Sirna for inhibiting FXII gene expression, conjugate thereof, and use thereof

Abstract

The present invention relates to an siRNA and an siRNA conjugate which inhibit coagulation factor XII (FXII) gene expression, a pharmaceutical composition containing same, and the use thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand. The siRNA and the conjugate and pharmaceutical composition thereof can effectively treat and / or prevent diseases related to FXII gene overexpression.

Description

siRNA for inhibiting expression of FXII gene, conjugates thereof and uses thereof TECHNICAL FIELD

[0001] The present application relates to siRNAs and siRNA conjugates that inhibit expression of the coagulation factor XII (FXII) gene, pharmaceutical compositions comprising the same and uses thereof. BACKGROUND

[0002] Coagulation factor XII (also known as FXII, F12 or Hageman factor), a serine protease that is primarily expressed in the liver and found in the blood, has a dual function in the intrinsic coagulation pathway and in the kallikrein-kinin system, which plays a role in inflammation, blood pressure control, coagulation and pain. The active form of coagulation factor XII binds to and cleaves coagulation factor XI in the coagulation cascade and prekallikrein in the kallikrein-kinin system, producing the active forms FXI and kallikrein, respectively.

[0003] Coagulation factor FXII is a protease involved in pro-coagulation in the coagulation pathway in the body, which does not participate in physiological hemostasis, is involved in fibrinolysis and formation of pathological thrombi; thus, FXII plays an important role in the propagation phase of pathological clot formation, but is not required for normal hemostasis. This paradigm shift that separates thrombus formation and hemostasis has led to the concept of anticoagulation using drugs with minimal side effects of bleeding, i.e. the development of FXIIa and FXIa inhibitors, as the next generation of anticoagulants. It has now been demonstrated that prophylactic use of FXIIa inhibitors has both anticoagulant and anti-inflammatory efficacy with minimal side effects of bleeding. Silasi et al. found that by inhibiting FXII function, activation of complement and inflammatory cytokines was reduced, preserving organ function and survival rates in baboons challenged with heat-inactivated Staphylococcus aureus (HI-SA) (Blood. 2021 Jul 15; 138(2): 178-189.). These findings suggest potential benefits of prophylactic treatment with FXIIa inhibitors in patients at increased risk of pathological coagulation and inflammatory responses. Such indications include prevention of sepsis or mitigation of severe infections.

[0004] In addition, coagulation factor XII is also one of the key targets for treating hereditary angioedema (HAE). HAE is a rare disease characterized by recurrent episodes of severe swelling. The most common areas of the body to swell are the extremities, face, intestines, and airways. Episodes can be spontaneous or triggered by physical trauma or stress. Laryngeal (airway) edema can be life-threatening because it can cause suffocation death. Most of the drugs on the market are selected to be given when HAE episodes occur, and if the gene expression can be silenced at the genetic level to block the generation of coagulation factor XII, the occurrence of HAE can be effectively inhibited.

[0005] The present application aims to provide siRNA and siRNA conjugates, pharmaceutical compositions and uses, which can selectively and effectively inhibit the expression of FXII gene, so as to achieve the purpose of disease treatment. SUMMARY

[0006] The present application provides an siRNA capable of inhibiting the expression of FXII gene, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences:

[0007] (1) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2;

[0008] (2) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 28, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 29.

[0009] In one embodiment, the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, the length of the nucleotide sequences V and VI is 0 to 3 nucleotides, the nucleotide sequence V is linked to the 3' end of the sense strand to form a 3' overhang of the sense strand, and / or the nucleotide sequence VI is linked to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In a preferred embodiment, the length of the nucleotide sequence V or VI is 2 nucleotides. In a preferred embodiment, the nucleotide sequence V is identical to or has nucleotide difference with the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA. In a preferred embodiment, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

[0010] In one embodiment, at least one of the nucleotides in the sense strand or the antisense strand is a modified nucleotide, and / or at least one of the phosphate groups is a phosphate group with a modification; preferably, the phosphate group with a modification is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom.

[0011] In one embodiment, the siRNA comprises a sense strand that does not comprise 3' overhang nucleotides.

[0012] In one embodiment, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0013] In one embodiment, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0014] In some embodiments, neither the 5' end nor the 3' end of the sense strand is linked to an inverted abasic deoxyribose residue. In other embodiments, only the 5' end of the sense strand is linked to an inverted abasic deoxyribose residue. In other embodiments, only the 3' end of the sense strand is linked to an inverted abasic deoxyribose residue. In other embodiments, the 5' end and the 3' end of the sense strand are linked to an inverted abasic deoxyribose residue, respectively.

[0015] In some preferred embodiments, the inverted abasic deoxyribose residue is linked to the 3' terminal nucleotide and / or the 5' terminal nucleotide of the sense strand by a phosphodiester bond, a phosphorothioate group, or other internucleoside linkage.

[0016] In one embodiment, the modified nucleotides are selected from 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, nucleotide analogs, or a combination of any two or more thereof.

[0017] In one embodiment, the modified nucleotides are selected from 2'-F modified nucleotides, 2'-0-CH3modified nucleotides, 2'-0-CH2-CH2-0-CH3modified nucleotides, 2'-0-CH2-CH=CH2modified nucleotides, 2'-CH2-CH2-CH=CH2modified nucleotides, 2'-deoxy modified nucleotides, nucleotide analogs, or a combination of any two or more thereof.

[0018] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide or a non-fluoro modified nucleotide.

[0019] In a preferred embodiment, in the sense strand, in the 5' to 3' direction, 2'-fluoro modified nucleotides are at positions 5, 7, 8, and 9, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 9, 10, and 11, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 8, 9, and 10, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 8, 9, 10, and 11, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9, and 11, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9, 11, 13, and 15, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 9, 11, and 13, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 8, and 9, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 9, 11, and 13, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 9, and 11, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 11, and 13, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 7, 11, 12, and 13, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 11, and 16, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 11, and 17, with the remaining positions being non-fluoro modified nucleotides; or, 2'-fluoro modified nucleotides are at positions 3, 7, 11, 16, and 17, with the remaining positions being non-fluoro modified nucleotides; and / or

[0020] In the antisense strand, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 8, 9, 14, and 16, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2 and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 5, 7, and 14; or, 2'-fluoro-modified nucleotides are at positions 2, 7, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 7, 10, 12, 14, and 16, and the remaining positions are non-fluoro-modified nucleotides.

[0021] In one embodiment, each non-fluoro-modified nucleotide is independently selected from one of the group consisting of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced with a non-fluoro group, the nucleotide analog selected from one of the group consisting of pseudouracil, an iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA.

[0022] In one embodiment, each nucleotide in the sense strand and in the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or a combination of any two or more thereof.

[0023] In a preferred embodiment, in the sense strand, 2'-fluoro-modified nucleotides are at positions 5, 7, 8, and 9, and the remaining positions are 2'-methoxy-modified nucleotides, in the 5' to 3' direction; or, 2'-fluoro-modified nucleotides are at positions 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, and 10, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13, and 15, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8, and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16, and 17, and the remaining positions are 2'-methoxy-modified nucleotides; and / or

[0024] In the antisense strand, in the direction from 5' to 3', 2'-fluoro-modified nucleotides are at positions 2, 6, 8, 9, 14, and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2 and 14, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and position 6 is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 5, 7, and 14; or, 2'-fluoro-modified nucleotides are at positions 2, 7, and 14, positions 5 and 12 are 2'-deoxy-modified nucleotides, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 7, 10, 12, 14, and 16, position 5 is a 2'-deoxy-modified nucleotide, position 6 is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides.

[0025] In some embodiments, when neither the 5' end nor the 3' end of the sense strand is linked (invAb), the siRNA has a 5' end to 3' end direction of,

[0026] (1) the sense strand comprises a phosphorothioate group at a position shown as follows:

[0027] between the 1st and 2nd nucleotides from the 5' end of the sense strand; and

[0028] between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and

[0029] between the 1st and 2nd nucleotides from the 3' end of the sense strand; and

[0030] between the 2nd and 3rd nucleotides from the 3' end of the sense strand;

[0031] or,

[0032] (2) the sense strand comprises a phosphorothioate group at a position shown as follows:

[0033] between the 1st and 2nd nucleotides from the 5' end of the sense strand; and

[0034] between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and

[0035] when only the 3' end of the sense strand is linked to one (invAb), the sense strand comprises a phosphorothioate group at the positions shown below, in the 5' end to 3' end direction:

[0036] between the 1st and 2nd nucleotides from the 5' end of the sense strand; and

[0037] between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and

[0038] between the 3' end of the sense strand (invAb) and the 1st nucleotide from the 3' end.

[0039] In some embodiments, the siRNA comprises, in the 5' end to 3' end direction of the antisense strand, a phosphorothioate group at the positions shown below:

[0040] between the 1st and 2nd nucleotides from the 5' end of the antisense strand; and

[0041] between the 2nd and 3rd nucleotides from the 5' end of the antisense strand; and

[0042] between the 1st and 2nd nucleotides from the 3' end of the antisense strand; and

[0043] between the 2nd and 3rd nucleotides from the 3' end of the antisense strand.

[0044] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, a GNA modified nucleotide, a 2'-deoxy modified nucleotide, or a combination of any two or more thereof.

[0045] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are at positions 5, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, the 5' end nucleotide of the antisense strand being linked to a 5'-trans vinyl phosphonate group.

[0046] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0047] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 8, 9, and 10 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0048] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group.

[0049] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 8, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0050] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0051] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0052] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 11, 13, and 15 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0053] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 11, 13, and 15 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0054] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0055] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 9, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0056] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate group.

[0057] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0058] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 11, 12, and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0059] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 11, and 16 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0060] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 11, and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0061] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 11, 16, and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0062] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, with the 6 position being a GNA modified nucleotide, and the remaining positions being 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group.

[0063] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group.

[0064] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 5, 7, and 14, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group.

[0065] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group.

[0066] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0067] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0068] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0069] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0070] In the present application, in the description of the modification method of the sense strand or the antisense strand of siRNA, when the base is "T", the person skilled in the art understands that the base "T" means deoxyribonucleic acid, without modification of the base "T" by 2'-F, 2'-O-CH3, nucleotide derivatives, etc., according to the general understanding.

[0071] In one embodiment, the application provides an siRNA selected from Table 1 ; preferably, the siRNA is selected from N-ER-FY009151M53, N-ER-FY009204M53, N-ER-FY009204M9.

[0072] The application also provides an siRNA conjugate comprising an siRNA of the application and a conjugate group conjugated to the siRNA (as shown below, the double helix structure represents the siRNA and the conjugate group is attached to the 3' end of the sense strand of the siRNA):

[0073] In the conjugate structure described above, X can be selected from O or S, and in one embodiment, X is O.

