Preparation and application of small interfering nucleic acid for inhibiting expression of coagulation factor xi and conjugate thereof

Abstract

This invention relates to the preparation and application of an siRNA that inhibits the expression of coagulation factor XI (F11) and its GalNAc conjugate. The siRNA molecule, with the QLS0429 sequence as its core, exhibits F11 expression inhibitory activity in vitro and in hFXI mice after chemical modification and GalNAc conjugation. This invention also provides the use of the siRNA, its conjugate, and pharmaceutical compositions in the preparation of drugs for reducing FXI expression or activity, treating and / or preventing FXI-related thrombotic diseases, thromboembolic diseases, or ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of small nucleic acid drugs and medicinal chemistry, specifically relating to small interfering nucleic acids (siRNA) targeting coagulation factor XI (FXI / F11) mRNA, chemically modified siRNA, liver-targeting GalNAc conjugates, pharmaceutical compositions thereof, and pharmaceutical uses. More specifically, this invention provides an siRNA sequence with QLS0429 as its core and its modified form, wherein the antisense strand of the siRNA can target the region from position 265 to 283 of human F11 mRNA (GenBank accession number NM_000128.4) and inhibit F11 expression.

[0002] Compared to traditional anticoagulants, siRNAs targeting FXI reduce FXI protein production by decreasing F11 mRNA expression in hepatocytes, potentially reducing some bleeding-related risks while maintaining antithrombotic effects. GalNAc conjugation technology can promote siRNA entry into hepatocytes via ASGPR-mediated delivery, thereby improving hepatic targeted delivery efficiency and in vivo drug efficacy persistence. Background Technology

[0003] Thrombotic diseases are one of the leading causes of death and disability worldwide, encompassing various clinical manifestations such as deep vein thrombosis, pulmonary embolism, and ischemic stroke. While traditional anticoagulation therapy can prevent thrombosis to some extent, it often comes with an increased risk of bleeding, especially for patients requiring long-term anticoagulation. Finding a balance between effective anticoagulation and controlling bleeding risk has always been a major challenge in clinical medicine.

[0004] As a key regulator of the intrinsic coagulation pathway, the mechanism of action of plasma coagulation factor XI (FXI) in thrombosis has received widespread attention in recent years. FXI is mainly synthesized by the liver and secreted into the bloodstream, occupying a relatively upstream position in the coagulation cascade. When the vascular endothelium is damaged, FXI is activated by activated factor XII (FXIIa) or thrombin, which in turn activates factor IX, initiating the intrinsic coagulation pathway. Notably, although patients with FXI deficiency exhibit mild coagulation dysfunction, they rarely experience spontaneous bleeding. This clinical observation provides important theoretical support for FXI as a target for antithrombotic drugs.

[0005] In recent years, with the continuous development and improvement of RNA interference technology, siRNA drugs have shown great application potential in the field of gene therapy. Compared with traditional small molecule drugs and protein drugs, siRNA has unique advantages such as flexible design, high specificity, and long-lasting effect. Through complete complementary binding with target mRNA, siRNA can induce RNA-induced silencing complex (RISC)-mediated mRNA degradation, thereby achieving precise regulation of specific gene expression at the post-transcriptional level.

[0006] However, the clinical application of siRNA drugs still faces many challenges, the most important of which include: (1) naked siRNA molecules are easily degraded by nucleases in the bloodstream, resulting in a very short half-life; (2) siRNA molecules carry a negative charge, making it difficult for them to cross the cell membrane and enter target cells; and (3) they lack tissue specificity, which may produce off-target effects in non-target organs. To address these issues, researchers have developed a variety of chemical modification strategies and delivery systems.

[0007] Chemical modification is an important means of improving the stability and biological activity of siRNA. Commonly used modification methods include 2'-O-methyl modification, 2'-fluorine modification, and phosphate thioate modification. These modifications can not only improve the resistance of siRNA to nucleases, but also regulate the binding affinity of siRNA to the RISC complex, optimizing the gene silencing effect. Simultaneously, by introducing modifying groups at specific positions on the siRNA molecule, immunogenicity can be reduced, lowering the risk of non-specific immune responses.

