 12 Rat pretoxicology (SER3-motif 3-S-ligand 1)

[0575] This study observed the potential acute toxicity and severity of a single subcutaneous injection of SER3-motif 3-s-ligand 1 into Sprague Dawley (SD) rats, providing a preliminary understanding of the toxicity of the test substance and offering a reference for subsequent non-clinical and clinical trials. The sequence of SER3-motif 3-s-ligand 1 is shown below:

[0576] Sense strand: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-s-ligand 1 (SEQ ID NO: 221-s-ligand 1), where -s-ligand 1 indicates that the 3' terminal nucleotide of the sense strand is linked to ligand 1 at the 3' position via a phosphate thioester bond;

[0577] Antonym chain: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222).

[0578] The specific steps are as follows:

[0579] Twenty qualified SPF-grade SD rats (half male and half female) were randomly divided into two groups: a solvent control group (sodium chloride injection) and a test substance group (SER3-motif 3-s-ligand 1). The first administration day was defined as day 1 (D1). On D1, animals in the test substance group received a single subcutaneous injection of the corresponding test substance at a dose of 5 mL / kg, while the solvent control group received the same volume of sodium chloride injection. Toxicological observation indicators in this study included mortality, clinical observation (cage-side observation and detailed clinical observation), administration site observation (local irritation), body weight and weight changes, clinicopathology (D3 and D14), and dissection of all animals on D14. For planned dissection, blood was collected from the abdominal aorta after anesthesia (to detect coagulation and blood biochemical indicators). Animals were euthanized by exsanguination and gross necropsy were performed, and the liver and kidneys were preserved for histopathological examination.

[0580] Under the conditions of this experiment, Sprague Dawley (SD) rats were administered a single subcutaneous injection of 250 mg / kg of SER3-motif 3-s-ligand 1. The animals tolerated the drug well. The main changes related to the test substance were variations in blood biochemical and coagulation parameters, and organ damage to the liver and kidneys. This was reflected in clinicopathological examinations of changes in TP and ALB, FIB and TT, as well as histopathological findings in the liver including hepatocyte vacuolation, single hepatocyte necrosis, and mitotic figures, and in the kidneys, basophilic granular deposition in the renal tubules. The safety profile was deemed good.

[0581] Furthermore, further experiments in PHH cells (human primary liver cells) confirmed that SER3-motif 3-p-ligand 2 (sequence shown in Example 10) did not show off-target effects even at concentrations up to 500 nM (working concentration), indicating that it has a low off-target rate.

[0582] The above examples demonstrate that the SER3-motif 3 sequence has a high inhibition rate and safety against the SERPINC1 gene.

[0583] The sequences used in the above embodiments of this application are shown in the following sequence list. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence list may represent DNA sequences or RNA sequences, and when they represent RNA base sequences, the "T" and "U" may be used interchangeably.

[0584] Sequence Appendix

Claims

1. A nucleic acid molecule for inhibiting SERPINC1 gene expression via RNAi, comprising or composed of complementary sense and antisense strands, wherein the sense strand is 18-25 nt in length and the antisense strand is 20-27 nt in length. Each nucleotide independently contains one or more modifications selected from the following: 2'-O-methyl nucleotide modification, 2'-fluoronucleotide modification, 2'-deoxynucleotide modification, and phosphate thioester modification; Furthermore, the nucleic acid molecule contains at least one desialyl glycoprotein receptor (ASGPR) ligand linked to the sense strand; in, The nucleic acid molecule comprises 18-21 (e.g., 18, 19, 20, or 21) consecutive sense strands of any dsRNA as shown in the sequence listing SER3, SER1, or SER2, or SER4 to SER100, and 20-23 (e.g., 20, 21, 22, or 23) consecutive antisense strands of the any dsRNA.

