CN-121109397-B - SiRNA, modifier and conjugate for targeted regulation of FXI gene expression and application thereof

Abstract

The present disclosure provides siRNA, modifications, conjugates and uses thereof that target modulation of the expression of the coagulation undecylenic Factor (FXI) gene. The present disclosure designs a series of siRNAs based on FXI messenger ribonucleic acid (mRNA) sequences and makes alternate modifications and uses a specific set of modification templates. Cell and animal experiment results show that the oligonucleotide sequences modified by alternative modification and specific template can obviously inhibit the expression of FXI gene and can be used for developing anticoagulants.

Inventors

• Huang Zeao
• SONG GENGSHEN
• YANG SHUO
• XU CHEN
• Ji Guangshen
• YU FEI
• WU YUCHENG
• TIAN ZHIKANG

Assignees

• 北京悦康科创医药科技股份有限公司

Dates

Publication Date

20260508

Application Date

20251113

Priority Date

20250508

Claims (9)

1. 1. An siRNA duplex comprising a sense strand and an antisense strand forming a reverse complementary duplex region, the siRNA duplex comprising any one of the oligonucleotide duplex selected from the group consisting of a pair of the following sense strand and antisense strand: (1) The sequence of the sense strand is Cms-Ams-Gm-Am-Um-Gf-Am-Uf-Um-Cm-Um-Am-Um-Cm-Am; and the antisense strand has a sequence of UmsEVP-Gfs-Am-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Um-Gms-Ams-Am; (2) The sequence of the sense strand is Cms-Ams-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Cm-Um-Am-Uf-Am-Um-Cm-Am; and the antisense strand has a sequence of UmsEVP-Gfs-Af-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Um-Gms-Ams-Am; (3) The sequence of the sense strand is Ams-Gms-Gm-Am-Um-Gf-Am-Uf-Um-Cm-Um-Am-Um-Cm-Am; and the antisense strand has a sequence of UmsEVP-Gfs-Am-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Ums-Gms-Am; (4) The sequence of the sense strand is Cm-Am-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Am-Um-Am-Ums-Cms-dAinV; and the antisense strand has a sequence of UmsEVP-Gfs-Af-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Cms-Ums-Gm; (5) The sequence of the sense strand is Cm-Am-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Am-Um-Am-Ums-Cms-dAinV; and the antisense strand has a sequence of UmsEVP-Gfs-Af-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Um-Gms-Ams-Am; (6) The sequence of the sense strand is Gms-Gms-Am-Um-Gm-Am-Uf-Um-Uf-Um-Cf-Um-Am-Um-Am; and the antisense strand has a sequence of UmsEVP-Ufs-Uf-Gm-Am-Uf-Am-Um-Am-Gm-Am-Af-Am-Uf-Cm-Am-Um-Cm-Cms-Ums-Gm; Wherein m represents 2 '-O-methyl modification, f represents 2' -fluoro modification, s represents phosphorothioate linkage between the nucleotide at the position and the adjacent nucleotide at the right side, EVP represents 5 '-vinyl- (E) -phosphonate, inV represents that the linkage at the 3' -end of the sequence is changed from natural 3'-5' to 3'-3'.
2. 2. A conjugate for reducing expression of FXI, comprising the siRNA duplex of claim 1, and a conjugate group attached thereto.
3. 3. The conjugate of claim 2, wherein the conjugate group is attached to the 3 '-end or the 5' -end of the sense strand of the oligonucleotide and/or, The conjugate group is a GalNAc derivative attached with a divalent or trivalent branched linker arm.
4. 4. The conjugate of claim 2, wherein the conjugate group is: 。
5. 5. The conjugate of any one of claims 2-4, wherein the conjugate comprises any one of the oligonucleotide duplex selected from the group consisting of the following sense strand and antisense strand pairings: (1) The sequence of the sense strand is Cms-Ams-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Cm-Um-Am-Uf-Am-Um-Cm-Am-G101; and the antisense strand has a sequence of UmsEVP-Gfs-Am-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Um-Gms-Ams-Am; (2) The sequence of the sense strand is Cms-Ams-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Cm-Um-Am-Uf-Am-Um-Cm-Am-G101; and the antisense strand has a sequence of UmsEVP-Gfs-Af-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Um-Gms-Ams-Am; (3) The sequence of the sense strand is G101-Cm-Am-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Uf-Um-Cm-Um-Um-Am-Am-Ums-Cms-dAinV, and the sequence of the antisense strand is UmsEVP-Gfs-Af-Um-Am-Uf-Am-Am-Gm-Am-Am-Uf-Cm-Af-Um-Cm-Cms-Ums-Gm; (4) The sequence of the sense strand is G101-Cm-Am-Gm-Gm-Am-Um-Gf-Am-Uf-Um-Uf-Um-Cm-Um-Um-Am-Ums-Cms-dAinV, and the sequence of the antisense strand is UmsEVP-Gfs-Af-Um-Am-Uf-Am-Am-Gm-Am-Am-Uf-Cm-Af-Um-Um-Cm-Um-Gms-Ams-Am; (5) The sequence of the sense strand is Ams-Gms-Gm-Am-Um-Gf-Am-Uf-Um-Cm-Um-Am-Um-Cm-Am-G101; and the antisense strand has a sequence of UmsEVP-Gfs-Am-Um-Am-Uf-Am-Gm-Am-Uf-Cm-Af-Um-Cm-Ums-Gms-Am; (6) The sequence of the sense strand is Gms-Gms-Am-Um-Gm-Am-Uf-Um-Uf-Um-Cf-Um-Am-Um-Uf-Cm-Am-G101; and the antisense strand has a sequence of UmsEVP-Ufs-Uf-Gm-Am-Uf-Am-Um-Am-Gm-Am-Af-Am-Uf-Cm-Am-Um-Cm-Cms-Ums-Gm.
6. 6. A nucleic acid protein composition comprising the double-stranded region of the siRNA duplex of claim 1, and a nuclease; Or the nucleic acid protein composition comprises the antisense strand of the duplex region of the siRNA duplex of claim 1, and a nuclease.
7. 7. A pharmaceutical composition comprising the siRNA duplex of claim 1, the conjugate of any one of claims 2-5, or the nucleic acid protein composition of claim 6, and a pharmaceutically acceptable carrier.
8. 8. Use of the siRNA duplex according to claim 1, the conjugate according to any one of claims 2 to 5, the nucleic acid protein composition according to claim 6 or the pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment of a disease associated with FXI gene expression, said disease associated with FXI gene expression being a thrombotic disease.
9. 9. Use of the siRNA duplex according to claim 1, the conjugate according to any one of claims 2-5, the nucleic acid protein composition according to claim 6 or the pharmaceutical composition according to claim 7 in the preparation of an anticoagulant drug.

