Nucleic acid molecule inhibiting f11 gene expression

Abstract

The present invention relates to a nucleic acid molecule inhibiting factor 11 (FXI) gene expression in a cell by means of RNAi, comprising a sense sequence and an antisense sequence complementary to each other, or consisting of a sense sequence and an antisense sequence complementary to each other. The FXI inhibition rate of the nucleic acid molecule is significantly superior to that of other small nucleic acid molecules which inhibit the expression of FXI by means of RNAi.

Description

A nucleic acid molecule for inhibiting F11 gene expression

[0001] This application claims priority to prior applications, including patent application number 202410114595.7, filed with the State Intellectual Property Office of China on January 26, 2024, entitled "A Nucleic Acid Molecule for Inhibiting F11 Gene Expression," and patent application number 202410568807.9, filed with the State Intellectual Property Office of China on May 9, 2024, entitled "GalNAc Derivatives and Oligonucleotide Conjugates Thereof." The entire contents of both prior applications are incorporated herein by reference. Technical Field

[0002] The present application relates to the field of RNAi, and in particular to a nucleic acid molecule capable of inhibiting F11 gene expression through RNAi and its use. Background Art

[0003] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. dsRNA is typically 19 to 30 bp in length and is one of the important tools in RNAi technology. In natural organisms, after entering cells, longer dsRNA can be specifically recognized by the Dicer enzyme and cleaved into small RNA fragments (i.e., siRNA) of approximately 21 to 23 nucleotides in length. The resulting dsRNA fragments unwind into single strands and form complexes with certain proteins (referred to as RISC). RISC can bind to mRNA in the cell that is complementary to the antisense strand of the dsRNA and cleave the mRNA, causing it to be degraded, preventing protein synthesis and resulting in gene "silencing." In industrial production, people prefer to chemically synthesize dsRNA and modify it to further improve the stability and effectiveness of dsRNA drugs.

[0004] Thrombi are blood clots that restrict blood flow within blood vessels. They can occur in either the arterial or venous circulation and are the common pathological basis for most myocardial infarctions, ischemic strokes, and venous thromboembolism (VTE). Currently, the main targets of commonly used anticoagulants are thrombin and FX (or coagulation factor 10), which are equally important in hemostasis and thrombosis. Attempting to achieve anticoagulant effects by inhibiting either of these enzymes inevitably compromises hemostasis. This conflict limits the therapeutic efficacy of anticoagulants and means that patients at high risk of bleeding may not be suitable for anticoagulant therapy. New anticoagulants are clinically needed that can simultaneously reduce bleeding risk while providing anticoagulation, and coagulation factor XI (FXI) has emerged as a new anticoagulant target. Compared to FX, FXI (or coagulation factor FXI or F11) plays a supporting role in hemostasis but plays an essential role in thrombosis. Even in patients with severe FXI deficiency, spontaneous bleeding, central nervous system bleeding, or gastrointestinal bleeding is rarely observed.

[0005] Application Overview

[0006] The present application provides a nucleic acid molecule that inhibits factor XI (FXI) gene expression in cells by RNAi, including compositions, preparations, and uses thereof. The present application examples have sufficient data to show that preferred nucleic acid molecules of the present application include, but are not limited to, dsRNA molecules numbered 3, 3', 5, 8, 9, 9', 14, 14', 15, 19, 24, 24', 26, 26', 50, 70, 70', and 82, shRNA molecules whose structures and sequences include the aforementioned dsRNA molecules, precursor molecules of the shRNA molecules or dsRNA molecules, and modified shRNA molecules or dsRNA molecules. Among them, preferred modifications include, but are not limited to, modification E and modification E-1 described in detail in the present application examples.

[0007] Specifically, the present application relates to a nucleic acid molecule comprising or consisting of a sense sequence and an antisense sequence that are complementary to each other, and the antisense sequence comprises a polynucleotide sequence that is complementary to FXI gene mRNA, wherein:

[0008] When the antisense sequence hybridizes with the FXI gene mRNA at a maximum complementarity rate, complementary region 1 is formed by complementary base pairs in the antisense sequence and the FXI gene mRNA sequence;

[0009] When the antisense sequence hybridizes with the sense sequence at a maximum complementarity rate, complementary region 2 is formed by complementary paired base pairs in the antisense sequence and the sense sequence;

[0010] wherein complementary region 1 and complementary region 2 have at least 18, 19 or 20 identical base pairs,

[0011] The complementary region 1 has a base pair number of 15 to 35 bp, and the complementary region 1 includes the following in the FXI gene mRNA sequence:

[0012] Nucleotides 418 to 437 or 18 or 19 consecutive nucleotides therein,

[0013] Nucleotides 1141 to 1160 or 18 or 19 consecutive nucleotides therein;

[0014] Nucleotides 363 to 382 or 18 or 19 consecutive nucleotides therein,

[0015] Nucleotides 372 to 391 or 18 or 19 consecutive nucleotides therein, nucleotides 411 to 430 or 18 or 19 consecutive nucleotides therein,

[0016] Nucleotides 421 to 440 or 18 or 19 consecutive nucleotides therein,

[0017] Nucleotides 422 to 441 or 18 or 19 consecutive nucleotides therein,

[0018] Nucleotides 425 to 444 or 18 or 19 consecutive nucleotides therein, nucleotides 465 to 484 or 18 or 19 consecutive nucleotides therein,

[0019] Nucleotides 470 to 489 or 18 or 19 consecutive nucleotides therein,

[0020] Nucleotides 471 to 490 or 18 or 19 consecutive nucleotides therein, nucleotides 518 to 537 or 18 or 19 consecutive nucleotides therein;

[0021] The position numbers of nucleotides in the FXI gene mRNA sequence are the numbers of the corresponding nucleotides in the reference sequence SEQ ID NO: 244.

[0022] The "18 or 19 consecutive nucleotides" of a nucleotide sequence may be 19 consecutive nucleotides starting from the first nucleotide or the second nucleotide at the 5' end of the nucleotide sequence and including the first nucleotide or the second nucleotide, which are consistent with the nucleotide sequence in the nucleotide sequence, or may be 18 consecutive nucleotides starting from the first nucleotide, the second nucleotide or the third nucleotide at the 5' end of the nucleotide sequence and including the first nucleotide, the second nucleotide or the third nucleotide, which are consistent with the nucleotide sequence in the nucleotide sequence.

