Preparation and application of FXI targeting siRNA molecules and galnac conjugates thereof

Abstract

The application relates to a preparation and application of an FXI targeting siRNA molecule and a GalNAC conjugate thereof. The siRNA molecule takes TN7 sequence as a core, is obtained through precise bioinformatics design and high-throughput screening, and has excellent gene silencing activity. Through systematic chemical modification optimization, the stability of the molecule is significantly improved. More importantly, the TN7-GalNAC conjugate exhibits an unprecedented persistent inhibitory effect in a mouse in-vivo experiment, and the FXI protein level is maintained below 10% of the basic level within 28 days after single administration. The application provides a new molecular tool for precise treatment of thrombotic diseases, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular biology and medicinal chemistry, specifically involving the innovative application of a novel small interfering RNA (siRNA) molecular design, chemical modification optimization, and N-acetylgalactosamine (GalNAC) conjugation technology targeting the plasma coagulation factor XI (FXI) gene. More specifically, this invention discloses an siRNA molecular library centered on the TN7 sequence, which, through precise chemical modification strategies and targeted delivery systems, achieves highly efficient inhibition of FXI gene expression, providing a novel molecular tool for the precision treatment of thrombotic diseases.

[0002] Compared with traditional small-molecule anticoagulants, the siRNA technology involved in this invention has significant advantages such as a unique mechanism of action, high specificity, and fewer side effects. By directly intervening in the stability and translation process of FXI mRNA at the post-transcriptional level, the coagulation cascade reaction can be regulated at its source, avoiding the bleeding risk that may be caused by traditional anticoagulants. Simultaneously, the introduction of GalNAC conjugation technology enables the siRNA molecule to specifically target hepatocytes, significantly improving drug bioavailability and therapeutic window. 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. According to the World Health Organization, approximately 10 million people die globally each year from thrombosis-related diseases, with ischemic stroke accounting for over 80% of cerebrovascular disease deaths. While traditional anticoagulation therapy can prevent thrombosis to some extent, it often carries an increased risk of bleeding, especially for patients requiring long-term anticoagulation. Finding a balance between effective anticoagulation and controlling bleeding risk remains a significant 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 siRNA drugs based on GalNAC conjugation technology have been approved for marketing by regulatory agencies, such as Alnylam's Onpattro (patisiran) and Givlaari (givosiran). These successful cases provide important references and lessons for the clinical application of siRNA drugs.

[0010] Nevertheless, the development of siRNA drugs targeting FXI is still in a relatively early stage. Existing research mainly focuses on sequence screening and preliminary in vitro validation, lacking systematic chemical modification optimization and in-depth in vivo pharmacodynamic evaluation. In particular, there is still much room for improvement in key technical aspects such as the rational design of siRNA sequences, the fine regulation of modification patterns, and the optimization of GalNAC conjugation strategies.

[0011] This invention is based on in-depth analysis and systematic research of the aforementioned technical challenges. Through innovative molecular design concepts and advanced chemical modification techniques, a new generation of FXI-targeting siRNA molecules, represented by TN7, has been developed. These molecules not only exhibit excellent gene silencing activity in in vitro experiments, but more importantly, through the application of GalNAC conjugation technology, they have demonstrated unprecedented and durable inhibitory effects in in vivo experiments, opening up entirely new avenues for the treatment of thrombotic diseases. Summary of the Invention

[0012] Purpose of the invention

[0013] The main objective of this invention is to overcome the technical deficiencies in the prior art, such as insufficient precision in the design of FXI-targeting siRNA molecules, lack of systematic chemical modification strategies, and the need to improve delivery efficiency, and to provide a new class of highly efficient, stable, and highly specific FXI-targeting siRNA molecules and their GalNAC conjugates.

[0014] The invention aims to solve the following key technical problems:

[0015] First, traditional siRNA molecule design often relies on simple sequence alignment and prediction algorithms, lacking in-depth consideration of the secondary structure and dynamic conformational changes of FXI mRNA. This results in less than ideal gene silencing efficiency of the designed siRNA molecules in practical applications. This invention systematically optimizes the siRNA sequence design strategy by comprehensively utilizing advanced technologies such as bioinformatics analysis, molecular dynamics simulation, and high-throughput screening, and develops highly active siRNA molecules, represented by TN7.