[0074] In one embodiment, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand has or forms a blunt end, and the 3' end of the antisense strand has 1-3 overhanging nucleotides extending out of the double-stranded region;

[0075] or,

[0076] In the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand forms a blunt end.

[0077] In one embodiment, the conjugate group is selected from:

[0078] In one embodiment, the siRNA conjugate is selected from Table 2; preferably, the siRNA conjugate is selected from N-ER-FY009151M53L96, N-ER-FY009204M53L96, N-ER-FY009204M9L96.

[0079] The application also provides a pharmaceutical composition comprising an siRNA of the application, or an siRNA conjugate of the application, and a pharmaceutically acceptable carrier.

[0080] The application also provides a kit comprising an siRNA of the application, or an siRNA conjugate of the application, or a pharmaceutical composition of the application.

[0081] The application also provides an siRNA of the application, or an siRNA conjugate of the application, or a pharmaceutical composition of the application, for use in therapy.

[0082] The present application also provides use of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application for preparing a medicament for inhibiting the expression of FXII gene.

[0083] The present application also provides use of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application for preparing a medicament for preventing and / or treating a disease related to overexpression of FXII gene.

[0084] In one embodiment, the disease is multiple sclerosis, atherosclerosis, Alzheimer's disease, hereditary angioedema, sepsis, deep vein thrombosis, community-acquired pneumonia, thrombotic inflammation of COVID-19, microscopic polyangiitis, neurogenic inflammation, cerebral vascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis, colitis, pulmonary fibrosis, lung injury, liver fibrosis, and other yet unidentified related conditions, pathologies or syndromes.

[0085] The present application also provides a method for inhibiting the expression of FXII gene, comprising contacting a cell expressing FXII with a therapeutically effective amount of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application, or administering to a subject in need thereof.

[0086] The present application also provides a method for treating and / or preventing a disease related to overexpression of FXII gene, comprising administering to a subject in need thereof a therapeutically effective amount of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application. Beneficial effects

[0087] The siRNA, the pharmaceutical composition and the siRNA conjugate provided by the present application show excellent inhibitory activity on the expression of FXII gene in in vitro cell experiments, and have good potential for treating diseases related to overexpression of FXII gene. For example, the siRNA and the conjugate thereof disclosed by the present application can reduce the expression of FXII mRNA in the liver, have low toxic and side effects, good plasma stability, and good clinical application prospect. The siRNA conjugate of the present application has a high inhibitory rate on the expression of hFXII protein in humanized mice, and has good long-acting property.

[0088] The siRNA and the siRNA conjugate provided by the present application show good inhibitory effect on the FXII gene in human hepatoma cells Hep3B. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 is the effect of the siRNA conjugate on the expression amount of hFXII protein in Example 4.

[0090] Figure 2 is the result of the inhibition rate of FXII at various concentrations of N-ER- FY009204M53L96 in Example 5.

[0091] Figure 3 is the differential gene that meets |log2(FoldChange)|>1 & padj.<0.05 of N-ER- FY009204M53L96 at 40nM concentration in Example 5.

[0092] Figure 4 is the differential gene that meets |log2(FoldChange)|>1 & padj.<0.05 of N-ER- FY009204M53L96 at 200nM concentration in Example 5.

[0093] Figure 5 is the change of blood flow velocity of mice in Example 7. DETAILED DESCRIPTION

[0094] DEFINITIONS

[0095] Throughout the specification, unless specifically stated otherwise, in the art of technology, “G”, “C”, “A”, “T” and “U” generally represent the bases of guanine, cytosine, adenine, thymine, uracil, respectively, but it is also generally known in the art that “G”, “C”, “A”, “T” and “U” each generally also represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively, which is a common way of expression in representing deoxyribonucleic acid sequences and / or ribonucleic acid sequences, therefore in the context of the present disclosure, the meaning of “G”, “C”, “A”, “T”, “U” includes various possible cases as described above, “nucleotides”, “ribonucleic acids” and “ribonucleotides” are used interchangeably in the present application, “deoxyribonucleotides” and “2’-deoxyribonucleotides” are used interchangeably in the present application. Lowercase letters a, u, c, g: represent 2’-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2’-fluoro modified nucleotides; Lowercase letter s represents phosphorothioate linkage between the two nucleotides adjacent to the left and right of the letter s; EVP: represents that the nucleotide adjacent to the right of the EVP is a 5’-trans vinyl phosphonate nucleotide (i.e. 5’-trans vinyl phosphonate group); (underlined + bold + italic): represents GNA modified nucleotide; Base represents base, for example A, U, G, C or T; (invAb): represents inverted abasic deoxyribose residue; “dG”, “dC”, “dA”: represents 2’-deoxy modified ribonucleotide at the corresponding position in the motif sequence.

[0096] In the foregoing and hereinafter, the "2'-fluoro modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced with a fluorine. The "non-fluoro modified nucleotide" refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group. These nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced with a non-fluorine group are well known to those skilled in the art, and can be selected from one of a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxy modified nucleotide.

[0097] "Alkyl" includes straight-chain, branched-chain, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, n-pentyl, cyclohexyl, and the like. Exemplarily, "C1-6alkyl" means a group containing 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight chain, branched chain, or cyclic form.

[0098] "Alkoxy" refers herein to an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propyloxy, isopropyloxy, n-butyloxy, t-butyloxy, n-pentyloxy, and the like.

[0099] "Nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. Such as pseudouracil (Ψ), an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide.

[0100] Pseudouracil (Ψ) refers to: a natural structural analog of a uracil nucleoside, in which the ribose is connected to the C5 of the pyrimidine ring instead of the N1 of the uracil, and its structural formula is as follows:

[0101] BNA refers to a constrained or inaccessible nucleotide. A BNA can contain a five-, six-, or seven-membered ring bridged structure with a "fixed" C3'-endo sugar conformation. The bridge is typically incorporated at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, and the like, wherein LNA is represented by Formula (1), ENA is represented by Formula (2), and cET BNA is represented by Formula (3):

[0102] Base represents a base, such as A, U, G, C, or T.

[0103] Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is represented by Formula (4) and GNA is represented by Formula (5):

[0104] In the above Formula (4) and Formula (5), Base represents a base, such as A, U, G, C, or T, and R is selected from H, OH, or alkoxy (O-alkyl).

[0105] Iso-nucleotides refer to compounds in which the position of the base on the ribose ring is altered, such as compounds in which the base is moved from the 1'-position to the 2'-position or 3'-position of the ribose ring, such as represented by Formula (6) or (7):

[0106] In the above Formula (6)-(7) compounds, Base represents a base, such as A, U, G, C, or T; and R is selected from H, OH, F, or a non-fluoro group as described above.

[0107] In some embodiments, the nucleotide analog is selected from one of pseudouracil, iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, a GNA modified nucleotide, a 2'-deoxy modified nucleotide, or a combination of any two or more thereof. In some preferred embodiments, each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, and herein and hereafter, the 2'-methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl of the ribosyl group is replaced with a methoxy group.

[0108] The "2'-methoxy modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl of the ribosyl group is replaced with a methoxy group. The "phosphorothioate group" refers to a phosphorothioate group in which one of the oxygen atoms in the phosphodiester linkage of the phosphate group is replaced with a sulfur atom.

[0109] The "phosphorothioate group" refers to the following formula:

[0110] The "5'-phosphonucleotide" refers to the structure of the following formula:

[0111] In the context of the present specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or, in RNA, uracil (U)); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner.

[0112] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "essentially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0113] In the above and below, a nucleotide sequence has "nucleotide difference" with another nucleotide sequence, if the base type of the nucleotide at the same position in the former is different from that in the latter, e.g., if the nucleotide base in the latter is A, and the corresponding nucleotide base in the former at the same position is U, C, G or T, it is recognized that there is nucleotide difference between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference is also recognized to occur at a position, if an abasic nucleotide or its equivalent is substituted for the nucleotide at that position.

[0114] In the above and below, "overhang" means one or more unpaired nucleotides that protrude from a duplex structure of an siRNA when one 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the siRNA, i.e., there is no nucleotide overhang. A "blunt-ended" siRNA is one that is double-stranded throughout its length, i.e., there is no nucleotide overhang at either end of the molecule. "Double-stranded region" means the region of an siRNA where the sense strand and the antisense strand are complementary to form a double-stranded region of the siRNA.

[0115] In the foregoing and hereinafter, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of the pentose sugar, which is the predominant type of nucleotide found free in organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of the pentose sugar, which can include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-phosphorothioate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-phosphorothioate, 2'-deoxythymidine-3'-phosphorothioate. The definition can apply to modified or unmodified nucleotide phosphoramidite monomers. The terms "iRNA," "RNAi agent," "iRNA agent," "RNA interference agent" used in the context of the disclosure herein are used interchangeably herein to refer to the term as defined herein includes siRNA and mediates target RNA transcript cleavage via the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of a target gene in a cell, such as a cell of a subject, e.g., a mammalian subject.

[0116] As used herein, "2'-deoxy-modified nucleotide" or "2'-deoxy-modification" refers to the replacement of ribonucleotides A, U, C, G with the corresponding deoxyribonucleotides dA, dT, dC, dG in the process of modification. For example, in the 5' to 3' direction, the 5th nucleotide of the antisense strand of N-ER-FY009151 (SEQ ID NO: 29) is replaced with a deoxyribonucleotide, which means that the 5th ribonucleotide "G" is replaced with "dG"; similarly, the uracil ribonucleotide (U) is replaced with the corresponding deoxyribonucleotide (dT); the cytosine ribonucleotide (C) is replaced with the corresponding cytosine deoxyribonucleotide (dC); the adenine ribonucleotide (A) is replaced with the corresponding adenine deoxyribonucleotide (dA). Herein, "2'-deoxy-modified nucleotide" or "2'-deoxy-modification" is also referred to as "replacement of nucleotide with deoxyribonucleotide".

[0117] Throughout the present application specification, and in particular when describing the preparation of siRNAs, pharmaceutical compositions, or siRNA conjugates of the present application, the nucleoside monomers refer, unless otherwise specified, to modified or unmodified nucleoside phosphoramidite monomers used in the solid phase phosphoramidite synthesis according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid phase phosphoramidite synthesis is a method well known to those skilled in the art for use in RNA synthesis. All nucleoside monomers used in the present application are commercially available.

[0118] Throughout the present application specification, "conjugation" refers to the covalent linkage between two or more chemical moieties each having a specific function, unless otherwise specified; accordingly, "conjugate" refers to the compound formed by the covalent linkage between the chemical moieties. Further, "siRNA conjugate" refers to the compound formed by the covalent linkage of one or more chemical moieties having a specific function to an siRNA. The siRNA conjugate should be understood in context as a general term for a plurality of siRNA conjugates or an siRNA conjugate represented by a chemical formula. In the context of the present application specification, "conjugating molecule" should be understood as a specific compound that can be conjugated to an siRNA through a reaction to ultimately form an siRNA conjugate of the present application.