[0008] In terms of delivery systems, GalNAc conjugation technology represents the latest advancement in siRNA drug delivery. GalNAc is a natural ligand for the desialylate glycoprotein receptor (ASGPR) on the surface of hepatocytes, which is highly expressed on hepatocytes but expressed at relatively low levels in other tissues. By conjugating siRNA to GalNAc molecules, specific delivery of siRNA to the liver can be achieved, significantly increasing drug accumulation in the target organ while reducing distribution in other tissues, thereby improving therapeutic efficacy and reducing side effects.

[0009] Currently, several oligonucleotide drugs and GalNAc-conjugated small nucleic acid drugs have been approved for marketing by regulatory agencies, demonstrating the feasibility of chemical modification and liver-targeted delivery strategies in the development of small nucleic acid drugs. It should be noted that the delivery systems of different marketed drugs are not the same. For example, patisiran uses lipid nanoparticle delivery, while givasiran, lumasiran, inclisiran, and vutrisiran employ GalNAc-related liver-targeted delivery strategies.

[0010] Numerous published studies and patents have been filed for RNAi drugs targeting FXI / F11. Therefore, verifiable technical contributions are still needed in terms of specific target sequences, chemical modification patterns, conjugation structures, and in vitro and in vivo efficacy data.

[0011] This invention, based on the screening and chemical modification optimization of the human F11 mRNA target region, yielded FXI-targeting siRNA molecules, represented by QLS0429, and their GalNAc conjugates. These molecules demonstrated good F11 expression inhibitory activity in in vitro F11 mRNA inhibition experiments, human primary hepatocyte free uptake experiments, and hFXI mouse in vivo experiments. Summary of the Invention

[0012] The purpose of this invention is to provide an FXI / F11 siRNA and its conjugate that has a well-defined target region, can be chemically modified, and can be delivered via GalNAc to the liver, in order to solve the problem that the efficacy, stability, and liver-targeted delivery efficiency of candidate sequences in the prior art still need further optimization.

[0013] The invention aims to solve the following key technical problems: First, existing siRNA candidate sequences usually require screening and experimental verification on specific target regions. This invention identifies the region from position 265 to 283 of human F11 mRNA as the target region and screens out candidate siRNA sequences represented by QLS0429.

[0014] Second, existing siRNA chemical modification schemes may affect activity, stability, and the risk of immune stimulation. This invention designs various 2'-O-methyl, 2'-fluorine, and thiophosphate modification combinations around QLS0429, and further constructs GalNAc conjugates to improve the feasibility of in vivo application.

[0015] I. siRNA for inhibiting coagulation factor XI expression. In one aspect, this invention relates to an siRNA for inhibiting coagulation factor XI (F11) expression, said siRNA comprising a sense strand and an antisense strand, the sense strand consisting of the sequence shown in SEQ ID NO:1, and the antisense strand consisting of the sequence shown in SEQ ID NO:2, wherein the antisense strand is complementary to the target region 265-283 of human F11 mRNA (GenBank accession number NM_000128.4) and is capable of reducing F11 mRNA expression.

[0016] In some embodiments, the sense strand is 17-21 nucleotides long, and the antisense strand is 17-23 nucleotides long. In other embodiments, the sense strand and / or antisense strand may be a nucleotide sequence that differs from the sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2 by no more than 3 nucleotides, or a nucleotide sequence comprising at least 15 consecutive nucleotides of the sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0017] In some embodiments, the sense strand and / or antisense strand further comprises a dangling sequence at the 3' end, the two dangling sequences being 1-3 nucleotides in length, the two dangling sequences being complementary to the nucleotides at the corresponding positions of the target mRNA, or the two dangling sequences being independently selected from UU and dTdT, where "dT" represents thymine deoxyribonucleotide.