Description

SiRNA, modifier and conjugate for targeted regulation of FXI gene expression and application thereof Technical Field The present disclosure is in the field of biological medicine, and in particular relates to siRNA, modifications, conjugates, and uses thereof for targeted modulation of FXI gene expression, e.g., in modulation of FXI gene expression or in anticoagulation. Background The coagulation cascade is completed by the intrinsic coagulation pathway and the extrinsic coagulation pathway together. Wherein the intrinsic coagulation pathway comprises coagulation factors FXI and FXII. In the classical model, after exposure to collagen, RNA or polyphosphate (polyphophates), FXII is activated, FXIIa further activates downstream FXI, producing FXIa, which, through downstream FIX and FX, among other coagulation factors, ultimately produces thrombin through the common pathway, which initiates fibrin formation while activating FXI, forming positive feedback amplifying the clotting process. Thrombosis is initiated primarily by an endogenous pathway. The extrinsic coagulation pathway is usually initiated by tissue factor (Tissure Factor, TF), activates FVII, and in turn activates the common pathway, playing an important role in the hemostatic process. At the time of angiogenic injury, FVII or FVIIa in the blood binds to tissue factor TF from extravascular sources, and the FVIIa-TF complex converts FX to FXa, which then converts a limited amount of prothrombin (Prothrombin) to Thrombin (Thrombin). During hemostasis, FXI is converted to FXIa by thrombin, which subsequently causes clot consolidation by activating FIX and its downstream pathways, but the overall effect is relatively small. Taken together, the important molecular FXI of the intrinsic coagulation pathway plays an important role in regulating the physiological process of thrombosis, but hardly participates in the case processes of hemorrhage and the like which are mainly regulated by the extrinsic pathway, so that the FXI can be used as a new target point of antithrombotic treatment. Among the anticoagulants currently on the market, vitamin K Antagonists (VKA) mainly inhibit prothrombin and FX, heparin and oral anticoagulant (dotc) mainly inhibit FXa and thrombin, and these several drugs may inhibit the hemostatic function associated with the extrinsic coagulation pathway while inhibiting thrombosis, thus there is a certain bleeding risk during treatment. In contrast, the target FXI in this study has little effect in hemostasis and plays a fundamental role in thrombosis, so that hemostasis and thrombosis can be effectively separated, and bleeding risk can be reduced while anticoagulation is performed. In addition, the full-modified siRNA drug coupled with GalNAc has good long-term effect, can realize one-half-year administration, has curative effect continuously, and can obviously improve the compliance of patients. Disclosure of Invention Problems to be solved by the invention: Among the anticoagulants commonly used in clinic, vitamin K antagonists mainly inhibit prothrombin and FX, heparin and oral anticoagulant (dotc) mainly inhibit FXa and thrombin, and these several drugs may inhibit the hemostatic function associated with the extrinsic coagulation pathway while inhibiting thrombosis, thus there is a certain bleeding risk during treatment. In addition, many of the anti-coagulant small molecule inhibitors under investigation require daily dosing and have low patient compliance. Again, for patients with impaired kidney function, drugs metabolized through the kidneys have a greater impact on kidney function. There is therefore an urgent need to develop anticoagulant drugs that reduce the risk of bleeding and improve patient compliance. The solution is as follows: the present disclosure couples N-acetylgalactosamine (GalNAc) by designing a series of siRNA sequences against FXI mRNA sequences and performing specific template modifications thereto. The present disclosure screens out a number of alternative and specific modification sequences having a significant inhibitory effect on FXI gene expression by modifying the designed siRNA sequences. In one aspect, the present disclosure provides an siRNA duplex comprising an oligonucleotide duplex consisting of a sense strand and an antisense strand pairing. In another aspect, the present disclosure provides a conjugate for reducing expression of FXI comprising an siRNA duplex described above, and a conjugate group attached thereto. In another aspect, the present disclosure provides a nucleic acid protein composition comprising a double-stranded region or an antisense strand of a double-stranded region of an siRNA duplex described above, and a nuclease. In another aspect, the present disclosure provides a recombinant vector comprising a nucleic acid molecule encoding the above siRNA duplex. In some embodiments, the vector backbone of the recombinant vector is selected from the group consisting of a recombinant