[0023] In some embodiments, the sense sequence is equal to the antisense sequence. In some embodiments, the sense sequence and / or antisense sequence further comprise overhanging nucleotides outside the complementary region 2. In some embodiments, the sense sequence is longer than the antisense sequence. In some embodiments, the antisense sequence is longer than the sense sequence. In some embodiments, the sense sequence is 1, 2, 3, or 4 nucleotides longer than the antisense sequence. In some embodiments, the antisense sequence is 1, 2, 3, or 4 nucleotides longer than the sense sequence. In some embodiments, the sense sequence further comprises 1 or 2 nucleotides on the 5' end side and / or the 3' end side of the complementary region 2. In some embodiments, the antisense sequence further comprises 1 or 2 nucleotides on the 5' end side and / or the 3' end side of the complementary region 2. In some embodiments, the sense sequence and antisense sequence further comprise 1 or 2 nucleotides on the 5' end side and / or the 3' end side of the complementary region 2. In some embodiments, when the antisense sequence hybridizes to the sense sequence at maximum complementarity, the sense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the antisense sequence between the first nucleotide and the last nucleotide in the complementary region 2. In some embodiments, when the antisense sequence hybridizes to the sense sequence at maximum complementarity, the antisense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the sense sequence between the first nucleotide and the last nucleotide in the complementary region 2. In some embodiments, the overhang nucleotides are two and are located in the antisense sequence, adjacent to the 5' end of the complementary region 2, and the sense sequence does not include an overhang nucleotide. In some embodiments, the antisense sequence contains only 0, 1, or 2 nucleotides outside the complementary region 1, and the sense sequence contains only 0, 1, or 2 nucleotides outside the complementary region 2. In some embodiments, when the antisense sequence hybridizes to the FXI gene mRNA sequence at maximum complementarity, the antisense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the FXI gene mRNA sequence between the first nucleotide and the last nucleotide in the complementary region 1. In some embodiments, when the antisense sequence hybridizes with the FXI gene mRNA sequence at maximum complementarity, the FXI gene mRNA sequence does not contain nucleotides that are not complementary to the antisense sequence between the first nucleotide and the last nucleotide of complementary region 1. In some embodiments, the 3' end of complementary region 2 is an AU base pair. In some embodiments, the 3' end of complementary region 2 consists of an A from the antisense sequence and a U (which may be written as T in this application) from the sense sequence. In some embodiments, the 3' end of complementary region 2 consists of an A from the sense sequence and a U (which may be written as T in this application) from the antisense sequence. In some embodiments, complementary region 1 and complementary region 2 are uninterrupted, i.e., continuous.In some embodiments, the complementary region 1 and / or the complementary region 2 is discontinuous, ie, interrupted by one or more bubbles (a non-complementary region formed by non-complementary nucleotides in each of the two sequences forming the complementary region).

[0024] In some embodiments, the number of base pairs in complementary region 1 is 15 to 35, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bp. In some embodiments, the number of base pairs in complementary region 2 is 15 to 35, such as 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bp. In some embodiments, complementary region 1 and complementary region 2 have 18, 19, 20, 21, 22, or 23 identical base pairs. In some embodiments, the length of the sense sequence and / or the antisense sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nt. In some embodiments, the sense sequence is 21nt in length and the antisense sequence is 23nt in length. In some embodiments, the nucleic acid molecule is composed of a sense sequence of 21nt in length and an antisense sequence of 23nt in length. In some embodiments, the nucleic acid molecule is composed of a sense sequence and an antisense sequence and a connecting chain (or shRNA loop), wherein the connecting chain connects the 3' end nucleotides of the sense sequence and the 5' end nucleotides of the antisense sequence. The selection of the connecting chain is consistent in the art, for example, with reference to the literature Jensen, Stig The selection was performed as described in Gaard Rask et al. "Functional selection of shRNA loops from randomized retroviral libr aries." PLoS one vol. 7, 8 (2012): e43095. doi: 10.1371 / journal.pone.0043095, which is incorporated herein by reference in its entirety.

[0025] In some embodiments, the sense sequence and / or antisense sequence further comprises 1 to 2 overhang nucleotides outside the complementary region 2.

[0026] In some embodiments, the antisense sequence comprises only 0, 1, or 2 nucleotides outside complementary region 1, and the sense sequence comprises only 0, 1, or 2 nucleotides outside complementary region 2.

[0027] In some embodiments, the 3' end of complementary region 2 is an AU base pair; in further embodiments, the 5' end A of the antisense strand is outside complementary region 1.

[0028] In some embodiments, the FXI gene mRNA sequence complementary to the antisense strand is transcribed from a mammalian FXI gene coding region. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human or a cynomolgus macaque. In some embodiments, the mRNA comprises the polynucleotide sequence set forth in SEQ ID NO: 244. In some embodiments, the antisense sequence comprises the polynucleotide sequence set forth in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201, or 213. In some embodiments, the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201 or 213, and an additional 1, 2, 3 or 4 nucleotides. In some embodiments, the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201 or 213, and an additional 1 to 2 nucleotides on the 5' and / or 3' side of the polynucleotide sequence. In some embodiments, the antisense sequence is a polynucleotide sequence as set forth in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201, or 213. In some embodiments, the sense sequence comprises a polynucleotide sequence as set forth in SEQ ID NO: 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82, or 94. In some embodiments, the antisense sequence consists of a polynucleotide sequence as set forth in SEQ ID NO: 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82, or 94 and an additional 1 to 4 (e.g., 2 or 3) nucleotides. In some embodiments, the antisense sequence is a polynucleotide sequence as shown in 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82 or 94 and an additional 1 to 2 nucleotides on the 5' and / or 3' side of the polynucleotide sequence. In some embodiments, the antisense sequence is a polynucleotide sequence as shown in 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82 or 94.

[0029] In some embodiments, the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:3, and the antisense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:122; the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:4, and the antisense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:123; the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:7, and the antisense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:126; the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:10, and the antisense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:129; the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:11, and the antisense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:130; the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:12, and the antisense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:131; the sense sequence comprises or is the polynucleotide sequence set forth in SEQ ID NO:132. NO:17, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:136; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:18, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:137; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:19, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:138; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:24, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:143; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:30, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:149; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:31, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:150; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: NO:33, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:152; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:34, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO:153;The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 81, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 200; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 82, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 201; or the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 94, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 213.