[0119] Various hydroxyl protecting groups can be used in the present application. In general, a protecting group renders a chemical functional group insensitive to particular reaction conditions, and can be added to and removed from that functional group in a molecule without substantially damaging the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, nonexclusive examples of hydroxyl protecting groups that can be used in the present application include monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl) xanthine-9-yl (Mox). In some embodiments, nonexclusive examples of hydroxyl protecting groups that can be used in the present application include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0120] The term "FXII gene overexpression related disease" is a disease or disorder associated with the synthesis of cholesterol, steroids and other lipids. The term "FXII gene overexpression related disease" includes a disease, disorder or condition that would benefit from a reduction in FXII expression (i.e. "FXII-related disease"). Such diseases are typically associated with coagulation and inflammation. Non-limiting examples of FXII gene abnormal expression related diseases include: multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation in COVID-19 (contributing to pro-coagulant and pro-inflammatory responses in COVID-19), microscopic polyangiitis (MPA), neurogenic inflammation, cerebral vascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis, among others (Miroslava Didiasova et al. Factor XII in coagulation, inflammation and beyond. Cell Signal. 2018 Nov;51:257-265. doi: 10.1016 / j.cellsig.2018.08.006. Epub 2018 Aug 15; Kerstin et al. The Coagulation Factors Fibrinogen, Thrombin, and Factor XII in Inflammatory Disorders - A Systematic Review. Front Immunol. 2018 Jul 26;9: 1731. doi: 10.3389 / fimmu.2018.01731. eCollection 2018 Gobel K et al. Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells. Nat Commun. 2016;7(l):l 1626.; Sandra Vorlova et al. Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice. Thromb Haemost. 2017 Jan 5;117(l):176-187.; Daria Zamolodchikov et al. Activation of the factor XII-driven contact system in Alzheimer's disease patient and mouse model plasma. Proc Natl Acad Sci U S A. 2015 Mar 31;112(13):4068-73.; Longhurst HJ et al. Hereditary angioedema: an update on causes, manifestations and treatment. Br J Hosp Med (Lond). 2019;80(7):391-398.; Jansen PM, et al. Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase. Blood. 1996; 87(6):2337-2344.; Pixley RA, et al. The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons. J Clin Invest. 1993; 91(1):61-68.; Yan Meng, et al. FXII regulates the formation of deep vein thrombosis via the PI3K / AKT signaling pathway in mice Int J Mol Med. 2021 May; 47(5): 87.; Kristin Ehrlich, et al. Sex-specific differences in plasma levels of FXII, HK, and FXIIa-C1-esterase inhibitor complexes in community-acquired pneumonia. Am J Physiol Lung Cell Mol Physiol. 2021 Oct 1; 321(4): L764-L774.; Hanna Englert, et al. Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation. EBioMedicine.2021 May; 67: 103382; Luo HJ, The role and mechanism of FXII / uPAR pathway in NETs formation in microscopic polyangiitis. University of Electronic Science and Technology of China, 2019; Stavrou EX, et al. Factor XII and uPAR upregulate neutrophil functions to influence wound healing. J Clin Invest, 2018, 128(3):944-959.; Gobel K, et al. Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells. Nature Communications, 2016, 7: 11626.; Katrin F, et al. Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases. Arteriosclerosis, Thrombosis, and Vascular Biology Volume 37, Issue 1, January 2017, Pages 13-20; C Maas and T Renné, Coagulation factor XII in thrombosis and inflammation. Blood, Volume 131, Issue 17, 26 April 2018, Pages 1903-1909).

[0121] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0122] The term “subject,” as used herein, refers to any animal, such as a mammal or a marsupial. Subjects of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, rabbits, or any species of poultry.

[0123] As used herein, "treatment" refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding the fact that the subject can still be afflicted with the disorder.

[0124] As used herein, "prevention" refers to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To achieve "prophylactic benefit," an siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the subject can not yet be diagnosed with the disease.

[0125] siRNA

[0126] The present application relates to an siRNA capable of inhibiting the expression of a FXII gene. The siRNA of the present application contains nucleotide groups as the basic structural units, which are known to those skilled in the art to contain a phosphate group, a ribose group, and a base. The siRNA that is generally active, i.e., functional, is about 12-40 nucleotides in length, and in some embodiments, about 15-30 nucleotides in length.

[0127] The siRNA of the present application contains a sense strand and an antisense strand, each of the nucleotides in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I and the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. In some embodiments, the length of the double-stranded region is 15-30 nucleotide pairs. In other embodiments, the length of the double-stranded region is 17-23 nucleotide pairs. In other embodiments, the length of the double-stranded region is 19-21 nucleotide pairs. In yet other embodiments, the length of the double-stranded region is 19 or 21 nucleotide pairs.

[0128] In some embodiments, the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, the length of the nucleotide sequence V and VI is 0 to 3 nucleotides, the nucleotide sequence V is linked to the 3' end of the sense strand to form a 3' overhang of the sense strand, and / or the nucleotide sequence VI is linked to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In some embodiments, the length of the nucleotide sequence V or VI is 2 nucleotides. In a preferred embodiment, the nucleotide sequence V is identical to or has nucleotide difference with the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA. In other embodiments, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

[0129] It has been found that different modification strategies have a distinct impact on the stability, biological activity and cytotoxicity of siRNA. For example, CN201010106762.1 studies various chemical modification strategies for siRNA, and confirms that seven modification methods are effective. Compared with unmodified siRNA, the siRNA obtained by one of the modification methods not only improves the stability of blood, but also maintains the inhibitory activity of unmodified siRNA.

[0130] The nucleotides in the siRNA of the present application are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the present application is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present application are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA of the present application to inhibit the expression of the FXII gene.

[0131] In some embodiments, the siRNA of the present application contains at least one modified nucleotide. In the context of the present application, the term "modified nucleotide" refers to a nucleotide or a nucleotide analog in which the ribose group 2' position hydroxyl group is replaced by other groups, or a nucleotide with a modified base. The modified nucleotide does not significantly weaken or lose the function of the siRNA to inhibit gene expression. For example, the modified nucleotide disclosed in J. K. Watts, G. F. Deleavey, and M. J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected.

[0132] In some embodiments, at least one of the nucleotides in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and / or at least one of the phosphate groups is a phosphate group with a modification. In other words, at least a portion of the phosphate groups and / or the ribose groups in the sugar-phosphate backbone of at least one of the single strands of the sense strand and the antisense strand is a phosphate group with a modification and / or a ribose group with a modification. In some embodiments, the phosphate group with a modification is a phosphorothioate group, in which one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom.

[0133] In some embodiments, the siRNA comprises a sense strand that does not comprise 3' overhanging nucleotides; that is, the sense strand of the siRNA can have 3' overhanging nucleotides, and the 3' overhanging nucleotides of the sense strand are excluded to form a blunt end.

[0134] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to each other to form a double-stranded region, the 3' end of the sense strand does not have overhanging nucleotides, and a nucleotide sequence V is added to the 3' end of the sense strand as overhanging nucleotides. Then, when the nucleotide sequence V added to the 3' end of the sense strand is chemically modified, the nucleotide sequence V is excluded, and the sense strand of the siRNA forms a blunt end.

[0135] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to each other to form a double-stranded region, the 3' end of the sense strand has overhanging nucleotides extending out of the double-stranded region, and the overhanging nucleotides at the 3' end of the sense strand are excluded to form the nucleotide sequence of the sense strand, and the sense strand of the siRNA forms a blunt end.

[0136] In some embodiments, the 5' end nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group. In some embodiments, the 5' end nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0137] When the 5' end nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, the following structure is formed:

[0138] wherein Formula (8) shows the structure of a 5' terminal nucleotide linked to a 5' phosphate group, Formula (9) shows the structure of a 5' terminal nucleotide linked to a 5' phosphate derivative group (EVP), and Formula (10) shows the structure of a 5' terminal nucleotide linked to a 5' phosphate derivative group (5' methylene phosphate group), wherein Base represents a base, such as A, U, G, C, or T. R' is a hydroxyl group or various groups known to one skilled in the art, for example, the modified nucleotide after substitution can be a 2'-fluoro (2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxy modified nucleotide.

[0139] In some embodiments, the 5' terminal nucleotide of the sense strand or the antisense strand is not linked to a 5' phosphate group, a 5' phosphate derivative group, or (invAb) (i.e., the ribose group of the 5' terminal nucleotide of the sense strand or the antisense strand is a 5' hydroxyl group), which has the following structure:

[0140] wherein Base represents a base, such as A, U, G, C, or T. R is a hydroxyl group or hydrogen or various groups known to one skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0141] Exemplary modified nucleotides have the following structures:

[0142] wherein Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribose group is substituted with R. The hydroxyl group at the 2' position of the ribose group can be substituted with various groups known to one skilled in the art, for example, the modified nucleotide after substitution can be a 2'-fluoro (2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxy modified nucleotide.

[0143] In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0144] In some embodiments, the 2'-substituted alkoxy-modified nucleotide is a 2'- methoxyethoxy (2'-0-CH2-CH2-0-CH3) modified nucleotide, a 2'-0-CH2-CH=CH2 modified nucleotide, and the like.

[0145] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2- CH2-CH=CH2 modified nucleotide, and the like.

[0146] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each of the nucleotides in the sense strand and the antisense strand of the siRNA provided herein is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide. In some embodiments, each of the non-fluoro-modified nucleotides is a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or a combination of any two or more thereof; the 2'-methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl of the ribosyl group is replaced with a methoxy group.

[0147] In some embodiments, neither the 5' nor the 3' end of the sense strand is attached (invAb), or only the 5' end of the sense strand is attached with one (invAb), or only the 3' end of the sense strand is attached with one (invAb), or the 5' and 3' ends of the sense strand are each attached with one (invAb); (invAb) is not counted as a site in the siRNA.

[0148] In some embodiments, the sense strand can comprise one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more of the terminal ends of the nucleotide sequence of the siRNA disclosed herein. In some cases, a capping residue can provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverted deoxyabasic residue (invAb) is added as a capping residue. In some embodiments, the capping residue occurs at the 3' end of the sense strand or at both the 5' and 3' ends of the sense strand.