[0018] II. Modified siRNA for inhibiting coagulation factor XI (F11) expression. In a second aspect, the present invention relates to a modified siRNA for inhibiting coagulation factor XI (F11) expression, comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially anti-complementary to form a double-stranded region; wherein the sense strand and the antisense strand are selected from a group of sequences shown in i)-iv), each of the sense strand and the antisense strand independently consisting of the nucleotide sequences shown in i)-iv), or nucleotide sequences differing from the shown nucleotide sequences by no more than 3 nucleotides, or nucleotide sequences comprising at least 15 consecutive nucleotides of the shown nucleotide sequences: i) QLS0429m1: 5'-CmsAmsCmCmCmAmAfGmAfUmGfUmUmUmAmCmUmsCmsUm-3' (SEQ ID NO: 3); 5'-AmsGfsAmGmUmAmAfAmCmAmUmCfUmUfGmGfGmUmGmsGmsUm-3' (SEQ ID NO: 4); ii) QLS0429m2: 5'-CmsAmsCmCmCmAmAfGfAfUmGmUmUmUmAfCmUmsCmsUm-3' (SEQ ID NO: 5); 5'-AmsGfsAfGmUmAfAmAmCmAfUmCmUmUfGmGfGmUmGmsGmsUm-3' (SEQ ID NO:6); iii) QLS0429m3: 5'-CmsAmsCmCmCmAmAfGfAfUmGmUmUmUmAmCmUmCmUm-3' (SEQ ID NO: 7); 5'-AmsGfsAmGmUmAfAmAmCmAmUmCmUmUfGmGfGmUmGmGmUm-3' (SEQ ID NO: 8); iv) QLS0429m4: 5'-CmsAmsCmCmCfAmAfGfAfUmGmUmUmUmAmCmUmsCmsUm-3' (SEQ ID NO:9); 5'-AmsGfsAmGfUmAmAmAmCmAmUmCmUmUfGmGfGmUmGmsGmsUm-3' (SEQ ID NO:10).

[0019] In some embodiments, the length of the sense strand is 17-21 modified nucleotides, and the length of the antisense strand is 17-23 modified nucleotides.

[0020] In some embodiments, the sense strand and / or antisense strand further comprises a dangling sequence at the 3' end, the two dangling sequences being 1-3 nucleotides in length; the dangling sequences may be complementary to the corresponding position of the target mRNA, or may be independently selected from UU and dTdT, where dT represents thymine deoxyribonucleotide.

[0021] III. siRNA conjugates. In a third aspect, the present invention relates to an siRNA conjugate containing the aforementioned siRNA and a liver-targeting conjugate covalently linked to the siRNA; preferably, the conjugate is a GalNAc ligand, more preferably a trivalent GalNAc ligand L96, and is attached to the 3' end of the positive strand.

[0022] IV. Pharmaceutical Compositions, Pharmaceutical Uses, and Treatment Methods. This invention also provides a pharmaceutical composition comprising the aforementioned siRNA or siRNA conjugate, and a pharmaceutically acceptable carrier.

[0023] The present invention also provides the use of the siRNA or siRNA conjugate and pharmaceutical composition described above in the preparation of medicaments for the treatment and / or prevention of thrombotic diseases and / or ischemic stroke.

[0024] The present invention also provides a method for inhibiting the expression of coagulation factor XI (F11) gene in cells in vitro, comprising contacting cells expressing the F11 gene with an effective amount of the aforementioned siRNA, siRNA conjugate and / or pharmaceutical composition. Attached Figure Description

[0025] Figure 1 .GalNAc represents the structure of compound L96; Figure 2 A schematic diagram of the siRNA conjugate with GalNAc. Detailed Implementation Plan

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. In this disclosure, FXI mRNA refers to mRNA having the sequence shown in GenBank accession number NM_000128.4. Further, unless otherwise specified, the term "target gene" as used in this disclosure refers to the gene that transcribes the aforementioned FXI mRNA, and the term "target mRNA" refers to the aforementioned FXI mRNA.