[0030] In some embodiments, the sense sequence consists of the polynucleotide sequence of SEQ ID NO:3 and an additional 1, 2, 3 or 4 nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO:122 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO:4 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO:123 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO:7 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO:126 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO:10 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO:127 and an additional 1 to 4 (e.g., 2 or 3) nucleotides. NO: 129, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 11, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 130, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 12, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 131, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 17, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 136, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 137, and 1 to 4 (e.g., 2 or 3) additional nucleotides. wherein the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 137 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 19 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 138 and 1 to 4 (e.g., 2 or 3) additional nucleotides;The sense sequence consists of the polynucleotide sequence of SEQ ID NO: 24 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 143 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 30 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 149 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 31 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 150 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 33 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: NO: 152, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 34, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 153, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 58, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 177, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 81, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 200, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 58, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 177, and 1 to 4 (e.g., 2 or 3) additional nucleotides. The present invention further comprises the polynucleotide sequence of SEQ ID NO: 82 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 201 and 1 to 4 (e.g., 2 or 3) additional nucleotides; or the sense sequence consists of the polynucleotide sequence of SEQ ID NO: 94 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 213 and 1 to 4 (e.g., 2 or 3) additional nucleotides.

[0031] In some embodiments, the sense sequence consists of the polynucleotide sequence of SEQ ID NO:3 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 122. The sense sequence consists of the polynucleotide sequence of SEQ ID NO:4 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 123. The sense sequence consists of the polynucleotide sequence of SEQ ID NO:7 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 126. The sense sequence consists of the polynucleotide sequence of SEQ ID NO:10 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 129. The sense sequence consists of the polynucleotide sequence of SEQ ID NO:11 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: The polynucleotide sequence of SEQ ID NO: 130 is a sense sequence consisting of the polynucleotide sequence of SEQ ID NO: 12 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 131. The sense sequence consists of the polynucleotide sequence of SEQ ID NO: 17 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 136. The sense sequence consists of the polynucleotide sequence of SEQ ID NO: 18 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 137. The sense sequence consists of the polynucleotide sequence of SEQ ID NO: 19 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 138. The sense sequence consists of the polynucleotide sequence of SEQ ID NO: 24 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence of SEQ ID NO: The sense sequence of the polynucleotide sequence shown in SEQ ID NO: 143 consists of the polynucleotide sequence shown in SEQ ID NO: 30 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 149, the sense sequence consisting of the polynucleotide sequence shown in SEQ ID NO: 31 and 1 to 4 (e.g., 2 or 3) additional nucleotides,and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 150; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 33 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 152; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 34 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 153; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 58 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 81 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 200; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: The antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 201, or the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 94, and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 213.

[0032] In some embodiments, the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 3 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 122 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 4 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 123 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 7 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 126 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 10 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 129 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 11 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: NO: 130 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 12 and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 131 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 17 and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 136 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 18 and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 137 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 19 and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 138 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 24 and the antisense sequence consists of the polynucleotide sequence of SEQ ID NO: 25. wherein the sense sequence is the polynucleotide sequence set forth in SEQ ID NO:30, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO:149, and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence set forth in SEQ ID NO:31, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO:150, and 1 to 4 (e.g., 2 or 3) additional nucleotides;The sense sequence is the polynucleotide sequence set forth in SEQ ID NO:33, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO:152 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence set forth in SEQ ID NO:34, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO:153 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence set forth in SEQ ID NO:58, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO:177 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence set forth in SEQ ID NO:81, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO:200 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence set forth in SEQ ID NO:82, and the antisense sequence consists of the polynucleotide sequence set forth in SEQ ID NO: The sense sequence is the polynucleotide sequence shown in SEQ ID NO: 94 and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 213 and 1 to 4 (e.g., 2 or 3) additional nucleotides.

[0033] In some embodiments:

[0034] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 34, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 153;

[0035] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 33, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 152;

[0036] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 3, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 122;

[0037] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 4, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 123;

[0038] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 7, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 126;

[0039] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 10, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 129;

[0040] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 11, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 130;

[0041] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 12, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 131;

[0042] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 17, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 136;

[0043] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 18, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 137;

[0044] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 19, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 138;

[0045] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 24, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 143;

[0046] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 30, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 149;

[0047] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 31, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 150;

[0048] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 81, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 200;

[0049] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 82, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 201; or

[0050] The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 94, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 213.

[0051] In some embodiments, the nucleic acid molecule is a dsRNA or shRNA. In some embodiments, the dsRNA is an siRNA. In some embodiments, one or more nucleotides in the nucleic acid molecule are chemically modified. In some embodiments, the chemical modification makes the nucleic acid molecule more stable in a cell or in vivo environment. In some embodiments, the chemical modification prolongs the in vivo and / or in vitro half-life of the nucleic acid molecule. In some embodiments, the chemical modification comprises any one or more selected from the following, or consists of one or more modifications selected from the following:

[0052] Locked nucleic acid modification, open ring or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification (2'-OM e), 2′-O-allyl modification, 2'-C-alkyl modification, 2'-C-allyl modification, 2'-fluoro modification (2'-F), 2'-deoxy modification (d), phosphorothioate modification (s), 2'-amino-modification, morpholino modification, phosphoramidate modification, methylphosphonate modification, tetrahydropyranyl modification, 1,5-anhydrohexitol modification, 5'-vinyl phosphate modification, and cyclohexenyl modification.

[0053] In some embodiments, the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the nucleotides at positions 2, 14, and 16 of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the nucleotides at positions 2, 14, and 16 of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and / or the nucleotides at positions 2, 14, and 16 of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.

[0054] In some embodiments, the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the 2nd, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the 2nd, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and / or the 2nd, 14th, and 16th nucleotides of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.

[0055] In some embodiments, the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and / or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.

[0056] In some embodiments, the nucleic acid molecule comprises a motif selected from any one of the following:

[0057] (1) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm

[0058] (2) sense sequence: NmNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm; and

[0059] (3) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

[0060] Wherein, N in each sequence from 5' to 3' independently represents the nucleotide at the position of the sequence in which it is located, Nm represents a 2'-O-methyl modified ribonucleotide, and Nf represents a 2'-fluoro modified ribonucleotide; it should be understood that this scheme does not limit whether each nucleotide contains or does not contain other modifications besides the marked modifications.