2. The nucleic acid molecule according to claim 1, comprising or composed of complementary sense and antisense strands, wherein the sense strand has a length of 18-25 nt and the antisense strand has a length of 20-27 nt. Each nucleotide independently contains one or more modifications selected from the following: 2'-O-methyl nucleotide modification, 2'-fluoronucleotide modification, 2'-deoxynucleotide modification, and phosphate thioester modification; Furthermore, the nucleic acid molecule contains at least one desialyl glycoprotein receptor (ASGPR) ligand linked to the sense strand; in, The nucleic acid molecule: (a) The sense chain sequence comprises 18 consecutive nucleotides of SEQ ID NO:

1. The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 2; (b) The sense chain sequence comprises 18 consecutive nucleotides of SEQ ID NO:

3. The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 4; (c) The sense chain sequence comprises 18 consecutive nucleotides of SEQ ID NO:

5. The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 6; (d) The sense chain sequence comprises 18 consecutive nucleotides as specified in SEQ ID NO:

7. The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 8; (e) The sense chain sequence comprises 21 consecutive nucleotides of SEQ ID NO:

9. The antisense strand base sequence comprises 23 consecutive nucleotides of SEQ ID NO: 10; (f) The sense base sequence comprises 18 consecutive nucleotides as specified in SEQ ID NO:

11. The antisense base sequence comprises 20 consecutive nucleotides of SEQ ID NO: 12; or (g) The sense base sequence comprises 18 consecutive nucleotides as defined in SEQ ID NO:

13. The antisense strand base sequence comprises 20 consecutive nucleotides of SEQ ID NO:

14.

3. A nucleic acid molecule for inhibiting SERPINC1 gene expression via RNAi, comprising or composed of complementary sense and antisense strands, wherein the sense and antisense strands are each independently 15-30 nt in length, and wherein each nucleotide independently comprises one or more modifications selected from: Locked nucleic acid modification, ring-opening or non-locked nucleic acid modification, morpholino modification, tetrahydropyran modification, 1,5-dehydrated hexadiol modification, cyclohexenyl modification, 2'-methoxyethoxy modification (2'-MOE), 2'-O-methyl modification (2'-OMe), 2'-O-allyl modification, 2'-C-alkyl modification, 2'-C-allyl modification, 2'-fluorinated modification (2'-F), 2'-deoxy modification, 2'-amino modification, thiophosphate modification, aminophosphate modification, methylphosphonate modification, 5'-phosphate modification, 5'-phosphate mimic modification, and invAb (reverse debased deoxyribonucleotide) modification; Furthermore, the 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the other nucleotides in the antisense strand is substituted with methoxy or hydrogen. Furthermore, the antisense strand and sense strand of the nucleic acid molecule respectively comprise 15-21 consecutive nucleotides of the sense or antisense strand base sequence of any dsRNA of SER3, SER1, SER2, SER4 to SER-100 as shown in the sequence listing.

4. The nucleic acid molecule according to claim 3, characterized in that, Its nucleic acid molecules have one of the following characteristics in their sense and antisense strands: (a) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

1. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO: 2; (b) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

3. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO: 4; (c) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

5. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO: 6; (d) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

7. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO: 8; (e) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

9. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO: 10; (f) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

11. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO: 12; or (g) The sense chain sequence comprises 18-21 consecutive nucleotides of SEQ ID NO:

13. The antisense strand base sequence comprises 20-23 consecutive nucleotides of SEQ ID NO:

14.

5. The nucleic acid molecule according to claim 3 or 4, wherein the complementary region between the antisense strand and the sense strand is 15-21 bp in length.

6. The nucleic acid molecule according to any one of claims 3-5, wherein the sense strand is 18-23 nt in length and the antisense strand is 20-25 nt in length; optionally, wherein the sense strand is 18-21 nt in length and the antisense strand is 20-23 nt in length; optionally, the sense strand is 21 nt in length and the antisense strand is 23 nt in length.

7. The nucleic acid molecule according to any one of claims 3-6, wherein: (a) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 1, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 1 starting from the 3' end. The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 2, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 2 starting from the 5' end; (b) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 3, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 3 starting from the 3' end. The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 4, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 4 starting from the 5' end; (c) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 5, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 5 starting from the 3' end. The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 6, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 6 starting from the 5' end; (d) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 7, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 7 starting from the 3' end. The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 8, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 8 starting from the 5' end; (e) The sense sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 9, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 9 starting from the 3' end. The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 10, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 10 starting from the 5' end; (f) The sense chain sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 11, optionally comprising at least 19 consecutive nucleotides of SEQ ID NO: 11 starting from the 3' end. The antisense base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 12, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 12 starting from the 5' end; or (g) The sense chain sequence comprises at least 19 consecutive nucleotides of SEQ ID NO: 13, and optionally comprises at least 19 consecutive nucleotides of SEQ ID NO: 1 starting from the 3' end. The antisense strand base sequence comprises at least 21 consecutive nucleotides of SEQ ID NO: 14, optionally comprising at least 21 consecutive nucleotides of SEQ ID NO: 14 starting from the 5' end.