[0027] In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, each base on one strand pairs with a base on the other strand in a complementary manner. In DNA, adenine (A) pairs with thymine (T); in RNA, adenine (A) pairs with uracil (U); and guanine (G) pairs with cytosine (C). Correspondingly, "mismatch" refers to a base pairing at a corresponding position that is not complementary.

[0028] In the foregoing and hereinafter, particularly in the description of the preparation methods of siRNA, siRNA-containing pharmaceutical compositions, or siRNA conjugates of this disclosure, unless otherwise specified, the nucleoside monomer refers to a modified or unmodified nucleoside phosphorus amide monomer used in phosphorus amide solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphorus amide solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0029] In the context of this disclosure, unless otherwise stated, "conjugation" means the covalent connection between two or more chemical parts, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection between the respective chemical parts. Further, "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts having a specific function to siRNA.

[0030] As used herein, “treatment” refers to a method of obtaining a beneficial or desired outcome, including but not limited to treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby resulting in observed improvement in the subject.

[0031] As used herein, “prevention” refers to methods for obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” siRNA, siRNA conjugates, or pharmaceutical compositions may be given to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0032] Example 1: Design and Synthesis of QLS0429 siRNA Molecule The design of the QLS0429 sequence was based on bioinformatics analysis and experimental screening of human FXI / F11 mRNA (GenBank accession number NM_000128.4). First, the F11 mRNA sequence was scanned using the siRNA design algorithm to identify potential target regions; then, combined with target accessibility and off-target risk analysis, target sequences located at positions 265-283 of human F11 mRNA were screened.

[0033] The human target sequence of QLS0429 is located at positions 265-283 of human F11 mRNA (NM_000128.4), corresponding to the sequence 5'-CACCCAAGAUGUUUACUCU-3'. This region is a perfect match on human F11 mRNA and is suitable for evaluating the inhibitory activity of human F11 expression. Cross-species homology should be based on the specific species' F11 transcript; whether it applies to non-human primates and rodents should not be inferred solely from the human sequence.

[0034] The QLS0429 siRNA molecule was synthesized using a standard solid-phase phosphoramide chemical synthesis method, with the specific steps as follows: (1) Synthesis of the positive strand: Synthesized on a DNA synthesizer in the 3'→5' direction, the sequence is shown in SEQ ID NO:1. Standard RNA phosphoramide monomers were used during the synthesis process, and a dTdT hanger was added to the 3' end in the final step; (2) Synthesis of antisense strand: The same solid-phase synthesis method was used. The sequence is shown in SEQ ID NO:2. The 5' end is phosphorylated and dTdT hanger is added to the 3' end. (3) Purification and annealing: The synthesized single-stranded RNA was purified to a purity of over 95% by HPLC, and then heated at 95°C for 5 minutes in annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl), and then slowly cooled to room temperature to form double-stranded siRNA.

[0035] QLS0429: 5'-CACCCAAGAUGUUUACUCU-3' (SEQ ID NO: 1); 5'-AGAGUAAACAUCUUGGGUG-3' (SEQ ID NO: 2).

[0036] Example 2: Design and synthesis of modified siRNA Based on a deep understanding of the siRNA-RISC interaction mechanism, we chemically modified QLS0429. The modification principles are as follows: (1) The passenger strand can withstand more modifications without affecting its activity; (2) The 5' end and central region of the guide strand are crucial for RISC binding, and modifications should be made with caution; (3) The 3' end overhang area can be heavily modified to improve stability.