[0061] In some embodiments, the above embodiments further include at least one phosphorothioate modification between the first and second nucleotides, and between the second and third nucleotides of the 5' sense sequence; and at least one phosphorothioate modification between the first and second nucleotides, between the second and third nucleotides, between the penultimate and second to last, and between the penultimate and third nucleotides of the 5' antisense sequence.

[0062] In some embodiments, the nucleic acid molecule comprises a motif selected from any one of the following (1)-(9):

[0063] (1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0064] In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNfNmNmNmNmNmNmNmNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0065] (2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0066] In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0067] (3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0068] In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, and the antisense sequence is NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0069] (4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0070] In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0071] (5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0072] In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0073] (6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0074] In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, and the antisense sequence is NmsNfsNmNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0075] (7) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0076] In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0077] (8) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0078] In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

[0079] (9) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0080] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, and the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate.

[0081] In all motifs of the present application, Nm represents a 2'-O-methyl modified ribonucleotide, Nf represents a 2'-fluoro modified ribonucleotide, and s represents a phosphorothioate modification.

[0082] In some embodiments, the nucleic acid molecule is linked to a ligand having organ targeting, such as a liver targeting ligand. In some embodiments, the nucleic acid molecule is linked to at least one asialoglycoprotein receptor (ASGPR) ligand. In some embodiments, the organ targeting ligand is linked to the 5' end or the 3' end of the sense sequence. In some embodiments, the ASGPR ligand is one or more GalNAc derivatives connected by a divalent or trivalent branched structure. In some embodiments, the GalNAc derivative comprises the following structure:

[0083] In some embodiments, the GalNac derivative is L96, and the structure of L96 is shown below:

[0084] In some embodiments, the GalNac derivative is ligand 1, and the structure of ligand 1 is shown below:

[0085] (X=S - or O - )(Formula II);

[0086] wherein the The terminator represents a link to the 3' end of the sense sequence or antisense sequence of the nucleic acid molecule; preferably, the link is via a phosphodiester bond or a phosphorothioate diester bond.

[0087] Further preferably, the nucleic acid molecule has a modification motif as any one of (1) to (6):

[0088] (1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96; Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0089] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0090] (2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmsNmsNm;

[0091] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-Ligand 1; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than Ligand 1, 2'-F, 2'-OMe and phosphorothioate;

[0092] (3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0093] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0094] (4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0095] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-Ligand 1; the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than Ligand 1, 2'-F, 2'-OMe and phosphorothioate;

[0096] (5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0097] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0098] (6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmsNmsNm;

[0099] In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-Ligand 1; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than Ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0100] In some embodiments, the nucleic acid molecule comprises a 5' phosphate or 5' phosphate mimetic modification, such as a 5'-terminal phosphate or phosphate mimetic on the antisense strand; preferably, the 5' phosphate mimetic is a 5'-VP modification.

[0101] In some embodiments, the nucleic acid molecule has the following modification motif:

[0102] GN-E20VP:

[0103] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1

[0104] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0105] In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNms-Ligand 1

[0106] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0107] In some embodiments, the nucleic acid molecule has the following modification motif:

[0108] GN-E04VP:

[0109] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

[0110] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0111] In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-Ligand 1

[0112] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0113] In some embodiments, the nucleic acid molecule has the following modification motif:

[0114] GN-E05VP:

[0115] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

[0116] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0117] In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-Ligand 1

[0118] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0119] In some embodiments, the nucleic acid molecule has the following modification motif:

[0120] LN-E20VP:

[0121] Justice sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96

[0122] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0123] In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNm-L96

[0124] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0125] In some embodiments, the nucleic acid molecule has the following modification motif:

[0126] LN-E04VP:

[0127] Justice sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0128] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0129] In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0130] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0131] In some embodiments, the nucleic acid molecule has the following modification motif:

[0132] LN-E05VP:

[0133] Justice sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0134] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0135] In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0136] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0137] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided herein is a sense sequence: SEQ ID NO: 58 and an antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has any of the modification patterns listed above.

[0138] In some embodiments, the naked nucleic acid sequence specifically provided herein is a sense sequence: SEQ ID NO: 58 and an antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

[0139] GN-E20:

[0140] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0141] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0142] GN-E20VP:

[0143] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0144] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0145] GN-E04:

[0146] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0147] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0148] GN-E04VP:

[0149] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0150] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0151] GN-E05:

[0152] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0153] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

[0154] GN-E05VP:

[0155] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0156] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

[0157] In some embodiments, the naked nucleic acid sequence specifically provided herein is a sense sequence: SEQ ID NO: 58 and an antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

[0158] GN-E20:

[0159] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0160] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0161] GN-E20VP:

[0162] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0163] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0164] GN-E04:

[0165] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0166] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0167] GN-E04VP:

[0168] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0169] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0170] GN-E05:

[0171] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0172] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate; or

[0173] GN-E05VP:

[0174] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0175] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0176] In some embodiments, the naked sequence of the nucleic acid molecule provided herein is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having any of the modification patterns listed above.

[0177] In some embodiments, the naked sequence of the nucleic acid molecule provided herein is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having the modification pattern shown below:

[0178] GN-E20:

[0179] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0180] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0181] GN-E20VP:

[0182] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0183] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0184] GN-E04:

[0185] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0186] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0187] GN-E04VP:

[0188] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0189] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0190] GN-E05:

[0191] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0192] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

[0193] GN-E05VP:

[0194] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0195] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

[0196] In some embodiments, the naked nucleic acid sequence specifically provided herein is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), and has the following modification pattern:

[0197] GN-E20:

[0198] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0199] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0200] GN-E20VP:

[0201] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

[0202] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0203] GN-E04:

[0204] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0205] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0206] GN-E04VP:

[0207] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0208] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

[0209] GN-E05:

[0210] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0211] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate; or

[0212] GN-E05VP:

[0213] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

[0214] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0215] In some embodiments, the naked nucleic acid sequence specifically provided herein is a sense sequence: SEQ ID NO: 58 and an antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

[0216] LN-E20:

[0217] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0218] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0219] LN-E20VP:

[0220] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0221] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0222] LN-E04:

[0223] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0224] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0225] LN-E04VP:

[0226] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0227] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0228] LN-E05:

[0229] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0230] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

[0231] LN-E05VP:

[0232] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0233] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

[0234] In some embodiments, the naked nucleic acid sequence specifically provided herein is a sense sequence: SEQ ID NO: 58 and an antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

[0235] LN-E20:

[0236] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0237] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0238] LN-E20VP:

[0239] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0240] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0241] LN-E04:

[0242] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0243] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0244] LN-E04VP:

[0245] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0246] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0247] LN-E05:

[0248] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0249] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate; or

[0250] LN-E05VP:

[0251] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0252] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0253] In some embodiments, the naked sequence of the nucleic acid molecule provided herein is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having the modification pattern shown below:

[0254] LN-E20:

[0255] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0256] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0257] LN-E20VP:

[0258] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0259] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0260] LN-E04:

[0261] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0262] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0263] LN-E04VP:

[0264] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0265] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0266] LN-E05:

[0267] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0268] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

[0269] LN-E05VP:

[0270] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0271] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

[0272] In some embodiments, the naked nucleic acid sequence specifically provided herein is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), and has the following modification pattern:

[0273] LN-E20:

[0274] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0275] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0276] LN-E20VP:

[0277] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0278] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0279] LN-E04:

[0280] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0281] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0282] LN-E04VP:

[0283] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0284] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0285] LN-E05:

[0286] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0287] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate; or

[0288] LN-E05VP:

[0289] Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0290] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0291] In addition, the present application also provides a precursor of the aforementioned nucleic acid molecule, which may be shRNA or dsRNA.

[0292] The second aspect of the present application provides a second nucleic acid molecule, which can be transcribed into a dsRNA or shRNA precursor in a cell, and the dsRNA or shRNA is the nucleic acid molecule of the aforementioned first aspect. In some embodiments, the second nucleic acid molecule is a circular or linear nucleic acid molecule. In some embodiments, the second nucleic acid molecule is a repackaged or linear plasmid. In some embodiments, the nucleic acid molecule belongs to an artificially constructed viral genome, which can be selected from, for example, lentiviral vectors or other retroviral vectors, adenoviral vectors, AAV vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors. In some embodiments, the nucleic acid molecule belongs to a cellular genome, such as a nuclear genome, mitochondrial nucleic acid, or cytoplasmic free nucleic acid.

[0293] The third aspect of the present application further provides a nucleic acid delivery body comprising the nucleic acid molecule of the aforementioned first aspect, or the second nucleic acid molecule of the aforementioned second aspect. In some embodiments, the nucleic acid delivery body is a liposome, lipid nanoparticle or other polymer, endosome, exosome or vesicle.

[0294] Also provided are viral particles comprising the second nucleic acid molecule of the aforementioned second aspect. In some embodiments, the viral particles are enveloped viruses or togavirus particles. In some embodiments, the viral particles are pseudovirions. In some embodiments, the viral particles are AAV, baculovirus, poxvirus, herpes virus, alphavirus, lentivirus, or other retroviruses.

[0295] Also provided is a cell comprising the second nucleic acid molecule of the second aspect. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, such as a stem cell, such as a hematopoietic stem cell, a mesenchymal stem cell, or the like.

[0296] The present application also provides a pharmaceutical composition comprising the nucleic acid molecule or salt thereof of the first aspect and an acceptable carrier or diluent. In some embodiments, the pharmaceutical composition is used to treat or prevent a thromboembolic complication or coagulopathy in a subject, wherein the thromboembolic complication is preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0297] The fourth aspect of the present application further provides uses of the nucleic acid of the first aspect, the second nucleic acid of the second aspect, the nucleic acid delivery body of the third aspect, and the viral particles, cells, and pharmaceutical compositions.

[0298] For example, the present application provides the use of the nucleic acid molecule or salt thereof of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, or the cell for preparing a medicament for preventing or treating thromboembolic complications or coagulation disorders in a subject. In some embodiments, the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0299] The present application also provides a method for treating or preventing thromboembolic complications or coagulation disorders in a subject, the method comprising administering to a subject in need thereof an effective amount of the nucleic acid molecule of the first aspect or a salt thereof, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, the cell, or the pharmaceutical composition. In some embodiments, the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0300] Also provided is a compound for treating or preventing thromboembolic complications or coagulation disorders in a subject, comprising the nucleic acid molecule or salt thereof of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, the cell, or the pharmaceutical composition. In some embodiments, the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0301] The present application also provides use of the nucleic acid molecule or a salt thereof according to the first aspect or the second nucleic acid molecule according to the second aspect for inhibiting the expression of factor 11.

[0302] The present application also provides use of the nucleic acid molecule or a salt thereof of the first aspect or the second nucleic acid molecule of the second aspect for preparing a drug for inhibiting FXI expression in a subject.

[0303] The present application also provides a compound for inhibiting FXI expression in a subject, the compound comprising the nucleic acid molecule or a salt thereof of the first aspect, and a pharmaceutically acceptable carrier or diluent. The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the scope of the technical concept of the present application, a variety of simple variations can be made to the technical solution of the present application, including combining the various technical features in any other suitable manner. These simple variations and combinations should also be regarded as the contents disclosed in the present application and fall within the scope of protection of the present application. The aspects and embodiments of the present application described herein include aspects and embodiments of "comprising", "consisting of" and "essentially consisting of..." BRIEF DESCRIPTION OF THE DRAWINGS

[0304] FIG1 shows the results of high-throughput screening of naked dsRNA molecules (5 nM) in HepG2 cells.

[0305] FIG2 shows the relative levels of FXI mRNA in HepG2 cells when 5 nM and 1 nM candidate dsRNA or Yangshen dsRNA naked nucleic acid molecules were administered, respectively.

[0306] FIG3 shows the inhibition rate of FXI mRNA in HepG2 cells at a concentration of 1 nM by dsRNA modified with modification E.

[0307] FIG4 shows the inhibition rate of FXI mRNA by GalNAc-coupled dsRNA at different concentrations.

[0308] FIG5 shows the results of target protein inhibition after in vivo administration of different GalNAc-conjugated dsRNAs.