8. The nucleic acid molecule according to any one of claims 3-7, wherein: (a) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 1, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 2; (b) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 3, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 4; (c) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 5, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 6; (d) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 7, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 8; (e) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 9, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO: 10; (f) The sense strand sequence comprises the base sequence shown in SEQ ID NO: 11, and the antisense strand sequence comprises the nucleotide sequence shown in SEQ ID NO: 12; or (g) The sense strand base sequence comprises the base sequence shown in SEQ ID NO: 13, and the antisense strand base sequence comprises the nucleotide sequence shown in SEQ ID NO:

14.

9. The nucleic acid molecule according to any one of claims 1-8, wherein: The 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the other nucleotides in the antisense strand is substituted with methoxy groups; or The 2' position of the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, is substituted with fluorine, and the 2' position of the nucleotides at positions 5 and 7 is substituted with hydrogen. The 2' position of the other nucleotides in the antisense strand is substituted with methoxy groups.

10. The nucleic acid molecule according to any one of claims 1-9, wherein its sense strand is: The 2' position of the 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups. The 2' position of the 7th, 9th and 11th nucleotides starting from the 5' end is replaced by fluorine, the 2' position of the 10th nucleotide is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups. The 2' position of the nucleotides at positions 7, 9, 10, 11, and 12, starting from the 5' end, is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups. The 2' position of the nucleotides at positions 7, 9, 10, and 11 starting from the 5' end is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy groups. The 2' position of the nucleotides corresponding to the 11th and 13th positions from the 5' end of the antisense strand is replaced by fluorine, the 2' position of the nucleotide corresponding to the 12th position from the 5' end of the antisense strand is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups. The 2' position of the nucleotides corresponding to the 11th, 13th and 15th positions from the 5' end of the antisense strand is replaced by fluorine, the 2' position of the nucleotide corresponding to the 12th position from the 5' end of the antisense strand is replaced by hydrogen, and the 2' position of the other nucleotides is replaced by methoxy groups. The 2' position of the nucleotides at positions 10, 11, 12, 13, and 15, starting from the 5' end of the antisense strand, is substituted with fluorine, while the 2' position of all other nucleotides is substituted with methoxy groups; or The 2' position of the nucleotides at positions 11, 12, 13, and 15, starting from the 5' end of the antisense strand, is replaced by fluorine, while the 2' position of the other nucleotides is replaced by methoxy.

11. The nucleic acid molecule according to any one of claims 1-10, comprising or not comprising a phosphate ester or phosphate ester mimicry modification at the 5' end of the antisense strand; preferably, the 5' phosphate ester mimicry is a 5'-VP modification.

12. The nucleic acid molecule according to any one of claims 1-11, wherein it comprises phosphate thioester modification at the following positions: Between the first and second nucleotides starting at the 5' end of the sense strand, Between the second and third nucleotides starting from the 5' end of the sense strand, Between the first and second nucleotides starting at the 5' end of the antisense strand, Between the second and third nucleotides starting from the 5' end of the antisense strand, Between the first and second nucleotides starting from the 3' end of the antisense strand, and Between the second and third nucleotides starting from the 3' end of the antisense strand, Alternatively, any other two nucleotides may be linked by a phosphate ester bond.

13. The nucleic acid molecule according to any one of claims 1-12, comprising at least one desialylate glycoprotein receptor (ASGPR) ligand linked to the positive strand.

14. The nucleic acid molecule of claim 13, wherein the ASGPR ligand is attached to the 5' end and / or the 3' end of the sense strand; optionally, the ASGPR ligand is attached to the 3' end of the sense strand.

15. The nucleic acid molecule according to claim 13 or 14, wherein the ASGPR ligand is a divalent or trivalent GalNAc ligand; optionally, wherein the GalNAc ligand is L96 as shown in Formula I, or a ligand as shown in Formula II or Formula III: Among them, Equations I to III These represent the 3' end of the sense or antisense strand of the nucleic acid molecule, respectively; preferably, they are linked to the 3' position of the 3' end nucleotide via a phosphodiester bond or a thiophosphate diester bond.