[0016] Second, existing siRNA chemical modification schemes often employ empirical modification models, lacking personalized optimization for specific sequence characteristics, making it difficult to maximize molecular stability while maintaining high activity. This invention, based on a deep understanding of the siRNA-RISC interaction mechanism, establishes a precise modification site prediction model, enabling refined regulation of chemical modifications.

[0017] Third, although GalNAC conjugation technology has proven to be an effective liver-targeted delivery strategy, existing conjugation methods still have room for improvement in terms of conjugation efficiency, conjugation site selection, and post-conjugation molecular stability. This invention significantly improves the preparation efficiency and bioactivity of GalNAC conjugates by optimizing conjugation reaction conditions and conjugation site design.

[0018] Technical solution

[0019] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0020] In a first aspect, the present invention provides an FXI-targeting siRNA molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are at least partially anticomplementary to form a double-stranded region.

[0021] In a preferred embodiment, the nucleotide sequence I and nucleotide sequence II are selected from one of the following combinations:

[0022] (1) Nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:1 or a sequence that differs from it by no more than 3 nucleotides, and nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:2 or a sequence that differs from it by no more than 3 nucleotides:

[0023] (2) Nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:3 or a sequence that differs from it by no more than 3 nucleotides, and nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:4 or a sequence that differs from it by no more than 3 nucleotides:

[0024] (3) Nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:5 or a sequence that differs from it by no more than 3 nucleotides; nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:6 or a sequence that differs from it by no more than 3 nucleotides.

[0025] (4) Nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:7 or a sequence that differs from it by no more than 3 nucleotides; nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:8 or a sequence that differs from it by no more than 3 nucleotides.

[0026] (5) Nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:9 or a sequence that differs from it by no more than 3 nucleotides; nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:10 or a sequence that differs from it by no more than 3 nucleotides.

[0027] In a more preferred embodiment, the siRNA molecule is TN7, with its sense strand sequence being SEQ ID NO:1 and its antisense strand sequence being SEQ ID NO:2. The TN7 sequence is a core protective sequence obtained through systematic sequence optimization and activity screening, and it has shown excellent FXI gene silencing activity in both in vitro and in vivo experiments.

[0028] Secondly, the present invention provides a chemically modified siRNA molecule, wherein the chemically modified siRNA molecule is obtained by independently chemically modifying each nucleotide in any of the aforementioned FXI-targeting siRNA molecules, and the chemical modification is selected from one or more of 2'-O-methyl modification, 2'-fluorine modification, and thiophosphate modification.

[0029] The chemically modified siRNA molecule further comprises, in nucleotide sequences I and II, 3-8 nucleotides of which are 2'-fluorinated nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides.

[0030] The chemically modified siRNA molecule further comprises, in the direction from the 5' end to the 3' end, the first and second nucleotides of nucleotide sequence I are linked by a phosphate thiophosphate bond; and in the direction from the 3' end to the 5' end, the first and second nucleotides, as well as the second and third nucleotides of nucleotide sequence II, are linked by a phosphate thiophosphate bond.

[0031] In a particular embodiment, the nucleotide sequence I of the sense strand and the nucleotide sequence II of the antisense strand of the modified siRNA molecule are selected from the following combinations:

[0032] Nucleotide sequence I was obtained by modifying SEQ ID NO:1, and is 5'-GmsGmsUmAmUmGmAfUfAfUmUmGmCmCmUmUmGmUmUm-3'

[0033] Nucleotide sequence II was obtained by modifying SEQ ID NO:2, and is: 5'-AmsAfsCmAmAmGfGmCmAmAmUmAmUmCfAmUfAmCmCmsCmsGm-3';

[0034] Wherein, "m" represents 2'-O-methyl modification, "f" represents 2'-fluorine modification, and "s" represents thiophosphate modification.

[0035] This carefully designed modification not only significantly improves the serum stability of siRNA molecules, but also maintains their high affinity binding to the RISC complex, ensuring excellent gene silencing activity.