[0309] FIG6 shows the comparative results after in vivo administration of different GalNAc

[0310] FIG7 shows the comparison results of IC50 of different modified dsRNAs at the cell level in vitro

[0311] FIG8 shows the comparative results after in vivo administration of dsRNA with different modified motifs

[0312] Application Details

[0313] The present invention provides a nucleic acid molecule capable of initiating RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the coagulation factor FXI (or FX11) gene, a second nucleic acid molecule capable of transcribing the nucleic acid molecule, a delivery vehicle for the nucleic acid molecule and the second nucleic acid molecule, a virus or cell capable of transcribing the nucleic acid molecule, and uses of the nucleic acid molecule and the second nucleic acid molecule.

[0314] the term

[0315] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the technology belongs. All technical and patent disclosures cited herein are incorporated herein by reference in their entirety.

[0316] As used herein, "dsRNA" is double-stranded RNA. Since siRNA is a double-stranded RNA, the term "dsRNA" encompasses siRNA. dsRNA also includes double-stranded RNA that is longer than siRNA. The length greater than siRNA can refer to its sense strand being longer than siRNA, or its antisense strand being longer than siRNA, or its sense strand and antisense strand being longer than siRNA. After a double-stranded RNA that is generally longer than the siRNA sequence it contains enters the cell, it is broken down into siRNA by a type III endonuclease called Dicer. In some embodiments, the lengths of the two chains of the dsRNA are each independently 15 to 30 nt (in this application, "nt" refers to nucleotides). When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in RISC unwind the siRNA double helix. When bound to a target mRNA complementary to the antisense strand in the siRNA, one or more endonucleases in RISC cleave the target, inducing gene silencing. Typically, the majority of nucleotides in each chain of a dsRNA molecule are ribonucleotides, but this does not exclude the inclusion of one or more non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or both chains. In some embodiments, the dsRNA molecule does not include non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, a dsRNA may include one or more chemically modified nucleotides or may not include chemically modified nucleotides.

[0317] The terms "FXI," "coagulation factor FXI," and "Factor 11" are used interchangeably and are also referred to in the art as FXI or PTA. FXI may be mammalian-derived FXI. In some embodiments, FXI is primate-derived FXI. In some embodiments, FXI is human-derived FXI. In some embodiments, FXI is cynomolgus monkey-derived FXI. As used herein, "FXI gene mRNA" refers to mRNA encoding Factor 11 protein, which may be transcribed from FXI gene DNA and may be mature mRNA or pre-mRNA (Pre-mRNA), and thus may or may not contain introns. Because the Factor 11 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the Factor 11 gene mRNA sequence of the present application is intended to include all mRNA sequences transcribed from Factor 11 gene mutants. The human FXI gene mRNA sequence can be found, for example, in GenBank Accession No. GI:1732746318 (NM_000128.4). The rhesus macaque FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: ​​1622942384 (XM_015139652.2). The cynomolgus macaque FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: ​​2161917139 (XM_005556483.3). The mouse FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: ​​2293430447 (NM_028066.3). The rat FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: ​​2293332621 (NM_001411666.1). Other examples of FXI gene mRNA sequences are readily available using publicly available databases, such as GenBANK. Unless otherwise specified, the FXI gene mRNA uses SEQ ID NO: 244 (i.e., NM_000128.4) as the reference sequence, i.e., each nucleotide position in the FXI gene mRNA is numbered according to the nucleotide numbering in the 5' to 3' direction of the reference sequence SEQ ID NO: 244. As used herein, the term "reference sequence" is a standard sequence used for homologous sequence alignment and can be used to define the sequence of nucleotide positions in a homologous polynucleotide or polynucleotide sequence. For example, "the base positions in the FXI gene mRNA sequence are numbered according to the corresponding base positions in the reference sequence SEQ ID NO: 244" means that after the FXI gene mRNA sequence and the reference sequence have the same bases at as many positions as possible by introducing gaps or deleting nucleotides in the FXI gene mRNA sequence, the nucleotides in the reference sequence are numbered consecutively in sequence order, starting from the first nucleotide at the 5' end. The positions of the corresponding nucleotides in the FXI gene mRNA sequence and the reference sequence are defined by the same numbering by aligning the corresponding nucleotides.

[0318] As used herein, "complementarity" of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of nucleotides in the shorter of two nucleic acid molecules that can form hydrogen bonds (i.e., Watson-Crick base pairing) with the other nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, and 10 out of 10 are approximately 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For single bases or single nucleotides, according to the Watson-Crick base pairing rules, when A pairs with T or U, or C pairs with G or I, and vice versa, they are said to be complementary, paired, or matched; base pairings other than these are said to be non-complementary.

[0319] As used herein, "hybridization" of nucleic acids refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can include two chains forming a double-stranded structure, three or more chains forming a multi-stranded complex, a single self-hybridizing chain, or a combination thereof.

[0320] In this application, when referring to two polynucleotide sequences or two nucleic acid chains "hybridizing at maximum complementarity", it refers to a hybridization mode in which as many nucleotides as possible in the two chains are paired with each other by forming hydrogen bonds. When "hybridizing at maximum complementarity", one or more mismatches may be allowed to occur, and one or more bulges may be allowed to appear in one or more of the two chains. However, in some embodiments, the two chains "hybridized at maximum complementarity" may not have mismatches and bulges after hybridization. As used herein, "complementary region" refers to all base pairs paired by hydrogen bonds from the first base pair paired by hydrogen bonds at the 5' end to the last base pair paired by hydrogen bonds after hybridization of the two chains. The complementary region formed by the two hybridized chains may be continuous or intermittent. In this application, "complementary region 1" specifically refers to the complementary region consisting of the complementary base pairs in the antisense sequence and the FXI gene mRNA sequence when the antisense sequence hybridizes with the FXI gene mRNA at maximum complementarity; and "complementary region 2" refers to the complementary region consisting of the complementary base pairs in the antisense sequence and the sense sequence when the antisense sequence hybridizes with the sense sequence at maximum complementarity. Since both complementary regions 1 and 2 involve antisense sequences, the antisense sequence portions of complementary regions 1 and 2 typically have partially or completely identical nucleotide compositions. In this application, the antisense sequence and sense sequence are relative to a third sequence that is complementary to one of the sequences. For example, the antisense sequence and sense sequence in the "nucleic acid molecule that inhibits the expression of the factor 11 (FXI) gene in cells through RNAi" are relative to the third sequence, the FXI gene mRNA sequence. That is, the antisense sequence refers to the sequence in the nucleic acid molecule that has a complementary region with the FXI gene mRNA sequence, and the sense sequence refers to the sequence in the nucleic acid molecule that has at least 10 consecutive identical nucleotides with the FXI gene mRNA sequence. In addition, the 5' end of any complementary region in the present application refers to the position of the nucleotide or base pair in the complementary region that is closest to the 5' end of the sense strand or the third sequence, and the 5' end of the complementary region refers to the side or end relatively close to the 5' end of the sense strand or the third sequence. Similarly, the 3' end of any complementary region in the present application refers to the position of the nucleotide or base pair in the complementary region that is closest to the 3' end of the sense strand or the third sequence, and the 3' end of the complementary region refers to the side or end relatively close to the 3' end of the sense strand or the third sequence. In the present application, when describing the position of nucleotides on the same sequence or nucleoside chain, "close", "near" or "far" refers to the number of nucleotides between the two nucleotide positions.