[0149] In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5' end of the sense strand and to the 3' end of the sense strand. The inverted abasic deoxyribose residues can be linked via a phosphoester linkage, a phosphorothioate linkage, or other internucleoside linkage. In describing the modified site in a modification method, the (invAb) is not counted as the 1st site of the sequence. The chemical structure of the inverted abasic deoxyribose residue is shown below:

[0150] Formula B when (invAb) is at the 3' end of the siRNA; and Formula C when (invAb) is at the 5' end of the siRNA:

[0151] siRNA conjugate

[0152] The present application relates to a siRNA conjugate, which contains the above-mentioned siRNA and a conjugate group conjugated to the siRNA.

[0153] In the present application, the sense strand of the siRNA conjugate forms a double-stranded region of the siRNA conjugate with the antisense strand, and the 3' end of the sense strand of the siRNA conjugate forms a blunt end. In some embodiments, the 3' end of the sense strand of the siRNA conjugate has or forms a blunt end, and the 3' end of the antisense strand of the siRNA conjugate has 1-3 overhanging nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of the sense strand of the siRNA conjugate forms a blunt end, and the 3' end of the antisense strand of the siRNA conjugate forms a blunt end. In the present application, "the 3' end of the sense strand forms a blunt end" includes the case where the 3' end of the sense strand of the siRNA originally has a blunt end, and the case where the 3' end of the sense strand of the siRNA has an overhanging end, and the overhanging end is excluded to form a blunt end.

[0154] In some preferred embodiments, the siRNA conjugate is obtained by conjugating the siRNA with a conjugate group. In the siRNA, the sense strand is complementary to the antisense strand to form a double-stranded region of the siRNA, and the 3' end of the sense strand has a blunt end. The conjugate group is conjugated to the 3' end of the sense strand having a blunt end to form the siRNA conjugate.

[0155] Exemplarily, the siRNA has a sequence as shown in N-ER-FY009028M11, the 3' end of the sense strand of the siRNA has a blunt end, the sequence with 3' blunt end formed by excluding the overhanging nucleotide at the 3' end of the sense strand of the siRNA is used as the nucleotide sequence for connecting the L96 conjugating group, and thus, the sense strand of the siRNA conjugate N-ER-FY009028M11 L96 is gsasgaUfaUfGfGfuauagaacuaL96 (SEQ ID NO: 58), and the antisense strand is EVPusAfsguuCfuAfUfaccaUfaUfcucsTsT (SEQ ID NO: 4).

[0156] In some preferred embodiments, the 3' end of the sense strand of the siRNA has an overhanging nucleotide extending out of the double-stranded region, the sequence with 3' blunt end formed by excluding the overhanging nucleotide at the 3' end of the sense strand of the siRNA is used as the nucleotide sequence for connecting the conjugating group, and the conjugating group is connected at the 3' blunt end of the sense strand to form the siRNA conjugate. In some more preferred embodiments, when the 3' end of the sense strand has no overhanging nucleotide after the nucleotide sequence of the sense strand is complementary to the nucleotide sequence of the antisense strand to form the double-stranded region, a nucleotide sequence V is added at the 3' end of the sense strand as the overhanging nucleotide. The sequence with 3' blunt end formed by excluding the overhanging nucleotide at the 3' end of the sense strand of the siRNA is used as the nucleotide sequence for connecting the conjugating group, and the conjugating group is connected at the 3' blunt end of the sense strand to form the siRNA conjugate.

[0157] In some more preferred embodiments, when the 3' end of the sense strand has an overhanging nucleotide extending out of the double-stranded region after the nucleotide sequence of the sense strand is complementary to the nucleotide sequence of the antisense strand to form the double-stranded region, the sequence with 3' blunt end formed by excluding the overhanging nucleotide at the 3' end of the sense strand of the siRNA is used as the nucleotide sequence for connecting the conjugating group, and the conjugating group is connected at the 3' blunt end of the sense strand to form the siRNA conjugate.

[0158] Generally, the conjugating group comprises at least one targeting group which is pharmaceutically acceptable, or further comprises a linker, and the siRNA, the linker and the targeting group are sequentially connected. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugating group, for example, can be covalently conjugated to the conjugating group. The conjugation site of the siRNA and the conjugating group can be at the 3' end or the 5' end of the sense strand of the siRNA, can also be at the 5' end of the antisense strand, and can also be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugating group is at the 3' end of the sense strand of the siRNA.

[0159] In some embodiments, the conjugate group can be attached to the phosphate group, 2'-position hydroxyl group, or base of the nucleotide. In some embodiments, the conjugate group can also be attached to the 3'-position hydroxyl group, in which case the nucleotides are connected by 2'-5' phosphodiester bonds. When the conjugate group is attached to the end of the siRNA strand, the conjugate group is usually attached to the phosphate group of the nucleotide; when the conjugate group is attached to the internal sequence of the siRNA, the conjugate group is usually attached to the ribose sugar ring or base. Various attachment methods can be referred to in the literature: Muthiah Manoharan et. al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0160] In some embodiments, the siRNA and the conjugate group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the intracellular endosome, thereby allowing the siRNA to become free. For non-degradable conjugation, the conjugate group can be attached to the sense strand of the siRNA, thereby minimizing the impact of conjugation on the activity of the siRNA.

[0161] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

[0162] In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the following ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, poly-lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells, such as asialoglycoproteins, asialo sugar residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0163] In some embodiments, each ligand is independently selected from a ligand that binds to a cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a liver cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a human liver cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a liver surface asialoglycoprotein receptor (ASGPR). The classes of such ligands are known to those skilled in the art and generally function to bind to a specific receptor on the surface of a target cell, mediating delivery of the siRNA linked to the ligand to the target cell.

[0164] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

[0165] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands can be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and derivatives thereof, mannans and derivatives thereof, galactose and derivatives thereof, xylose and derivatives thereof, ribose and derivatives thereof, fucose and derivatives thereof, lactose and derivatives thereof, maltose and derivatives thereof, arabinose and derivatives thereof, fructose and derivatives thereof, and sialic acid.

[0166] In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, a-D-mannofuranose, β-D-mannofuranose, a-D-mannopyranose, β-D-mannopyranose, a-D-glucopyranose, β-D-glucopyranose, a-D-glucoruranose, β-D-glucoruranose, a-D-fructofuranose, a-D-fructopyranose, a-D-galactopyranose, β-D-galactopyranose, a-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-0-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L- glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-0-methyl-D-mannopyranose, 2-deoxy-2- sulfonamido-D-glucopyranose, N-glycolyl-a-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-0-acetyl-1-thio-6-0-trityl-a-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-0-acetyl-2-deoxy-1,5-dithio-a-D-glucoheptoside ethyl ester, 2,5-anhydro-D-allosonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other choices of the ligands can be found, for example, in CN105378082A, which is incorporated by reference in its entirety.

[0167] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that monovalent, divalent, trivalent, and tetravalent as used herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate formed by conjugation of the siRNA molecule to the conjugation group containing galactose or N-acetylgalactosamine as the targeting group is 1 : 1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described herein is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0168] The targeting group can be linked to the siRNA molecule via a suitable linker, which can be selected by a person skilled in the art according to the specific type of the targeting group. The linkers, the types of the targeting groups and the connection modes to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0169] Synthesis method of siRNA

[0170] The nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction by the conventional solid-phase phosphoramidite method in the art. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and capping. When the phosphate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, oxidation, and capping. When the phosphorothioate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, sulfurization, and capping. The nucleotide monomers are selected according to the target sequence in the present application, and the selected nucleotide monomers are the nucleotide monomers commonly used by a person skilled in the art, for example, the nucleotide monomers for synthesizing A can be but are not limited to selecting adenosine-3-phosphate. It should be understood that these monomers are connected to each other by 5'-3' phosphodiester bonds or 5'-3' phosphorothioate groups when they exist in an oligonucleotide, and when the last nucleotide at the 3' position is hydroxyl, for example, in the 5' to 3' direction, it is realized according to the conventional means in the art.

[0171] For example, the synthesis conditions of the siRNA of the present application can be as follows:

[0172] The deprotection conditions include: the reaction temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is selected from dichloromethane solution (3% V / V) of dichloroacetic acid, and the molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

[0173] The coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from 0.25M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), the molar ratio of the nucleic acid sequence connected on the solid support to the nucleotide monomer is 1:10, and the molar ratio of the nucleic acid sequence connected on the solid support to the coupling reagent is 1:65.

[0174] The oxidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the oxidation reagent is selected from 0.05M iodine tetrahydrofuran solution, and the molar ratio of the oxidation reagent to the nucleic acid sequence connected on the solid support in the coupling step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1.

[0175] The sulfuration reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfuration reagent is selected from the group consisting of hydroxylamine, and the molar ratio of the sulfuration reagent to the nucleic acid sequence connected on the solid carrier in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0176] The capping reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the capping reagent is selected from a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methyl imidazole pyridine / acetonitrile solution) with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected on the solid carrier is the molar ratio of acetic anhydride:N-methyl imidazole:the nucleic acid sequence connected on the solid carrier is 1:1:1.

[0177] After all the nucleotide monomers are connected, the nucleic acid sequence connected on the solid carrier is subjected to amination, purification, desalination in sequence to obtain siRNA sense and antisense strands, and finally the two strands are heated to anneal to obtain the product.

[0178] The methods of amination, purification, desalination and annealing are well known in the art. For example, amination is carried out by contacting the nucleotide sequence connected with the solid carrier with concentrated ammonia water; purification is carried out by chromatography; desalination is carried out by reverse phase chromatography; and annealing is carried out by gradually cooling the mixture of the sense and antisense strands in equimolar ratio under different stringent conditions.

[0179] The synthesized siRNA is shown in Table 1.

[0180] siRNA conjugate synthesis method

[0181] Taking the synthesis of L96 as an example:

[0182] In the first step, compound L96-A is obtained by reacting DMTr-L96 and succinic anhydride:

[0183] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine are added to dichloromethane, stirred at 25°C for 24 hours, then the reaction solution is washed with 0.5M triethylamine phosphate, the aqueous phase is washed with dichloromethane three times, the organic phases are combined and evaporated under reduced pressure to obtain the crude product. Then column chromatography is used to purify the pure product L96-A.

[0184] In the second step, L96-A is reacted with NH2-SPS to obtain L96-B:

[0185] Preparation process: L96-A, O-benzotriazol-tetramethyl urea hexafluorophosphate (HBTU) and diisopropyl ethylamine (DIPEA) were mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to obtain a uniform solution, and then added to the reaction solution. Ammonia methyl resin (NH2-SPS, 100-200 mesh) was added to the reaction solution, and the shaking table reaction was started at 25°C. After 18 hours of reaction, the filter cake was washed with dichloromethane and acetonitrile in sequence to obtain the filter cake. The obtained filter cake was subjected to cap reaction with CapA / CapB mixed solution to obtain L96-B, which is a solid phase carrier containing a conjugated molecule.