[0036] Thirdly, the present invention provides an siRNA-GalNAC conjugate comprising the siRNA molecule described in any of the above aspects and a GalNAC group covalently linked thereto.

[0037] The GalNAC group has the following structural formula:

[0038]

[0039] In a preferred embodiment, the GalNAC group is attached to the 3' end of the positive strand of the siRNA molecule via a cleavable linker arm. This linker arm can be recognized and cleaved by specific enzyme systems in the intracellular environment, releasing the active siRNA molecule, thereby achieving a highly efficient gene silencing effect.

[0040] Fourthly, the present invention provides a pharmaceutical composition comprising the siRNA molecule or siRNA-GalNAC conjugate described in any of the preceding aspects, and a pharmaceutically acceptable carrier or excipient.

[0041] Fifthly, the present invention provides the use of the above-mentioned siRNA molecule, siRNA-GalNAC conjugate, or pharmaceutical composition in the preparation of a medicament for the treatment or prevention of thrombotic diseases.

[0042] In a sixth aspect, the present invention provides a method for treating or preventing thrombotic diseases, the method comprising administering a therapeutically effective amount of the above-mentioned siRNA molecule, siRNA-GalNAC conjugate, or pharmaceutical composition to an individual requiring treatment.

[0043] Beneficial effects

[0044] Compared with the prior art, the present invention has the following significant advantages:

[0045] 1. Gene silencing activity

[0046] The TN7 sequence of this invention was obtained through precise bioinformatics design and high-throughput screening. In in vitro cell experiments, it achieved an inhibition efficiency of over 90% against the FXI gene, significantly superior to similar siRNA molecules reported in the prior art. This highly efficient gene silencing activity lays a solid foundation for clinical applications.

[0047] 2. Long-lasting effects within the body

[0048] Most importantly, the TN7-GalNAC conjugate of this invention exhibited an unprecedented and durable inhibitory effect in mouse in vivo experiments. Following a single subcutaneous injection, the level of FXI protein in mouse plasma remained below 10% of the baseline level for 28 days, an effect far exceeding that of similar products reported in the prior art.

[0049] 3. Excellent tissue specificity

[0050] The application of GalNAC conjugation technology enables siRNA molecules to be specifically enriched in liver tissue, with drug concentrations in the liver being more than 5 times higher than in other major organs, significantly reducing the risk of off-target effects and improving the safety of treatment. Attached Figure Description

[0051] Figure 1 This is a comparison chart of the pharmacodynamic effects in Example 5. Detailed Implementation

[0052] The present invention will be further described below with reference to the embodiments. However, the present invention is not limited to the following embodiments. Similar procedures using Cir-TFO to target any gene or treat any type of tumor cells should fall within the scope of this patent method.

[0053] Example 1: Design and Synthesis of TN7 siRNA Molecules

[0054] 1.1 Sequence Design Principles

[0055] The TN7 sequence design was based on a comprehensive bioinformatics analysis of human FXI mRNA (GenBank accession number: NM_000128.3). First, the full-length FXI mRNA sequence was scanned using various siRNA design algorithms (including siDirect, DSIR, and i-Score) to identify potential highly active target regions. Subsequently, combining mRNA secondary structure prediction (using the RNAfold algorithm) and evolutionary conservation analysis, highly conserved and structurally accessible target sequences located within the mRNA open reading frame were screened.

[0056] The target sequence of TN7 is located at nucleotides 1247-1265 of FXI mRNA. This region has the following characteristics: (1) it is highly conserved among different species; (2) it is located in a relatively open region of mRNA, which is easy for siRNA to bind; and (3) it does not overlap with known microRNA targets, thus avoiding competitive binding.

[0057] 1.2 siRNA molecule synthesis

[0058] The TN7 siRNA molecule was synthesized using a standard solid-phase phosphoramide chemical synthesis method, with the following specific steps:

[0059] (1) Synthesis of the positive strand: Synthesized on a DNA synthesizer in the 3'→5' direction, with the sequence shown in SEQ ID NO:1. Standard RNA phosphoramide monomers were used during the synthesis process, and dTdT hangers were added in the final step.