[0321] As used herein, "overhang nucleotides" are relative to complementary region 2, and are nucleotides located outside complementary region 2 in the sense sequence and / or antisense sequence nucleotides after the sense sequence and antisense sequence hybridize at maximum complementarity. In some embodiments, the overhang nucleotides are located only in the sense sequence, in some embodiments, the overhang nucleotides are located only in the antisense sequence, and in some embodiments, the overhang nucleotides are located in both the sense sequence and the antisense sequence. In some embodiments, the overhang nucleotides are only present on the 5' side of complementary region 2. In some embodiments, the overhang nucleotides are only present on the 5' side of complementary region 2 of the sense strand. In some embodiments, the overhang nucleotides are only present on the 5' side of complementary region 2 of the antisense strand. In some embodiments, the overhang nucleotides are only present on the 3' side of complementary region 2. In some embodiments, the overhang nucleotides are only present on the 3' side of complementary region 2 of the sense strand. In some embodiments, the overhang nucleotides are only present on the 3' side of complementary region 2 of the antisense strand. In some embodiments, the number of nucleotides in the overhang region on the same side (e.g., the 5' end or the 3' end) of the sense sequence or antisense sequence is no more than two (i.e., one or two). In this application, "on the 5' end side" and "on the 3' end side" are both used to describe the relative positional relationship between two sequences, two nucleotides, or one nucleotide and a sequence in the same polynucleotide sequence; wherein, "5' end" refers to the end of the polynucleotide sequence containing a 5' free phosphate group or a 5' free hydroxyl group, and "3' end" refers to the end of the polynucleotide sequence containing a free 3'-hydroxyl group or a 3'-phosphate group, and the sequence or nucleotide on the 3' end side of a sequence in the nucleic acid chain is closer to the 3' end of the nucleic acid chain than the sequence. For example, "on the 5' end side of complementary region 2" also contains a certain sequence or a certain nucleotide or nucleotides, which means that the "certain sequence or a certain nucleotide or nucleotides" are closer to the 5' end of the polynucleotide sequence (e.g., the antisense sequence or the sense sequence) in which they are located relative to the sequence of "complementary region 2."

[0322] The term "nucleotide", in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, is also understood herein to refer to related structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used, unless the context clearly indicates otherwise. For example, "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. A nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), a nucleotide isomer, or a nucleotide analog. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog can be a naturally occurring nucleotide (e.g., inosine, pseudouridine, etc.) or a non-naturally occurring nucleotide. Non-limiting examples of modifications on the sugar or base portion of a nucleotide include the addition (or removal) of an acetyl group, an amino group, a carboxyl group, a carboxymethyl group, a hydroxyl group, a methyl group, a phosphoryl group, and a thiol group, and the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino oligonucleotides. In some embodiments, the "nucleotides" of the present application do not include non-natural nucleotides with modified bases. In some embodiments, the "nucleotides" of the present application do not include nucleotides with modified bases. In the present application, "G", "C", "A", "T" and "U" generally represent nucleotides with guanine, cytosine, adenine, thymine and uracil as bases, respectively. Unless otherwise specified, "G", "C", "A", "T" and "U" represent nucleotides with no limitation on the modifications they contain, that is, they can be used to represent natural nucleotides or non-natural nucleotides. The non-natural nucleotides may contain ribose and / or modified bases, as long as the bases therein can still be paired with their naturally paired bases (i.e., paired according to the Watson-Crick principle) through hydrogen bonding. However, in the context of RNA and in RNA sequences, unless otherwise specified, "T" refers to uridine or uracil. It should be understood that throughout this application, "nucleotide," "nucleotide residue," and "base" are used interchangeably in contexts related to nucleotide sequences. Base pairs are measured in bp, with one bp being one base pair. Nucleotides are measured in nt, with one nt being one nucleotide.

[0323] As used herein, "internucleotide linkage" refers to the chemical bond (or linking group) between two adjacent nucleotides or between a nucleotide and a ligand. Unless otherwise specified, the chemical bond is a phosphate bond or a phosphoester bond. However, when an "s" is specifically indicated in a modification motif, i.e., the position of a phosphorothioate modification, only the internucleotide linkage at the position where "s" appears is a phosphorothioate bond, and all other unindicated internucleotide linkages are phosphate bonds. For example:

[0324] (1) Sense strand: NmNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNm, antisense strand: NmNfNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

[0325] Then the internucleotide linkages in (1) can be either phosphate bonds or phosphorothioate bonds;

[0326] And if it is: sense strand: CmsAmsGmAmGmUmUfAmUfCdGfAmGmGmCmAmCmAmUmUmAms-ligand 1,

[0327] Antisense strand: vp-UmsAfsAmUmGdUmGdCmCmUmCmGmAmUfAmAfCmUmCmUmGmsGmsCm; because it has a specially marked "s", the internucleotide linkages at all positions are phosphate bonds except for the following internucleotide linkages which are phosphorothioate linkages: the first and second internucleotide linkages from the 5' end of the sense strand, the internucleotide linkage between the 3'-terminal nucleotide of the sense strand and Ligand 1, the first and second internucleotide linkages from the 5' end of the antisense strand, and the first and second internucleotide linkages from the 3' end of the antisense strand. Here, the first internucleotide linkage refers to the chemical bond between the first and second nucleotides starting from one end (e.g., the 5' end or the 3' end); the second internucleotide linkage refers to the chemical bond between the second and third nucleotides starting from one end (e.g., the 5' end or the 3' end), and so on; the nth internucleotide linkage refers to the chemical bond between the nth and n+1th nucleotides or ligands starting from one end (e.g., the 5' end or the 3' end), and so on.