[0186] Third step, preparation of siRNA conjugate

[0187] L96-B was used as a solid phase carrier to synthesize siRNA according to the siRNA synthesis method described above, and siRNA antisense chains were synthesized according to the siRNA synthesis method described above, and annealing generated the siRNA conjugate of the present application.

[0188] The synthesized siRNA conjugate is shown in Table 2.

[0189] Pharmaceutical composition

[0190] The present application provides a pharmaceutical composition containing siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

[0191] The pharmaceutically acceptable carrier can be one or more of the carriers conventionally used in the field of siRNA administration, such as, but not limited to, a lipid nanoparticle (LNP), a magnetic nanoparticle (e.g., a nanoparticle based on Fe3O4 or Fe2O3), a carbon nanotube, mesoporous silicon, a calcium phosphate nanoparticle, polyethylenimine (PEI), a polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and derivatives thereof.

[0192] The content of the siRNA and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited and can be a conventional content of each component.

[0193] In some embodiments, the pharmaceutical composition can further include a pharmaceutically acceptable additional excipient, which can be one or more of various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable additional excipient can include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

[0194] The pH buffer can be a tris-hydroxymethyl aminomethane hydrochloride buffer having a pH of 7.5 to 8.5 and / or a phosphate buffer having a pH of 5.5 to 8.5, for example, a phosphate buffer having a pH of 5.5 to 8.5.

[0195] The protective agent can be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent can be 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

[0196] The osmotic pressure adjusting agent can be sodium chloride and / or potassium chloride. The content of the osmotic pressure adjusting agent is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm / kg). The content of the osmotic pressure adjusting agent can be readily determined by one skilled in the art depending on the desired osmotic pressure.

[0197] In some embodiments, the pharmaceutical composition can be a liquid formulation, such as an injection solution, or a lyophilized powder formulation that is mixed with a liquid excipient to prepare a liquid formulation for administration. The liquid formulation can be, but is not limited to, for subcutaneous, intramuscular, or intravenous injection, or can be, but is not limited to, for administration to the lung by nebulization, or for administration to other organ tissues (e.g., liver) by nebulization through the lung. In some embodiments, the pharmaceutical composition is for intravenous injection.

[0198] In some embodiments, the pharmaceutical composition can be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter can also be referred to as an organic amine), a helper lipid, and / or a pegylated lipid.

[0199] The following examples are intended to further illustrate the present application, but are not to be construed as limiting the same in any way.

[0200] Example

[0201] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description, but it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0202] The experimental techniques and experimental methods used in the present examples are all conventional techniques and methods, unless otherwise specified, for example, the experimental methods not specifically indicated in the following examples are generally performed according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through regular commercial channels.

[0203] Example 1 Preparation of siRNA

[0204] The siRNA molecules with the following sequences were synthesized by Tianlin Biotech (Shanghai) Co., Ltd.

[0205] Table 1 siRNAs and their sequences

[0206] wherein, capital letters "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively; lower case letters a, u, c, g: represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro modified nucleotides; lower case letter s represents a phosphorothioate linkage between the two nucleotides adjacent to the letter s on the left and right; EVP: represents that the nucleotide adjacent to the EVP on the right is a 5'-trans-vinyl phosphonate nucleotide; (underlined + bold + italic): represents a GNA modified nucleotide; (invAb): represents an inverted abasic deoxyribose residue; "dG", "dC", "dA": represents a ribonucleotide at the corresponding position in the motif sequence is 2'-deoxy modified.

[0207] The siRNA conjugates with the following sequences were synthesized by Tianlin Biotech (Shanghai) Co., Ltd.:

[0208] Table 2 siRNA conjugates and their sequences

[0209] wherein, L96 is connected to the 3' end of the sense strand in Table 1 or the 3' end of the sense strand forms a blunt end by a phosphodiester bond, and L96 is:

[0210] In Table 1 and Table 2, if the 5' terminal nucleotide of the sense strand, the modified sense strand and the modified sense strand connected with the conjugate group is not marked with EVP or (invAb) on the left side, it represents that the 5' terminal nucleotide is not connected with a 5' phosphate group, a 5' phosphate derivative group or (invAb) (i.e. the ribose group of the 5' terminal nucleotide is 5' hydroxyl), and its structure is shown as formula X:

[0211] wherein, Base represents a base, for example, A, U, G, C or T; R is hydroxyl or hydrogen or substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0212] In Tables 1 and 2, the 5' terminal nucleotide of the antisense strand and the modified antisense strand is represented by a 5' hydroxyl group if not labeled with EVP, and the structure is also shown in Formula X.

[0213] In Tables 1 and 2, the 3' position of the 3' terminal nucleotide of the sense strand and the modified sense strand is a hydroxyl group when the 3' terminal is not linked (invAb); in Tables 1 and 2, the 3' position of the 3' terminal nucleotide of the antisense strand and the modified antisense strand is a hydroxyl group.

[0214] Example 2 siRNA and siRNA conjugate inhibit FXII gene expression

[0215] 2.1 Experimental materials:

[0216] Hep3B cells were purchased from ATCC, item number HB-8064;

[0217] Transfection reagent was purchased from Invitrogen, item number 13778-150;

[0218] Opti-medium: serum-reduced medium, purchased from Gibco, item number 31985-070;

[0219] EMEM medium: ATCC, item number 30-2003;

[0220] RNA extraction kit, 96 Kit, item number QIAGEN-74106;

[0221] Fastking RT Kit (with gDNase), purchased from TianGen, item number KR116-02;

[0222] Phosphate Buffer Saline (PBS), purchased from Gibco, item number 10010-023;

[0223] GAPDH TaqMan probe primer was purchased from Thermo Hs99999905_m1;

[0224] F12 TaqMan probe primer was purchased from Thermo HS01557542_g1;

[0225] FastStart Universal Probe Master, purchased from Roche, item number 04914058001.

[0226] 2.2 Experimental methods:

[0227] 2.2.1 Hep3B cells were cultured in fresh EMEM medium in 96-well plates for 24 hours. The cultured cells were resuspended in EMEM medium without PS (penicillin-streptomycin mixture) to make a cell suspension with a density of 5.55 x 10 4 / mL, and were plated in 96-well plates at 90 μL of the cell suspension per well, i.e. 5000 cells per well.

[0228] 2.2.2 The dry powder of the siRNA and siRNA conjugate (collectively referred to as siRNA for the convenience of description in the description of the experiment in this example) to be tested was centrifuged at low temperature and high speed, and was then dissolved in ultra-pure distilled water (ULtra Pure Distilled Water) to prepare a 100 μM stock solution.

[0229] 2.2.3 Preparation of 20 nM siRNA dilution Z and 2 nM siRNA dilution W

[0230] (1) Preparation of 1 μM siRNA stock solution Q and 0.1 μM siRNA stock solution X:

[0231] a) 2 μL of the 100 μM siRNA stock solution prepared in step 2.2.2 above was added to 18 μL of ultra-pure distilled water to obtain a 10 μM siRNA dilution;

[0232] b) 2 μL of the 10 μM siRNA dilution prepared in step a) was added to 18 μL of ultra-pure distilled water to obtain a 1 μM siRNA stock solution Q;

[0233] c) 2 μL of the 1 μM siRNA stock solution Q prepared in step b) was added to 18 μL of ultra-pure distilled water to obtain a 0.1 μM siRNA stock solution X;

[0234] (2) 2 μL of each of the prepared siRNA stock solution Q and siRNA stock solution X was added to 98 μL of Opti-medium, respectively, to obtain 20 nM siRNA dilution Z and 2 nM siRNA dilution W, respectively.

[0235] 2.2.4 Transfection of Hep3B cells

[0236] (1) 3 μL of transfection reagent was added to 97 μL of Opti-medium to obtain a transfection reagent dilution; and (2) 2 μL of the prepared siRNA dilution Z and 2 μL of the prepared siRNA dilution W were added to 96 μL of the transfection reagent dilution, respectively, to obtain a transfection mixture; and ​The transfection reagent dilution was mixed with the 20 nM siRNA dilution Z prepared in step 2.2.3 at a volume ratio of 1 : 1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10 μL of the transfection mixture was added to the Hep3B cells cultured in step 2.2.1 in a 96-well plate (final volume 100 μL, and the concentration of siRNA in the system was 1 nM) to perform transfection.

[0237] (2) Take The transfection reagent dilution was mixed with the 20 nM siRNA dilution Z prepared in step 2.2.3 at a volume ratio of 1 : 1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10 μL of the transfection mixture was added to the Hep3B cells cultured in step 2.2.1 in a 96-well plate (final volume 100 μL, and the concentration of siRNA in the system was 1 nM) to perform transfection. The transfection reagent dilution was mixed with the 20 nM siRNA dilution Z prepared in step 2.2.3 at a volume ratio of 1 : 1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10 μL of the transfection mixture was added to the Hep3B cells cultured in step 2.2.1 in a 96-well plate (final volume 100 μL, and the concentration of siRNA in the system was 1 nM) to perform transfection. The transfection reagent dilution was mixed with the 20 nM siRNA dilution Z prepared in step 2.2.3 at a volume ratio of 1 : 1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10 μL of the transfection mixture was added to the Hep3B cells cultured in step 2.2.1 in a 96-well plate (final volume 100 μL, and the concentration of siRNA in the system was 1 nM) to perform transfection.

[0238] The above transfection was cultured for 48 hours; 2 repeats were set for each concentration (1 nM and 0.1 nM).

[0239] 2.2.5 Utilize 96 Kit instructions to extract total RNA from the Hep3B cells obtained in 2.2.4.

[0240] 2.2.6 Reverse transcription of the extracted total RNA to cDNA was performed using the Fastking RT Kit (with gDNase) kit according to the following steps:

[0241] a) Remove gDNA according to the following table using gDNA enzyme;

[0242] Table 3

[0243] 42°C, 2 min

[0244] b) Add the following reagents to the system obtained in step a) and perform reverse transcription

[0245] Table 4

[0246] 50°C, 15 min; 85°C, 5 s; 4°C, stand.

[0247] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0248] 2.2.7 Real-time PCR analysis was performed

[0249] a) Prepare the qPCR reaction mixture as shown in the table below, and keep all reagents on ice throughout the procedure;

[0250] Table 5

[0251] Table 5-1

[0252] b) Perform the qPCR procedure as described below

[0253] 50°C, 2 min, 95°C, 10 min;

[0254] 95°C, 15 sec; 60°C, 1 min (40 cycles).

[0255] 2.2.8 Results analysis

[0256] a) Use the Quant Studio 7 software to automatically calculate the Ct values using the default settings;

[0257] b) Calculate the relative expression of the gene using the following formula:

[0258] ΔCt = Ct(FXII gene) - Ct(GAPDH)

[0259] ΔΔCt = ΔCt (test sample group) - ΔCt (Mock group)

[0260] mRNA expression relative to Mock group = 2 -ΔΔCt .