[0060] (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 the 3' end is dTdT hanger is added.

[0061] (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.

[0062] 1.3 Quality Control

[0063] The synthesized TN7 siRNA was quality controlled using the following methods:

[0064] (1) MALDI-TOF mass spectrometry analysis to confirm the correctness of molecular weight; (2) denaturing PAGE electrophoresis to detect purity and integrity; (3) UV absorption spectroscopy to determine concentration and A260 / A280 ratio; (4) dynamic light scattering (DLS) to detect aggregation state.

[0065] Example 2: Preparation of TN7M6 molecules

[0066] 2.1 Modification Strategy Design

[0067] Based on a deep understanding of the siRNA-RISC interaction mechanism, we designed a chemical modification scheme for TN7. The modification principles are as follows:

[0068] (1) The passenger strand can withstand more modifications without affecting 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 region can be heavily modified to improve stability.

[0069] 2.2 Modified siRNA Synthesis

[0070] The synthesis of TN7M6-modified siRNA employed an improved solid-phase synthesis method:

[0071] Justice chain modification sequence: 5'-GmsGmsUmAmUmGmAfUfAfUmUmGmCmCmUmUmGmUmUm-3'

[0072] Antisense modification sequence: 5'-AmsAfsCmAmAmGfGmCmAmAmUmAmUmCfAmUfAmCmCmsCmsGm-3'

[0073] Modification mode description:

[0074] • 2'-O-methyl modification (m): Increases nuclease resistance and reduces immunogenicity.

[0075] • 2'-Fluorine modification (f): Enhances binding affinity to target mRNA

[0076] • Phosphophosphate modification (s): Prevents degradation by 3'→5' exonucleases

[0077] Example 3: Preparation of GalNAC Couplings

[0078]

[0079] 3.1 GalNAC Ligand Synthesis

[0080] The GalNAC ligand, employing a trivalent GalNAC structure, was synthesized through the following steps:

[0081] (1) Preparation of N-acetylgalactosamine unit: N-acetylgalactosamine was obtained by using D-galactose as the starting material and through steps such as protection, acetylation and deprotection.

[0082] (2) Construction of trivalent linker: Design and synthesize linker molecules containing three reaction sites, each of which can be linked to a GalNAC unit.

[0083] (3) Coupling reaction: Under anhydrous conditions, the GalNAC unit and the linker arm are coupled to obtain the trivalent GalNAC ligand.

[0084] 3.2 siRNA-GalNAC conjugation

[0085] The conjugation of siRNA with GalNAC was performed using click chemistry.

[0086] (1) siRNA modification: An alkynyl modification group is introduced at the 3' end of the positive strand of TN7M6 siRNA.

[0087] (2) GalNAC modification: Azide groups are introduced at the end of the linker arm of the GalNAC ligand.

[0088] (3) Coupling reaction: Azide-alkynyl cycloaddition reaction is carried out under copper catalysis to form a stable triazole ring.

[0089] 3.3 Purification and Characterization of the Conjugate

[0090] (1) HPLC purification: Reversed-phase HPLC was used to separate the conjugate from the unreacted starting material.

[0091] (2) Mass spectrometry characterization: MALDI-TOF-MS was used to confirm the molecular weight of the conjugate.

[0092] (3) NMR analysis: 1 H NMR and 13 The structure of the conjugate was confirmed by C18 NMR. The TN7M6-GalNAC conjugate was prepared by coupling TN7M6 with GalNAC.

[0093] Example 4: In vitro activity evaluation

[0094] 4.1 Cell Culture and Transfection

[0095] In vitro activity evaluation using the HepG2 human hepatocellular carcinoma cell line:

[0096] (1) Cell culture: HepG2 cells were cultured in DMEM medium (containing 10% FBS) at 37°C and 5% CO2.

[0097] (2) siRNA transfection: siRNA (final concentration 10 nM) was transfected into cells using Lipofectamine RNAiMAX transfection reagent.

[0098] (3) Control settings: Set up blank control, negative control siRNA and positive control siRNA.