[0037] Table 1. List of modified siRNA sequences QLS0429m1 SEQ ID NO:3 CmsAmsCmCmCmAmAfGmAfUmGfUmUmUmAmCmUmsCmsUm SEQ ID NO:4 AmsGfsAmGmUmAmAfAmCmAmUmCfUmUfGmGfGmUmGmsGmsUm QLS0429m2 SEQ ID NO:5 CmsAmsCmCmCmAmAfGfAfUmGmUmUmUmAfCmUmsCmsUm SEQ ID NO:6 AmsGfsAfGmUmAfAmAmCmAfUmCmUmUfGmGfGmUmGmsGmsUm QLS0429m3 SEQ ID NO:7 CmsAmsCmCmCmAmAfGfAfUmGmUmUmUmAmCmUmCmUm SEQ ID NO:8 AmsGfsAmGmUmAfAmAmCmAmUmCmUmUfGmGfGmUmGmGmUm QLS0429m4 SEQ ID NO:9 CmsAmsCmCmCfAmAfGfAfUmGmUmUmUmAmCmUmsCmsUm SEQ ID NO:10 AmsGfsAmGfUmAmAmAmCmAmUmCmUmUfGmGfGmUmGmsGmsUm Wherein: the uppercase letters “G”, “C”, “A”, and “U” typically represent ribonucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively; “dT” represents thymine deoxyribonucleotide; Am, Um, Cm, and Gm represent 2'-methoxy modified adenosine, 2'-methoxy modified uridine, 2'-methoxy modified cytosine, and 2'-methoxy modified guanosine, respectively; Af, Gf, Cf, and Uf represent 2'-fluoro modified adenosine, 2'-fluoro modified guanosine, 2'-fluoro modified cytosine, and 2'-fluoro modified uridine, respectively; the lowercase letter s indicates that the two nucleotides adjacent to the letter s are linked by thiophosphate groups.

[0038] Example 3: Preparation of QLS0429m1L96 GalNAc Coupling The modified siRNA molecules shown in Table 1 were synthesized using the solid-phase phosphoramide method according to the procedure in Example 1, with an L96-type GalNAc conjugate group introduced at the 3' end of the positive strand. The synthesis method of L96 can be found in the published literature by Jayaprakash K. Nair et al. (J. Am. Chem. Soc. 2014, 136, 16958-16961). Subsequently, the siRNA-L96 conjugate shown in Table 2 was prepared. Figure 1 GalNAc represents the structure of compound L96; Figure 2 This is a schematic diagram of the siRNA conjugate with GalNAc.

[0039] Table 2 List of GalNAc-conjugated modified siRNA sequences QLS0429m1L96 CmsAmsCmCmCmAmAfGmAfUmGfUmUmUmAmCmUmsCmsUm-L96 AmsGfsAmGmUmAmAfAmCmAmUmCfUmUfGmGfGmUmGmsGmsUm QLS0429m2L96 CmsAmsCmCmCmAmAfGfAfUmGmUmUmUmAfCmUmsCmsUm-L96 AmsGfsAfGmUmAfAmAmCmAfUmCmUmUfGmGfGmUmGmsGmsUm QLS0429m3L96 CmsAmsCmCmCmAmAfGfAfUmGmUmUmUmAmCmUmCmUm-L96 AmsGfsAmGmUmAfAmAmCmAmUmCmUmUfGmGfGmUmGmGmUm QLS0429m4L96 CmsAmsCmCmCfAmAfGfAfUmGmUmUmUmAmCmUmsCmsUm-L96 AmsGfsAmGfUmAmAmAmCmAmUmCmUmUfGmGfGmUmGmsGmsUm This indicates the modified siRNA conjugated to the conjugation group L96. The other letters in the sense and antisense strand sequences in Table 2 have the same meaning as the corresponding letters in Table 1.

[0040] Example 4: In vitro activity evaluation 4.1 Cell Culture and Transfection. In vitro cell viability was evaluated using the HepG2 human hepatocellular carcinoma cell line: (1) Cell culture: HepG2 cells were cultured in DMEM medium (containing 10% FBS) at 37°C and 5% CO2. (2) siRNA transfection: siRNA (final concentration 10 nM) was transfected into cells using Lipofectamine RNAiMAX transfection reagent.

[0041] 4.2 FXI mRNA level detection. Total RNA was extracted 48 hours after transfection, and the FXI mRNA level was detected by qRT-PCR: (1) RNA extraction: Total RNA was extracted using TRIzol reagent; (2) Reverse transcription: cDNA synthesis was performed using random primers; (3) qRT-PCR: SYBR Green dye was used, with GAPDH as the internal reference gene.