[0261] Inhibition rate (%) = (Mock group mRNA relative expression - test sample group mRNA relative expression) / Mock group mRNA relative expression x 100%

[0262] Mock group means: the group without siRNA or siRNA conjugate added compared to the test sample group.

[0263] 2.3 Experimental results

[0264] Select the concentrations of 0.1 nM and 1 nM for testing.

[0265] Table 6 Inhibition rate of siRNA of the present application

[0266] Note: “--” means that the data is not shown.

[0267] Table 6-1

[0268] As can be seen from Table 6 and Table 6-1, the siRNA of the present application can significantly inhibit the expression of FXII gene at 1 nM and 0.1 nM.

[0269] Example 3 Rat liver homogenate in vitro stability experiment

[0270] 3.1 Experimental reagents and consumables

[0271] Table 7

[0272] 3.2 Experimental steps

[0273] 3.2.1 Preparation of liver homogenate

[0274] 3.2.1.1 Preparation of grinding fluid

[0275] Table 8

[0276] 3.2.1.2 Tissue homogenate

[0277] The rat liver tissue (from SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was prepared into liver homogenate (concentration of 20 mg / mL) with grinding fluid at 100 mg:5 mL. After preparation, the grinding beads were added and placed in the homogenizer. The grinding parameters were set as follows.

[0278] Table 9

[0279] 3.2.2 Sample preparation

[0280] The siRNA conjugate sample was prepared into a solution of 1 mg / mL with enzyme-free water and was ready for use. The internal standard sample was prepared into a solution of 0.125 mg / mL with enzyme-free water.

[0281] 3.2.3 Sample incubation

[0282] (1) Add 250 μL of prepared liver homogenate in a 2 mL enzyme-free tube,

[0283] (2) Add 50 μL of siRNA conjugate sample solution based on step (1);

[0284] (3) The system is 300 μL of biological sample solution, vortex, and stand for 5 min;

[0285] (4) Divide into 2 tubes, 100 μL each;

[0286] (5) Incubate the system at 37°C for 48 h.

[0287] 3.2.4 Biological sample processing

[0288] Each 100 μL of biological sample system vortex, mix, add 300 μL clarity OTX lysis liquid (Clarity OTX Lysis-loading Buffer, purchased from Agilent- Finnigan, product number AL0-8579) vortex, stand for 30 min, add 100 μL of internal standard solution, vortex, stand for 5 min, centrifuge for 1 min, ready for use (total sample volume is about 500 μL).

[0289] 3.2.5 Solid phase extraction:

[0290] (1) Preparation of solid phase extraction reagent

[0291] Activator: take 200 mL of methanol into the mobile phase bottle, labeled as activator;

[0292] Equilibrium liquid: prepare 1M phosphate buffer solution 【877 mL of sodium dihydrogen phosphate (1.56 g / L) + 123 mL of disodium hydrogen phosphate (3.58 g / L) 】, dilute 100 times, adjust pH to 5.5 with phosphoric acid, and label as equilibrium liquid;

[0293] Rinse liquid: take 500 mL of equilibrium liquid into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust pH to 5.5 with phosphoric acid, mix well, and label as rinse liquid;

[0294] Eluent: weigh 7.9 g of ammonium bicarbonate into a 1 L mobile phase bottle, add 1 L of water, take 500 mL of ammonium bicarbonate solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust pH to 9 with sodium hydroxide, mix well, and label as eluent;

[0295] (2) The extraction steps are as follows:

[0296] Table 10

[0297] 3.2.6 Post-processing

[0298] Take the eluent (take twice, 600 μL each, a total of 1200 μL) into a 2 mL EP tube, vacuum concentrate for 6 hours at a speed of 1800 rpm; add 100 μL of mobile phase (initial ratio) to the concentrated sample for reconstitution, centrifuge at low speed for 2 min, and inject 10 μL of supernatant into high-resolution mass spectrometry. The antisense strand ratio of siRNA conjugate in this application is detected by LC-MS / MS method semi-quantitatively, and the calculation formula is: residual percentage = target AS strand molecular weight response intensity / sum of all molecular weight response intensities related to target AS strand × 100%. The metabolic results after in vitro incubation in rat liver homogenate for 48 hours are shown in the table below.

[0299] Table 11 siRNA conjugate antisense strand residual percentage

[0300] wherein the sequences of N-ER-FY009151M3L96, N-ER-FY009151M7L96, N-ER-FY009151M9L96 are shown in Table 12.

[0301] Table 12

[0302] wherein the capital letters "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively; the lower case letters a, u, c, g represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf represent 2'-fluoro modified nucleotides; EVP: represents that the nucleotide adjacent to the right of the EVP is a 5'-trans-vinyl phosphonate nucleotide; P1: represents that the nucleotide adjacent to the right of the P1 is a 5'-phosphate nucleotide. In Table 12, the ribose group of the 5' terminal nucleotide of the modified sense strand linked to the conjugate group is a 5' hydroxyl group, and its structure is shown as Formula X. The ribose group of the 5' terminal nucleotide of the modified antisense strand is a 5' hydroxyl group if it is not marked with EVP or P1, and its structure is also shown as Formula X.

[0303] wherein AS represents the antisense strand of the siRNA conjugate, and the greater the remaining amount of AS, the better the stability of the drug, and the better the long-acting property of the drug. As can be seen from Table 11, the siRNA conjugate of the present disclosure has excellent in vitro stability in rat liver homogenate.

[0304] Example 4 Inhibition of human FXII (hFXII) gene expression by siRNA conjugate in humanized mice

[0305] 6-8 week old C57BL / 6-hFXII mice (provided by Shanghai Southern Model Organism Technology Co., Ltd.) were introduced into the breeding facility and adaptively fed for 7 days. Then, the mice were administered with N-ER-FY009Y04, N-ER-FY009151M9L96, N-ER-FY009151M53L96, and N-ER-FY009204M53L96 (5 mice in each group) at a single dose of 1 mg / kg by single subcutaneous administration, respectively, with PBS solution as the solvent, and the administration volume was 5 μL / g. The same dose of PBS was used as the blank control group. On day 7, day 14, day 21, day 28, day 35, day 42, and day 49 after administration, the expression of serum hFXII protein was detected, and the inhibition rate of siRNA conjugate on the expression of hFXII protein was calculated (the calculation formula was: inhibition rate % = (1- average value of hFXII protein expression after administration / average value of hFXII protein expression before administration) * 100 %). The sequence of N-ER-FY009Y04 is shown in Table 13.

[0306] Table 13

[0307] wherein the capital letters "G", "C", "A", "T", and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively; the lowercase letters a, u, c, g represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf represent 2'-fluoro modified nucleotides; dA, dG, dC, dT represent deoxyribonucleotides; and the lowercase letter s represents a phosphorothioate linkage between the two nucleotides adjacent to the left and right of the letter s. In Table 13, the ribose group of the 5' terminal nucleotide of the modified sense strand connected with the conjugate group is a 5' hydroxyl group, and the structure is shown as formula X. The ribose group of the 5' terminal nucleotide of the modified antisense strand is also a 5' hydroxyl group, and the structure is also shown as formula X.

[0308] The results are shown in Table 14 and FIG. 1.

[0309] Table 14 Inhibition rate of siRNA conjugate on the expression of hFXII protein

[0310] As can be seen from Table 14 and FIG. 1, the siRNA conjugate of the present disclosure has a higher inhibition rate on the expression of hFXII protein in humanized mice, and has better long-acting property.

[0311] Example 5 Inhibition effect of siRNA conjugate on the expression of human FXII (hFXII) gene in humanized mice

[0312] 6-8 weeks old C57BL / 6-hFXII mice (provided by Shanghai Southern Model Organism Technology Co., Ltd.) were introduced into the breeding facility and adaptively fed for 7 days, then the mice were administered with N-ER-FY009204M9L96 (5 mice per group) at a single dose of 1 mg / kg by single subcutaneous administration, the solvent was PBS solution, the administration volume was 5 μL / g, and the same dose of PBS was used as the blank control group. On day 7, day 14, and day 21 after administration, the serum hFXII protein expression was detected, and the inhibition rate of siRNA conjugate on hFXII protein expression was calculated (the calculation formula was: inhibition rate % = (1- average value of hFXII protein expression after administration / average value of hFXII protein expression before administration) * 100 %). The results are shown in Table 15.

[0313] Table 15 Inhibition rate of siRNA conjugate on hFXII protein expression

[0314] As can be seen from Table 15, the siRNA conjugate of the present disclosure has a high inhibition rate on hFXII protein expression in humanized mice.

[0315] Example 6 In vitro off-target experiment

[0316] 6.1 Main reagents and consumables

[0317] InvitroGRO CP Medium, serum-free was purchased from Bioreclamation, item number IVT-S03316.

[0318] Fetal bovine serum was purchased from ExCellBio, item number: FSP500.

[0319] Penicillin / streptomycin was purchased from Hyclone, item number: SV30010.

[0320] Nuclease-free water was purchased from Invitrogen, item number: AM9932.

[0321] Ribonucleic acid extraction kit (miRNeasy Tissue / Cells Advanced Mini Kit) was purchased from Qiagen, item number: 217604.

[0322] Agilent ribonucleic acid analysis kit (Agilent RNA 6000 Nano Kit) was purchased from Agilent, item number: 5067-1511.

[0323] Transcriptome library construction kit (TruSeq Stranded Total RNA Library Prep Kit, Illumina) was purchased from Illumina, item number: RS-200-0002. Universal V10 RNA-seq Library Prep Kit for Illumina) was purchased from Vazyme, Cat No.: NR606-02.

[0324] Qubit 1X dsDNA HS Assay Kit was purchased from Thermo Fisher Scientific, Cat No.: Q33231.

[0325] High Sensitivity D1000 ScreenTape was purchased from Agilent, Cat No.: 5067-5584.

[0326] NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles) was purchased from Illumina, Cat No.: 20028312.

[0327] Human primary hepatocytes (PHH) (batch: JMJ) were provided by Shanghai Generex Pharmaceutical Development Co., Ltd.

[0328] 6.2 Main instruments

[0329] Biosafety cabinet (Lisen (Shanghai) Instruments Co., Ltd., Hfsafe-1800LCA2), Nano UV-Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific), Agilent 2100 bioanalyzer (Agilent 2100 Bioanalyzer, Agilent), thermal cycler (T100 thermal cycler, Bio-Rad), fluorescence instrument (Qubit 4 fluorometer, Thermo Fisher Scientific), Agilent 4200 automated electrophoresis system (Agilent 4200 TapeStation), sequencer (NovaSeq 6000 (Illumina)), full-automatic cell fluorescence analyzer (Rigel S2, Countstar).