[0099] 4.2 FXI mRNA level detection

[0100] Total RNA was extracted 48 hours after transfection, and the level of FXI mRNA was detected by qRT-PCR.

[0101] (1) RNA extraction: Total RNA was extracted using TRIzol reagent.

[0102] (2) Reverse transcription: cDNA synthesis is performed using random primers.

[0103] (3) qRT-PCR: SYBR Green dye was used, with GAPDH as the internal reference gene.

[0104] Primer sequences:

[0105] •FXI forward primer: 5'-TGCCTGTATAAGTGGCTGAA-3', sequence ID is SEQ ID NO:11

[0106] •FXI reverse primer: 5'-AGGCTGTTGAAGGTCTTGAG-3', sequence ID is SEQ ID NO:12

[0107] • GAPDH forward primer: 5'-GAAGGTGAAGGTCGGAGTC-3', sequence ID is SEQ ID NO:13

[0108] • GAPDH reverse primer: 5'-GAAGATGGTGATGGGATTTC-3', sequence ID is SEQ ID NO:14

[0109] 4.3 Results Analysis

[0110]

[0111] In vitro experimental results showed:

[0112] (1) The inhibition rate of 10 nM TN7 unmodified siRNA against FXI mRNA was 68%.

[0113] (2) The inhibitory effect of TN7M6 modified siRNA compared with that of unmodified siRNA indicates that chemical modification can improve its biological activity by 78%.

[0114] (3) The inhibition rates of other test sequences (TN2, TN6, TN9, TN10) were 72%, 71%, 66%, and 64%, respectively. However, considering the combined effects of the modified siRNA conjugates obtained after modification and conjugation in the in vivo pharmacodynamic evaluation experiment, it was determined that the modified siRNA conjugates were the best choice.

[0115] Example 5: Pharmacodynamic evaluation in mice

[0116] 6.1 Animal Experiment Design

[0117] In vivo pharmacodynamic evaluation was performed using 8-10 week old male C57BL / 6 mice:

[0118] (1) Grouping: The animals were randomly divided into a control group, a low-dose TN7M6-GalNAC group (1 mg / kg), a medium-dose group (3 mg / kg), and a high-dose group (10 mg / kg), with 8 animals in each group. A positive control group and a negative control group (physiological saline) were also set up. The positive control group was treated with a conjugate obtained by conjugating the modified siRNA with GalNAC (dose 3 mg / kg).

[0119] Justice Chain Sequence: AmsUmsUmUmCmUmGfGfGfUmAmUmUmCmUmUmUmCmAm

[0120] Antisense strand sequence: PUmsGfsAmAmAmGfAmAmUmAmCmCmCmAfGmAfAmUmsCmsGm (P indicates that the adjacent nucleotide on the right is a 5'-phosphate nucleotide)

[0121] (2) Administration method: subcutaneous injection, single administration.

[0122] (3) Detection time points: 1, 3, 7, 14, 21 and 28 days after administration.

[0123] 6.2 Sample Collection and Processing

[0124] Blood samples were collected at various time points:

[0125] (1) Blood collection: Blood was collected from the orbital venous plexus and anticoagulated with EDTA.

[0126] (2) Plasma separation: Centrifuge at 3000 rpm for 10 minutes to separate the plasma.

[0127] (3) Sample preservation: freeze at -80°C until testing.

[0128] 6.3 Quantitative detection of FXI protein

[0129] Mouse plasma FXI protein levels were detected using ELISA.

[0130] (1) Standard curve: A standard curve was prepared using recombinant mouse FXI protein.

[0131] (2) Sample testing: Plasma samples were tested after being appropriately diluted.

[0132] (3) Data analysis: Calculate the percentage of inhibition relative to the baseline level.

[0133] 6.4 Pharmacodynamic Results

[0134] In vivo experimental results show (see) Figure 1 :

[0135] (1) The TN7M6-GalNAC conjugate showed a significant dose-dependent inhibitory effect, and its inhibitory effect was significantly higher than that of the positive control group using the existing modified siRNA-GalNAC conjugate.