[0042] For each qPCR reaction system, 2 μL of cDNA was used as a template, and 5 μL of the reagents provided in the kit were added to prepare the qPCR reaction system. The PCR primers used to amplify the target gene FXI and the internal reference gene GAPDH were purchased from Thermo's website, with a final concentration of 0.25 μM for each primer. Finally, nuclease-free water was added, bringing the final PCR reaction volume to 10 μL. The reaction conditions were: 95°C pre-denaturation for 10 min, followed by 95°C denaturation for 15 seconds, annealing and extension at 60°C for 1 minute, and repeating the above denaturation, annealing and extension process for a total of 40 cycles.

[0043] Primer sequences: FXI forward primer: 5'-TGCCTGTATAAGTGGCTGAA-3', sequence ID is SEQ ID NO:11; FXI reverse primer: 5'-AGGCTGTTGAAGGTCTTGAG-3', sequence ID is SEQ ID NO:12; GAPDH forward primer: 5'-GAAGGTGAAGGTCGGAGTC-3', sequence ID is SEQ ID NO:13; GAPDH reverse primer: 5'-GAAGATGGTGATGGGATTTC-3', sequence ID is SEQ ID NO:14.

[0044] Table 3. Results of in vitro bioactivity assay QLS0429 SEQ ID NO:1 SEQ ID NO:2 77.62% 64.92% QLS0429m1 SEQ ID NO:3 SEQ ID NO:4 96.02% 75.20% QLS0429m2 SEQ ID NO:5 SEQ ID NO:6 73.79% 65.61% QLS0429m3 SEQ ID NO:7 SEQ ID NO:8 82.62% 76.94% QLS0429m4 SEQ ID NO:9 SEQ ID NO:10 79.26% 75.2% Example 5: Free uptake experiment of nucleic acid conjugates in human primary hepatocytes 5.1 Freedom of uptake assay. Cells were diluted to a density of 6*10^4 / ml using cell culture medium, and then seeded into 96-well plates at a density of 90 μl per well. The nucleic acid conjugate was diluted to a 10X stock solution with nuclease-free water, and 10 μl was added to each well of the 96-well plate. The 96-well plates containing the nucleic acid conjugate were incubated at 37°C for 48 h before detection. Intracellular RNA was extracted according to the standard procedure manual of the kit (Qiagen-74182).

[0045] 5.2 qPCR detection was performed according to the method in Example 4, and the experimental results are shown in Table 4.

[0046] Table 4. Freedom of uptake test of human liver cells QLS0429m1L96 75.7% 61.0% Example 6: In vivo pharmacodynamic evaluation In vivo pharmacodynamic evaluation was conducted using 8-10 week old male hFXI transgenic mice or animal models expressing human F11: (1) Grouping: The participants were randomly divided into a control group, a positive control group (3 mg / kg) of siRNA-GalNAc conjugate, and a negative control group (physiological saline). The positive control group was administered the following conjugate obtained by conjugating siRNA with GalNAc (dose 3 mg / kg): the sense strand sequence is shown in SEQ ID NO:15 (AmsUmsUmUmCmUmGfGfGfUmAmUmUmCmUmUmUmCmAm), and the antisense strand sequence is shown in SEQ ID NO:16 (PUmsGfsAmAmAmGfAmAmUmAmCmCmCmAfGmAfAmUmsCmsGm, where P indicates that the adjacent nucleotide on the right is a 5'-phosphate nucleotide). The positive control sequence is referred to SEQ ID NO:373 and 374 of CN118256498 A. (2) Administration method: subcutaneous injection, single administration.

[0047] (3) Detection time points: 1, 3, 7, 14, 21 and 28 days after administration.