[0330] 6.3 Medium formula

[0331] 20 mL cell culture medium preparation system containing 10% fetal bovine serum: 17.8 mL in vitro serum-free culture medium Invitro GRO CP Medium, serum-free (Bioreclamation, item number: IVT-S03316), 2 mL fetal bovine serum (ExCell Bio, item number: FSP500), 0.2 mL penicillin / streptomycin (Hyclone, item number: SV30010).

[0332] 6.4 Experimental procedure

[0333] 6.4.1 Cell plating

[0334] Conjugate N-ER-FY009204M53L96 enters human primary hepatocytes by free uptake, the process is as follows:

[0335] (1) Dilute conjugate N-ER-FY009204M53L96 with nuclease-free water to 10 times the final concentration (final concentration of 40 nM and 200 nM).

[0336] (2) Take one vial of cryopreserved human primary hepatocytes (PHH) from the liquid nitrogen tank, gently shake in a water bath to thaw the cryopreservation solution, and transfer the cells to cell culture medium containing 10% fetal bovine serum, adjust the final cell density to 6.7*10 5 cells / mL.

[0337] (3) Take 50 μL of conjugate in (1) to a collagen-coated 24-well cell plate, and add 450 μL of cell suspension in step (2), with a final cell density of 300,000 per well. The final concentration of N-ER-FY009204M53L96 is 40 nM and 200 nM, and each conjugate is tested in triplicate, with wells without conjugate as controls.

[0338] (4) After 72 hours of culture, wash each well with PBS once, then add 260 μL of lysis solution to lyse the cells for subsequent experiments.

[0339] 6.4.2 Next-generation sequencing

[0340] (1) Purify total RNA from cells according to the manufacturer's (Qiagen-217604_miRNeasy Tissue / Cells Advanced Mini Kit) guidelines.

[0341] (2) The concentration of total RNA was detected using NanoDrop One, and the RNA integrity was analyzed according to the manufacturer's (Agilent 2100 Bioanalyzer\Agilent-5067-1511_Agilent RNA 6000 Nano Kit) guidelines.

[0342] (3) Library preparation was performed according to the manufacturer's (Vazyme-NR606 Universal V10 RNA-seq Library Prep Kit for Illumina) guidelines.

[0343] (4) The library concentration was detected according to the manufacturer's (Thermo Fisher Scientific-Qubit 4 fluorometer\Thermo Fisher Scientific-Q33231_Qubit 1X dsDNA HS Assay Kit) guidelines, and the library fragment size was analyzed according to the manufacturer's (Agilent 4200 TapeStation\Agilent-5067-5584_High Sensitivity D1000 ScreenTape) guidelines.

[0344] (5) Library sequencing was performed according to the manufacturer's (Illumina-NovaSeq 6000\Illumina-20028312_NovaSeq 6000 S4 Reagent Kit v1.5(300 cycles)) guidelines.

[0345] This experiment is based on the second-generation sequencing technology to study the effect of human hepatocytes on the transcriptional expression after free uptake of the test product (N-ER-FY009204M53L96). The screening criteria for differential genes are set as |log2(FoldChange)|>1 & padj.<0.05.

[0346] 6.5 IC in PHH cells 50 Test

[0347] The determination concentration range (nM) of the siRNA conjugate N-ER-FY009204M53L96 to be tested is set as 500, 125, 31.25, 7.8125, 1.9531, 0.4883, 0.1221 and 0.03052 nM, and the siRNA conjugate to be tested enters the PHH cells by free uptake. The free uptake IC of N-ER-FY009204M53L96 in human hepatocytes is obtained 50The inhibition rate of each concentration on FXII was 0.21 nM. The inhibition rate results of each concentration on FXII are shown in Figure 2.

[0348] After sequencing N-ER-FY009204M53L96 at 40 nM and 200 nM concentrations, it was found that the |log2(FoldChange)| and padj values of the differential genes were quite different from the target gene FXII (as shown in Figures 3 and 4). In vitro PHH cell free uptake verification was performed on the differentially expressed genes that appeared, such as RADX, PCDHGB4 and G6PC1, and it was found that the inhibition rates of the three targets were all less than 50% at the maximum concentration of 500 nM, so N-ER-FY009204M53L96 had low off-target risk for RADX, PCDHGB4 and G6PC1. The IC 50 value of N-ER-FY009204M53L96 in PHH cells was 0.21 nM, and the two concentrations selected for off-target testing were 190 times and 952 times the IC 50 value, respectively. It can be seen that the safety window of the conjugate reaches more than 190 times, so the possibility of producing off-target risk in vivo is relatively low, and the safety is good, which has good drugability.

[0349] Example 7 SD Rat Subcutaneous Injection of N-ER-FY009151M53L96, N-ER-FY009204M9L96, N-ER-FY009204M53L96 Repeated Dose Toxicity Test

[0350] This test has a total of 3 groups, and each group of 15 female and male SD rats (from Guokecai Fuhebei Pharmaceutical Technology Co., Ltd.) in the main test group is subcutaneously injected with a solvent control (0.9% sodium chloride injection), 50 or 100 mg / kg of N-ER-FY009151M53L96, N-ER-FY009204M9L96, N-ER-FY009204M53L96, respectively, once every two weeks, for a total of 3 times, and the drug volume is 5 mL / kg. On D30, 10 animals of each group of females and males were planned for autopsy at the end of the dosing period, and on D84, the remaining recovery period animals were planned for autopsy. Satellite groups (with the same dosing as the main test group animals) were set up, with 5 female and male rats in each group for cytokine research blood sampling. In addition, TK groups (with the same dosing as the main test group animals) were set up, with 4 female and male rats in the solvent control group and 8 female and male rats in the test product group for TK research blood sampling.

[0351] The toxicity indicators of this test include: death / illness, general observation, body weight, food consumption, ophthalmic examination, clinical pathology indicators (hematology and coagulation indicators, serum biochemical indicators, immune function indicators, urine indicators, bone marrow smears), gross necropsy, organ weight, histopathological examination, and tissue distribution. Satellite group animals were collected blood samples at 4h and 24h after dosing on the adaptation period, D1 and D29 for cytokine analysis. TK group animals were collected blood samples at pre-dose and 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours post-dose on D1 and D29 for toxicokinetic analysis.

[0352] During the experiment, all animals survived to the scheduled autopsy, and no obvious abnormalities related to the test product were found in the general observation, body weight, food consumption, ophthalmic examination, cytokines, clinical pathology indicators (hematology, coagulation, serum biochemistry, immune function indicators, urine indicators, bone marrow smears), gross necropsy of all animals.

[0353] Example 8 FeCl3-induced inferior vena cava thrombosis model experiment in mice

[0354] 8.1 Dosing

[0355] F12 humanized mice (Shanghai South Model Organisms Technology Co., Ltd.) were evenly divided into blank control group, self-made drug group (N-ER-FY009204M53L96), positive control group (N-ER-FY009Y04) according to the pre-drug F12 expression, 5 in each group (3 males and 2 females), single subcutaneous dose of 3mg / kg, solvent is PBS solution, the volume of the dose is 5μL / g, and inferior vena cava thrombosis model is established on the 14th day after dosing.

[0356] 8.2 Inferior vena cava thrombosis model

[0357] After the experimental animals were anesthetized with isoflurane gas, the abdomen was shaved and disinfected with iodophor, and the veins between the kidneys and the iliac lumbar were bluntly dissected through the median incision of the abdomen. A miniature Puler flow probe (0.7PS probe, 3.2mm long, 2.7mm wide, and 0.70mm deep) was placed at the branch of the inferior vena cava near the renal vein, and the blood flow was monitored for 1min to stabilize the blood flow rate before modeling. A 10% FeCl3 solution was used to pre-saturate filter paper (2mm x 4mm), and the long side was placed on the distal inferior vena cava for 3min and then removed. From the start of FeCl3 application modeling, the blood flow was continuously monitored for 40min. The vascular occlusion time and coagulation factors APTT and PT were detected.

[0358] Vessel occlusion time detection method: after inferior vena cava thrombosis modeling, FeCl3 was applied to start timing, blood flow velocity was continuously monitored for 40 min, and blood flow velocity was continuously recorded by Acqknowledge 5.0.6 software, and blood flow velocity was reduced to 25% of the baseline to consider vessel occlusion.

[0359] Coagulation factor APTT and PT detection method: after the end of the vessel occlusion time detection test, blood was taken, 3.8% sodium citrate was anticoagulated, 3000 rpm centrifugation for 10 min, plasma was separated, and APTT and PT were detected.

[0360] 8.3 Results

[0361] Table 16 Inhibition rate of siRNA conjugate on hFXII protein expression

[0362] As can be seen from Table 16, the siRNA conjugate of the present disclosure has a high inhibition rate on hFXII protein expression in the inferior vena cava thrombosis model of humanized mice.

[0363] Table 17 Day 14 inferior vena cava thrombosis model experimental data

[0364] As can be seen from Table 17, the N-ER-FY009204M53L96 administration group did not appear to have vessel occlusion in the 40-minute experimental monitoring, the thrombosis rate was 0%, and the APTT time was also the longest, indicating that N-ER-FY009204M53L96 has a good anticoagulant effect. Figure 5 shows the change in blood flow velocity in the experimental monitoring, it can be seen that the blood flow velocity of the N-ER-FY009204M53L96 administration group has no obvious decrease within 40 minutes, and compared with the change of the PBS blank group, it also indicates the anticoagulant effect of N-ER-FY009204M53L96.

[0365] Example 9 siRNA conjugate cynomolgus monkey knockdown experiment

[0366] Three male washout cynomolgus monkeys (body weight > 8 kg) over 10 years old (provided by Suzhou United Immunological Laboratories Co., Ltd.) were put into the feeding facility for feeding. Blood sampling was performed to detect the target protein FXII content one week before administration (D-7). The cynomolgus monkeys were administered subcutaneously (injection site: shoulder bone / outer neck skin relaxation area) at a single dose of 3 mg / kg, and the administration day was defined as Day 0 (D0). Serum was collected every week after administration to detect the target protein FXII level.

[0367] Table 18 Inhibition rate (%) of siRNA conjugate on FXII protein expression

[0368] As can be seen from Table 18, N-ER-FY009204M53L96 has a higher inhibition rate on the expression of hFXII protein in cynomolgus monkeys, indicating that the inhibitory effect of the siRNA conjugate of the present application is superior to that of the prior art, and the inhibition time is longer, which is long-acting.