16. The nucleic acid molecule according to any one of claims 1-15, comprising a modification motif selected from any one of the following: (1) Motif 1 Sense chain: NmsNmsNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmNm, Antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (2) Motif 2 Sense strand: NmsNmsNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmNm, antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (3) Motif 3 Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNmNm, Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (4) Motif 4 Sense chain: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm, Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; and Furthermore, among them, Nm represents 2'-OMe modified ribonucleotide, Nf represents 2'-F modified ribonucleotide, s represents phosphate thioester modification, Nd represents 2' deoxyribonucleotide, and optionally when the nucleotide corresponding to Nd is U or T, Nd represents thymine deoxyribonucleotide (Td).

17. The nucleic acid molecule according to any one of claims 1-16, comprising a modification motif selected from any one of the following: (1) Motif 1-GalNAc Sensitive chain: NmsNmsNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNm-GalNAc ligand, Antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (2) Motif 2-GalNAc Sensitive chain: NmsNmsNmNmNmNmNmNmNfNdNfNmNmNmNmNmNmNmNmNmNm-GalNAc ligand, Antisense strand: NmsNfsNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (3) Motif 3-GalNAc Sensitive chain: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-GalNAc ligand, Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (4) Motif 4-GalNAc Sensitive chain: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-GalNAc ligand, Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; and Furthermore, among them, Nm represents a 2'-OMe modified ribonucleotide, Nf represents a 2'-F modified ribonucleotide, s represents a phosphate thioester modified nucleotide, Nd represents a 2' deoxyribonucleotide, and optionally when the nucleotide corresponding to Nd is U or T, Nd represents thymine deoxyribonucleotide (Td), and -GalNAc indicates that it is linked to the corresponding GalNAc ligand via a phosphate ester bond or a phosphate thioester bond.

18. The nucleic acid molecule according to any one of claims 1 to 17, wherein it is dsRNA.

19. The nucleic acid molecule according to any one of claims 1-18, wherein the characteristics of the nucleic acid molecule are selected from any one of the following: (a) The sense base sequence is shown in SEQ ID NO: 1, and the antisense base sequence is shown in SEQ ID NO: 2; (b) The sense base sequence is shown in SEQ ID NO: 3, and the antisense base sequence is shown in SEQ ID NO: 4; (c) The sense base sequence is as shown in SEQ ID NO: 5, and the antisense base sequence is as shown in SEQ ID NO: 6; (d) The sense base sequence is shown in SEQ ID NO: 7, and the antisense base sequence is shown in SEQ ID NO: 8; (e) The sense base sequence is shown in SEQ ID NO: 9, and the antisense base sequence is shown in SEQ ID NO: 10; (f) The sense base sequence is as shown in SEQ ID NO: 11, and the antisense base sequence is as shown in SEQ ID NO: 12; or (g) The sense base sequence is shown in SEQ ID NO: 13, and the antisense base sequence is shown in SEQ ID NO: 14; Furthermore, the nucleotide molecule has the following modification motif: 4-p-L96. or The nucleotide molecule has the following modification motif: 4-p-ligand 2. or The nucleotide molecule has the following modification motif: 3-p-L96. or The nucleotide molecule has the following modification motif: 3-p-ligand 2. Sense strand: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNm-p-ligand 2, antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNfNmNmNmNmNmsNmsNm; L96 is shown in Formula I, and ligand 2 is shown in Formula III.

20. A nucleic acid molecule that inhibits SERPINC1 gene expression via RNAi, wherein the molecule is an siRNA molecule or an RNAi molecule with a hairpin structure formed by linkers connecting the sense and antisense strands of the siRNA molecule, the nucleic acid molecule comprising: A sense chain comprising a nucleotide sequence identical to at least 19 nt of the following sequence except for the presence of 0, 1, or 2 different ribose modifications: GmCmCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm(SEQ ID NO: 215); and The antisense strand contains a nucleotide sequence that is identical to at least 19 nt of the following sequence except for the presence of 0, 1 or 2 different ribose modifications: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmCmUm (SEQ ID NO: 216); or A sense chain comprising a nucleotide sequence identical to at least 19 nt of the following sequence except for the presence of 0, 1, or 2 different ribose modifications: GmCmCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm(SEQ ID NO: 217); and The antisense strand contains a nucleotide sequence that is identical to at least 19 nt of the following sequence except for the presence of 0, 1 or 2 different ribose modifications: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmCmUm (SEQ ID NO: 216).