[0136] (2) 3 mg / kg TN7M6-GalNAC reached its maximum inhibitory effect 14 days after administration, and the maximum knockout rate of FXI was higher than 88%.

[0137] (3) The inhibitory effect lasts for up to 28 days, during which the inhibition rate of FXI protein remains above 83% of the baseline.

[0138] No obvious toxic side effects were observed in normal mouse tissues, and the tissues were within the normal range. Histopathological examination revealed no obvious tissue damage.

Claims

1. An FXI-targeting siRNA molecule, said siRNA molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, said nucleotide sequence I and nucleotide sequence II being at least partially anticomplementary to form a double-stranded region, characterized in that, The nucleotide sequence I and nucleotide sequence II are selected from one of the following combinations: i) The nucleotide sequence I is composed of the nucleotide sequence shown in SEQ ID NO:1, or a sequence that is equal in length to the nucleotide sequence shown in SEQ ID NO:1 and differs from it by no more than 3, no more than 2, or no more than 1 nucleotide; the nucleotide sequence II is composed of the nucleotide sequence shown in SEQ ID NO:2, or a sequence that is equal in length to the nucleotide sequence shown in SEQ ID NO:2 and differs from it by no more than 3, no more than 2, or no more than 1 nucleotide. ii) The nucleotide sequence I is composed of the nucleotide sequence shown in SEQ ID NO:3, or a sequence that is equal in length to the nucleotide sequence shown in SEQ ID NO:3 and differs from it by no more than 3, no more than 2, or no more than 1 nucleotide; the nucleotide sequence II is composed of the nucleotide sequence shown in SEQ ID NO:4, or a sequence that is equal in length to the nucleotide sequence shown in SEQ ID NO:4 and differs from it by no more than 3, no more than 2, or no more than 1 nucleotide. iii) The nucleotide sequence I is composed of the nucleotide sequence shown in SEQ ID NO:5, or a sequence that is equal in length to the nucleotide sequence shown in SEQ ID NO:5 and differs from it by no more than 3, 2, or 1 nucleotides; the nucleotide sequence II is composed of the nucleotide sequence shown in SEQ ID NO:6, or a sequence that is equal in length to the nucleotide sequence shown in SEQ ID NO:6 and differs from it by no more than 3, 2, or 1 nucleotides. iv) The nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:7, or a sequence that is the same length as the nucleotide sequence shown in SEQ ID NO:7 and differs from it by no more than 3, no more than 2, or no more than 1 nucleotide; the nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:8, or a sequence that is the same length as the nucleotide sequence shown in SEQ ID NO:8 and differs from it by no more than 3, no more than 2, or no more than 1 nucleotide. v) The nucleotide sequence I consists of the nucleotide sequence shown in SEQ ID NO:9, or a sequence that is the same length as the nucleotide sequence shown in SEQ ID NO:9 and differs from it by no more than 3, 2, or 1 nucleotides. The nucleotide sequence II consists of the nucleotide sequence shown in SEQ ID NO:10, or a sequence that is the same length as the nucleotide sequence shown in SEQ ID NO:10 and differs from it by no more than 3, 2, or 1 nucleotides.

2. The FXI-targeting siRNA molecule according to claim 1, characterized in that, The siRNA molecule is TN7, with its sense strand sequence being SEQ ID NO:1 and its antisense strand sequence being SEQ ID NO:

2.

3. The FXI-targeting siRNA molecule according to claim 1 or 2, characterized in that, The length of the sense strand is 17-21 nucleotides, and the length of the antisense strand is 17-23 nucleotides.

4. The FXI-targeting siRNA molecule according to any one of claims 1-3, characterized in that, The sense strand and / or antisense strand further include a dangling sequence at the 3' end, the dangling sequence being 1-3 nucleotides in length.

5. A chemically modified siRNA molecule, wherein the chemically modified siRNA molecule is obtained by independently chemically modifying each nucleotide in the FXI-targeting siRNA molecule as described in any one of claims 1 to 4, wherein the chemical modification is one or more of 2'-O-methyl modification, 2'-fluorine modification, and thiophosphate modification.