[0048] Blood samples were collected at various time points: (1) Blood collection: Blood was collected from the orbital venous plexus and anticoagulated with EDTA; (2) Plasma separation: Centrifuge at 3000 rpm for 10 minutes to separate the plasma; (3) Sample preservation: freeze at -80°C until testing.

[0049] Mouse plasma FXI protein levels were detected using ELISA. (1) Standard curve: A standard curve was prepared using recombinant mouse FXI protein; (2) Sample testing: Plasma samples were appropriately diluted before testing; (3) Data analysis: Calculate the percentage of inhibition relative to the baseline level.

[0050] The results of in vivo experiments are shown in Table 5. QLS0429m1L96(3mg / kg) 92.1% 2.4% 94.5% 0.2% 93.6% 0.7% 90.1% 1.1% PC-L96 (3mg / kg) 68.7% 8.0% 64.5% 11.2% 61.1% 5.8% 54.9% 7.6%

Claims

1. A small interfering nucleic acid (siRNA) for inhibiting the expression of coagulation factor XI (F11), comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially inversely complementary to form a double-stranded region; wherein, The sense strand consists of the nucleotide sequence 5'-CACCCAAGAUGUUUACUCU-3' shown in SEQ ID NO:1, and the antisense strand consists of the nucleotide sequence 5'-AGAGUAAACAUCUUGGGUG-3' shown in SEQ ID NO:

2. The antisense strand is complementary to the target region 265-283 of human F11 mRNA (GenBank accession number NM_000128.4) and can reduce the expression level of F11 mRNA in cells.

2. The siRNA according to claim 1, characterized in that, The sense strand and / or antisense strand further comprises a dangling sequence at the 3' end, each dangling sequence being 1-3 nucleotides in length, and the dangling sequence being selected from UU or dTdT, where dT represents thymine deoxyribonucleotide.

3. A modified siRNA for inhibiting the expression of coagulation factor XI (F11), comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are selected from any one of the following groups: (i) SEQ ID NO:3 and SEQ ID NO:4; (ii) SEQ ID NO:5 and SEQ ID NO:6; (iii) SEQ ID NO:7 and SEQ ID NO:8; (iv) SEQ ID NO:9 and SEQ ID NO:10; wherein, Am, Um, Cm, and Gm represent 2'-O-methyl modified A, U, C, and G nucleotides, respectively; Af, Uf, Cf, and Gf represent 2'-fluorine modified A, U, C, and G nucleotides, respectively; the letter 's' indicates that the two adjacent nucleotides are linked by a phosphate thioester.

4. The modified siRNA according to claim 3, characterized in that, The modified siRNA is QLS0429m1, which consists of the sense strand shown in SEQ ID NO:3 and the antisense strand shown in SEQ ID NO:

4.

5. The siRNA according to any one of claims 1-4, characterized in that, The sense strand is 19-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; and the sense strand and / or antisense strand contains 2'-O-methyl modification, 2'-fluorine modification, and / or thiophosphate linkage.

6. An siRNA conjugate comprising the siRNA of any one of claims 1-5 and a liver-targeting ligand covalently linked to the siRNA.

7. The siRNA conjugate according to claim 6, characterized in that, The liver-targeting ligand is an N-acetylgalactosamine (GalNAc) ligand capable of binding to the desialyl glycoprotein receptor (ASGPR), and the ligand is a trivalent GalNAc ligand L96 attached to the 3' end of the sense strand.

8. The siRNA conjugate according to claim 7, characterized in that, The siRNA conjugate is QLS0429m1L96, QLS0429m2L96, QLS0429m3L96 or QLS0429m4L96.

9. A pharmaceutical composition comprising siRNA of any one of claims 1-5 or siRNA conjugate of any one of claims 6-8, and a pharmaceutically acceptable carrier, diluent or excipient.

10. Use of the siRNA of any one of claims 1-5, the siRNA conjugate of any one of claims 6-8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for reducing the expression or activity of coagulation factor XI in a subject, said medicament for the treatment and / or prevention of thrombotic diseases, thromboembolic diseases, or ischemic stroke associated with coagulation factor XI.

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