21. The nucleic acid molecule according to claim 20, comprising (1) or (2): (1) The nucleoside sequence contains or is a sense strand of the following sequence: CmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm (SEQ ID NO: 219), and The nucleoside sequence contains or is an antisense strand of the following sequence: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCm (SEQ ID NO: 218); (2) The nucleoside sequence contains or is a sense strand of the following sequence: CmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm(SEQ ID NO: 220), and The nucleoside sequence contains or is the antisense strand of the following sequence: AmGfAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCm (SEQ ID NO: 218).

22. The nucleic acid molecule according to claim 20 or 21, wherein it contains a phosphothioester bond at at least one of the following positions: Between the first and second nucleotides starting at the 5' end of the sense strand, Between the second and third nucleotides starting from the 5' end of the sense strand, Between the first and second nucleotides starting at the 3' end of the sense strand, Between the second and third nucleotides starting from the 3' end of the sense strand, Between the first and second nucleotides starting at the 5' end of the antisense strand, Between the second and third nucleotides starting from the 5' end of the antisense strand, Between the first and second nucleotides starting from the 3' end of the antisense strand, and Between the second and third nucleotides starting from the 3' end of the antisense strand; The positions of the nucleotides are counted starting from the first nucleotide with a base at the end of each nucleotide; Or, when the first nucleotide at the 5' end and / or the 3' end is a baseless nucleotide, the counting of nucleotide positions does not exclude baseless nucleotides.

23. The nucleic acid molecule according to any one of claims 20-22, further comprising an ASGPR ligand, optionally said ASGPR ligand being attached to the 5' end and / or the 3' end of the sense strand; optionally, said GalNAc ligand is L96 as shown in Formula I, or a ligand as shown in Formula II or Formula III: Among them, Equations I to III These represent the 3' end of the sense strand or antisense strand of the nucleic acid molecule, respectively; preferably, they are connected to the 3' end via a phosphodiester bond or a thiophosphate diester bond.

24. The nucleic acid molecule according to any one of claims 20-23, comprising or not comprising 5' phosphate mimicry modification.

25. The nucleic acid molecule according to claim 20 or 23, comprising: (1) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCfUmCmUmAmUmGmUmCmUm-ASGPR ligand (SEQ ID NO: 221-ASGPR ligand); and Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222); or (2) Sensitive chain: GmsCmsCmGmAmGmAfUmGfAfCfCmUmCmUmAmUmGmUmCmUm-ASGPR ligand (SEQ ID NO: 223-ASGPR ligand); and Antonyms: AmsGfsAmCmAmUmAmGmAmGmGmUmCmAfUmCfUmCmGmGmCmsCmsUm(SEQ ID NO: 222); The -ASGPR ligand indicates that the ASGPR ligand is linked to the 3' position of the nucleotide via a phosphate ester bond or a thiophosphate ester bond.

26. A second nucleic acid molecule comprising a nucleic acid molecule according to any one of claims 1-25 and one or more additional nucleotides, optionally said additional nucleotides comprising one or more additional RNAi molecules.

27. A tautomer, stereoisomer, solvate, isotope derivative, or pharmaceutically acceptable salt of any nucleic acid molecule according to any one of claims 1-25 or the second nucleic acid molecule according to claim 26.

28. A pharmaceutical composition comprising a nucleic acid molecule or a salt thereof as described in any one of claims 1 to 25, or a second nucleic acid molecule as described in claim 26, or a tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt as described in claim 27, and a pharmaceutically acceptable carrier.

29. Use of any nucleic acid molecule or salt thereof according to any one of claims 1 to 25, a second nucleic acid molecule according to claim 26, a tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt according to claim 27, or a pharmaceutical composition according to claim 28, for inhibiting the expression of SERPINC1 in cells or in a subject.

30. Use of any nucleic acid molecule or salt thereof according to any one of claims 1 to 25, a second nucleic acid molecule according to claim 26, a tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt according to claim 27, or a pharmaceutical composition according to claim 28, in the preparation of a medicament for inhibiting SERPINC1 expression in a subject.

31. Use of any nucleic acid molecule or salt thereof according to any one of claims 1 to 25, a second nucleic acid molecule according to claim 26, a tautomer, stereoisomer, solvate, isotope derivative or pharmaceutically acceptable salt according to claim 27, or a pharmaceutical composition according to claim 28, in the preparation of a medicament for the prevention or treatment of SERPINC1-mediated diseases or symptoms; preferably, the disease or symptom is a bleeding disorder; more preferably, the disease or symptom is hemophilia; more preferably, the disease or symptom is hemophilia A or hemophilia B.

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