[0369] Example 10 Rat liver homogenate in vitro stability experiment of siRNA modification

[0370] Except that the conjugate in the sample preparation step is replaced by the modification, the experimental materials and steps in this example are the same as those in Example 3. The antisense strand ratio of the siRNA modification of the present disclosure is semi-quantitatively detected by LC-MS / MS method, and the metabolism result of the siRNA modification of the present application after in vitro incubation for 48 hours in rat liver homogenate is obtained.

[0371] This experiment shows that the siRNA modification of the present disclosure has excellent in vitro stability in rat liver homogenate.

[0372] The above examples of the present disclosure are only examples for clearly illustrating the present disclosure, and are not intended to limit the embodiments of the present disclosure. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. All embodiments are not required or possible to be exhausted here. Any modification, equivalent replacement and improvement made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

1. An siRNA for inhibiting the expression of a coagulation factor XII gene, the siRNA comprising a sense strand and an antisense strand, wherein each of the nucleotides in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences: (1) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 2; (2) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 28, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:

29.

2. The siRNA of claim 1, wherein the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, the nucleotide sequence V and VI have a length of 0 to 3 nucleotides, the nucleotide sequence V is linked to the 3' terminal end of the sense strand to form a 3' overhang of the sense strand and / or the nucleotide sequence VI is linked to the 3' terminal end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the nucleotide sequence V or VI has a length of 2 nucleotides; the nucleotide sequence V is identical to or has a nucleotide difference from the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

3. The siRNA of claim 1 or 2, wherein at least one of the nucleotides in the sense strand or the antisense strand is a modified nucleotide, and / or at least one of the phosphate groups is a phosphate group with a modification group; preferably, the phosphate group with a modification group is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom; and / or, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide; and / or, the terminal end of the sense strand is linked to an inverted abasic deoxyribose residue (invAb). the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

5. The siRNA of any one of claims 1-4, wherein, 4. The siRNA according to any one of claims 1-3, wherein, neither the 5' terminal nucleotide nor the 3' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue; or only the 5' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue; or only the 3' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue; or the 5' terminal nucleotide and the 3' terminal nucleotide of the sense strand are respectively linked to an inverted abasic deoxyribose residue; preferably, the inverted abasic deoxyribose residue is linked to the 3' terminal nucleotide and / or the 5' terminal nucleotide of the sense strand by a phosphodiester bond, a phosphorothioate group or other internucleoside linkage. ​ ​ 6. The siRNA of any one of claims 1-5, wherein the modified nucleotide is selected from a 2’-fluoro modified nucleotide, a 2’-alkoxy modified nucleotide, a 2’-substituted alkoxy modified nucleotide, a 2’-alkyl modified nucleotide, a 2’-substituted alkyl modified nucleotide, a 2’-deoxy modified nucleotide, a 2’-amino modified nucleotide, a 2’-substituted amino modified nucleotide, a nucleotide analog, or a combination of any two or more thereof; preferably, the modified nucleotide is selected from a 2’-F modified nucleotide, a 2’-O-CH3 modified nucleotide, a 2’-O-CH2-CH2-O-CH3 modified nucleotide, a 2’-O-CH2-CH=CH2 modified nucleotide, a 2’-CH2-CH2-CH=CH2 modified nucleotide, a 2’-deoxy modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

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

8. The siRNA of claim 7, wherein each non-fluoride-modified nucleotide is independently selected from one of a nucleotide or a nucleotide analogue in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced with a non-fluorine group, the nucleotide analogue being selected from one of pseudouracil, an iso-nucleotide, LNA, ENA, cET BNA, UNA and GNA.

9. The siRNA of any one of claims 1-8, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoride-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or a combination of any two or more thereof. Preferably, in the sense strand, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 5, 7, 8, and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, and 10, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13, and 15, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8, and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16, and 17, and the remaining positions are 2'-methoxy-modified nucleotides; and / or In the antisense strand, in the direction from 5' to 3', 2'-fluoro-modified nucleotides are at positions 2, 6, 8, 9, 14, and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2 and 14, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and position 6 is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 5, 7, and 14; or, 2'-fluoro-modified nucleotides are at positions 2, 7, and 14, positions 5 and 12 are 2'-deoxy-modified nucleotides, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 7, 10, 12, 14, and 16, position 5 is a 2'-deoxy-modified nucleotide, position 6 is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides.

10. The siRNA of any one of claims 1-9, wherein when neither the 5' end nor the 3' end of the sense strand is linked (invAb), in the direction from the 5' end to the 3' end of the sense strand, (1) the sense strand comprises a phosphorothioate linkage at: between the 1st and 2nd nucleotides from the 5' end of the sense strand; and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and between the 1st and 2nd nucleotides from the 3' end of the sense strand; and between the 2nd and 3rd nucleotides from the 3' end of the sense strand; or, (2) the sense strand comprises a phosphorothioate linkage at: between the 1st and 2nd nucleotides from the 5' end of the sense strand; and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; when only the 3' end of the sense strand is linked (invAb), in the direction from the 5' end to the 3' end of the sense strand, the sense strand comprises a phosphorothioate linkage at: between the 1st and 2nd nucleotides from the 5' end of the sense strand; and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and between the 3' end of the sense strand (invAb) and the 1st nucleotide from the 3' end of the sense strand.

11. The siRNA of any one of claims 1-10, wherein, in the direction from the 5' end to the 3' end of the antisense strand, the antisense strand comprises a phosphorothioate linkage at: between the 1st and 2nd nucleotides from the 5' end of the antisense strand; and between the 2nd and 3rd nucleotides from the 5' end of the antisense strand; and between the 1st and 2nd nucleotides from the 3' end of the antisense strand; and between the 2nd and 3rd nucleotides from the 3' end of the antisense strand. between the 1st and 2nd nucleotides from the 5' terminus of the antisense strand; and between the 2nd and 3rd nucleotides from the 5' terminus of the antisense strand.

12. The siRNA of any one of claims 1-11, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxy-modified nucleotide, or a combination of any two or more thereof; preferably, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 5, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans- vinylphosphonate group; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 8, 9, and 10 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans- vinylphosphonate group; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 8, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 2 and 14 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate or 5' phosphate derivative group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 7, 9, 11, and 13 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 3, 7, 8, and 9 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, the 5' terminal nucleotide of the antisense strand linked to a 5'-trans vinyl phosphonate group; or, in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 3, 9, 11, and 13 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate or 5' phosphate derivative group; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate group or 5' phosphate derivative group; or, 2'-fluoro modified nucleotides at positions 7, 11, 12, and 13 of the sense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate group or 5' phosphate derivative group; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, and 16 of the sense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate group or 5' phosphate derivative group; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, and 17 of the sense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate group or 5' phosphate derivative group; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, 16, and 17 of the sense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at all other positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand not linked to a 5' phosphate group or 5' phosphate derivative group; or 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the sense strand; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and a GNA modified nucleotide at position 6, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the sense strand; 2'-fluoro modified nucleotides at positions 2, 7, 10, and 14, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the sense strand; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or 2'-fluoro modified nucleotides at positions 3, 7, 8, and 9, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the sense strand; 2'-fluoro modified nucleotides at positions 2, 7, 10, and 14, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the sense strand; 2'-fluoro modified nucleotides at positions 2, 7, 10, and 14, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or 2'-fluoro modified nucleotides at positions 3, 7, 8, and 9, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the sense strand; 2'-fluoro modified nucleotides at positions 2, 7, and 14, 2'-deoxy modified nucleotides at positions 5 and 12, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction on the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 3, 7, 8, and 9 of the sense strand, and 2'-methoxy-modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 2, 3, 7, 10, 12, 14, and 16 of the antisense strand, a 2'-deoxy-modified nucleotide at position 5, a GNA-modified nucleotide at position 6, and 2'-methoxy-modified nucleotides at the remaining positions, the 5'-terminal nucleotide of the antisense strand being linked to a 5'-trans-vinylphosphonate group; or, in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy-modified nucleotides at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and 2'-methoxy-modified nucleotides at the remaining positions, the 5'-terminal nucleotide of the antisense strand being linked to a 5'-trans-vinylphosphonate group.

13. The siRNA according to claims 1-12, which is selected from the siRNAs of Table 1; preferably, the siRNA is selected from N-ER-FY009151M53, N-ER-FY009204M53, N-ER-FY009204M9.

14. An siRNA conjugate comprising an siRNA according to any one of claims 1-13 and a conjugation group conjugated to the siRNA.

15. The siRNA conjugate according to claim 14, wherein in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3'-end of the sense strand has or forms a blunt end, and the 3'-end of the antisense strand has 1-3 overhanging nucleotides extending out of the double-stranded region; or, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3'-end of the sense strand forms a blunt end, and the 3'-end of the antisense strand forms a blunt end.

16. The siRNA conjugate of claim 15, wherein the conjugate group is selected from:

17. The siRNA conjugate according to any one of claims 14-16, which is selected from the siRNA conjugates of Table 2; preferably, the siRNA conjugate is selected from N-ER-FY009151M53L96, N-ER-FY009204M53L96, N-ER-FY009204M9L96.

18. A pharmaceutical composition comprising an siRNA according to any one of claims 1-13, or an siRNA conjugate according to any one of claims 14-17, and a pharmaceutically acceptable carrier.

19. A kit comprising an siRNA according to any one of claims 1-13, or an siRNA conjugate according to any one of claims 14-17, or a pharmaceutical composition according to claim 18.

20. Use of the siRNA of any one of claims 1-13, or the siRNA conjugate of any one of claims 14-17, or the pharmaceutical composition of claim 18 for the manufacture of a medicament for inhibiting the expression of a FXII gene.

21. Use of the siRNA of any one of claims 1-13, or the siRNA conjugate of any one of claims 14-17, or the pharmaceutical composition of claim 18 for the manufacture of a medicament for preventing and / or treating a disease associated with overexpression of a FXII gene.

22. The use of claim 21, wherein the disease is multiple sclerosis, atherosclerosis, Alzheimer’s disease, hereditary angioedema, sepsis, deep vein thrombosis, community-acquired pneumonia, thrombotic inflammation of COVID-19, microscopic polyangiitis, neurogenic inflammation, cerebral vascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis, colitis, pulmonary fibrosis, lung injury, liver fibrosis, and other yet unidentified related conditions, pathologies or syndromes.

23. A method of inhibiting the expression of a FXII gene, comprising contacting a cell expressing FXII with a therapeutically effective amount of the siRNA of any one of claims 1-13, or the siRNA conjugate of any one of claims 14-17, or the pharmaceutical composition of claim 18, or administering to a subject in need thereof.

24. A method of treating and / or preventing a disease associated with overexpression of a FXII gene, comprising administering to a subject in need thereof a therapeutically effective amount of the siRNA of any one of claims 1-13, or the siRNA conjugate of any one of claims 14-17, or the pharmaceutical composition of claim 18.

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