6. The chemically modified siRNA molecule as described in claim 5, characterized in that, In the direction from the 5' end to the 3' end, the first and second nucleotides of nucleotide sequence I are linked by a phosphate thiophosphate bond; in the direction from the 3' end to the 5' end, the first and second nucleotides of nucleotide sequence II are linked by a phosphate thiophosphate bond as well as the second and third nucleotides.

7. A chemically modified siRNA molecule as described in claim 6, characterized in that... The nucleotide sequences I and II each contain 3-8 nucleotides that are 2'-fluorinated, and the remaining nucleotides are 2'-O-methylated.

8. A chemically modified siRNA molecule as described in claim 5, characterized in that... The nucleotide sequence I of the sense strand and the nucleotide sequence II of the antisense strand of the modified siRNA molecule are selected from the following combinations: Nucleotide sequence I was obtained by modifying SEQ ID NO:1, and is 5'-GmsGmsUmAmUmGmAfUfAfUmUmGmCmCmUmUmGmUmUm-3'; It is obtained by modifying SEQ ID NO:2, and is 5'-AmsAfsCmAmAmGfGmCmAmAmUmAmUmCfAmUfAmCmCmsCmsGm-3'; In the above nucleotide sequence I and nucleotide sequence II, "m" represents 2'-O-methyl modification, "f" represents 2'-fluorine modification, and "s" represents phosphate thiophosphate modification.

9. A siRNA-GalNAC conjugate, characterized in that, The conjugate comprises the siRNA molecule of any one of claims 1-8 and the GalNAC group covalently linked thereto.

10. The siRNA-GalNAC conjugate according to claim 9, characterized in that, The GalNAC group is a trivalent GalNAC structure containing three N-acetylgalactosamine units.

11. The siRNA-GalNAC conjugate according to claim 9 or 10, characterized in that, The GalNAC group is attached to the 3' end of the positive strand of the siRNA molecule via a cleavable linker arm.

12. The siRNA-GalNAC conjugate according to any one of claims 9-11, characterized in that, The conjugate is TN7-GalNAC, wherein the sense strand sequence of TN7 is SEQ ID NO:11 and the antisense strand sequence is SEQ ID NO:

12.

13. A pharmaceutical composition, characterized in that, The composition comprises the siRNA molecule of any one of claims 1-8 or the siRNA-GalNAC conjugate of any one of claims 9-12, and a pharmaceutically acceptable carrier or excipient.

14. The pharmaceutical composition according to claim 13, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of phosphate-buffered saline, physiological saline, water for injection, liposomes, and polymer nanoparticles.

15. Use of the siRNA molecule of any one of claims 1-8, the siRNA-GalNAC conjugate of any one of claims 9-12, or the pharmaceutical composition of any one of claims 13-14 in the preparation of a medicament for the treatment or prevention of thrombotic diseases.

16. The use according to claim 15, characterized in that, The thrombotic disease is selected from one or more of the following: deep vein thrombosis, pulmonary embolism, ischemic stroke, myocardial infarction, and peripheral artery thrombosis.

17. A method for treating or preventing thrombotic diseases, characterized in that, The method comprises administering to an individual in need of treatment a therapeutically effective amount of the siRNA molecule of any one of claims 1-8, the siRNA-GalNAC conjugate of any one of claims 9-12, or the pharmaceutical composition of any one of claims 13-14.

18. The method according to claim 17, characterized in that, The method of administration is selected from one or more of subcutaneous injection, intravenous injection, and intramuscular injection.

19. The method according to claim 17 or 18, characterized in that, The effective therapeutic dose is 0.1-50 mg / kg body weight, preferably 1-10 mg / kg body weight.

20. A method for inhibiting the expression of coagulation factor XI gene in cells, characterized in that, The method comprises contacting the cells with an effective amount of the siRNA molecule of any one of claims 1-8, the siRNA-GalNAC conjugate of any one of claims 9-12, or the pharmaceutical composition of any one of claims 13-14.

21. A reagent kit, characterized in that, The kit comprises the siRNA molecule of any one of claims 1-8, the siRNA-GalNAC conjugate of any one of claims 9-12, or the pharmaceutical composition of any one of claims 13-14.

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