[0328] As used herein, "overhang," "overhang," and "overhang sequence" are used interchangeably and refer to one or more unpaired nucleotides that extend beyond the duplex region at the end of a chain. A nucleotide overhang is typically formed when the 3' end of one chain extends beyond the 5' end of the other chain, or when the 5' end of one chain extends beyond the 3' end of the other chain. The length of the nucleotide overhang is typically between 1 and 6 nucleotides, between 1 and 5 nucleotides, between 1 and 4 nucleotides, between 1 and 3 nucleotides, between 2 and 6 nucleotides, between 2 and 5 nucleotides, or between 2 and 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a specific embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide.

[0329] The nucleotides in the overhang can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides in the overhang are 2'-modified nucleotides (e.g., 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides), deoxyribonucleotides, reverse nucleotides (e.g., reverse abasic nucleotides, reverse deoxyribonucleotides), or combinations thereof. For example, in one embodiment, the nucleotides in the overhang are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides in the overhang are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, or combinations thereof. In other embodiments, the overhang comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotides. In such embodiments, the UU dinucleotides can comprise ribonucleotides or modified nucleotides, such as 2'-modified nucleotides. In other embodiments, the overhang comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotides. When a nucleotide overhang is present in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (e.g., a polypyrimidine or polypurine sequence, such as UU, TT, AA, GG, etc.).

[0330] The nucleotide overhangs can be at the 5' end or the 3' end of one or both strands. For example, in one embodiment, the RNA molecule comprises nucleotide overhangs at the 5' end and the 3' end of the antisense strand. In another embodiment, the RNA molecule comprises nucleotide overhangs at the 5' end and the 3' end of the sense strand. In some embodiments, the RNA molecule comprises nucleotide overhangs at the 5" end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNA molecule comprises nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.

[0331] An RNA molecule may comprise a nucleotide overhang at one end of a double-stranded RNA molecule and a flat end at the other end. "Flat end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the duplex region. In some embodiments, the RNA molecule comprises a nucleotide overhang at the 3' end of the sense strand and a flat end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNA molecule comprises a nucleotide overhang at the 3' end of the antisense strand and a flat end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNA molecule comprises a flat end at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and the antisense strand have the same length, and the length of the duplex region is the same as that of the sense strand and the antisense strand (i.e., the molecule is double-stranded over its entire length). As used herein, "GalNAc" or "N-acetylgalactosamine": refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term "GalNAc" or "N-acetylgalactosamine" includes both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Preferably, the GalNAc compound of the present application is the β form, i.e., 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0332] As used herein, "VP modification" or "E-VP" refers to a phosphate mimic modification located at the 5' end of the antisense strand, with the specific structure being:

[0333] in:

[0334] Bx1 is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine;

[0335] T2 is a phosphate or phosphorothioate internucleoside linking group that connects the above-mentioned compound to the oligonucleic acid chain; and G is a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2.

[0336] As used herein, "3' end" refers specifically to the position of the first nucleotide or base pair at the 3' end of a single nucleotide sequence or a double-stranded polynucleotide. "5' end" refers specifically to the position of the first nucleotide or base pair at the 5' end of a single nucleotide sequence or a double-stranded polynucleotide.

[0337] As used herein, the term "nucleic acid molecule" may be used to refer to any molecule having a nucleotide sequence composed of two or more nucleotides linked by a phosphate bond, or a modified phosphate bond (eg, a phosphorothioate bond).

[0338] As used herein, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in a specific order. This polynucleotide chain can constitute a nucleic acid molecule or a segment of a chain of a nucleic acid molecule. Therefore, a "nucleotide sequence" can be represented by a specific polynucleotide sequence composed of various nucleotides (e.g., ATCG) (e.g., any of SEQ ID NOs: 1 to 100), or it can be represented by nucleotides from a certain position to a certain position in a sequence, such as "nucleotides from positions 363 to 382 in the FXI gene mRNA sequence." In this application, unless otherwise specified, in a sequence or nucleic acid molecule comprising "nucleotides from a certain position to a certain position in a sequence," the order of nucleotides from a certain position to a certain position is consistent with the order of the aforementioned nucleotides in the sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid molecule of RNA and DNA, and can also incorporate non-natural nucleotides or artificially modified nucleotides.

[0339] In addition, the REL or Relative expression level appearing in the drawings of this application refers to the relative expression level of mRNA. In the sequence context, N represents a ribonucleotide, dN represents a deoxyribonucleotide (DNA), Nm represents a 2'-O-Me modified nucleotide or a 2'-O-methyl modified nucleotide; Nf represents a 2'-F modified ribonucleotide or a 2'-fluoro modified ribonucleotide; s represents a thiophosphate modification, that is, a 5'-thio modified phosphate. When the modification is between two nucleotides or between a nucleotide and a ligand, it replaces the phosphate bond between natural nucleotides and can be called a thiophosphate bond. As used herein, unless otherwise specified, "phosphorothioate bond" and "phosphothiodiester bond" in this application can be used interchangeably; similarly, "phosphate bond" and "phosphodiester bond" can be used interchangeably.

[0340] As used herein, the term "about" refers to the typical error range for each value that is readily known to those skilled in the art. Reference to an "about" value or parameter herein includes (and describes) embodiments directed to that value or parameter itself. As used herein, when the term "about" precedes a numerical value, it means within a range of 10% above or below that numerical value. For example, "about 100" encompasses 90 and 110.

[0341] As used herein, the singular forms "a," "an," and "the" include plural forms unless otherwise indicated.

[0342] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0343] It should be understood that the present application includes various aspects, embodiments, and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Within the scope of the technical concept of the present application, the technical solution of the present application can be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the contents disclosed in the present application and all fall within the scope of protection of the present application.

[0344] Unless otherwise indicated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are well documented in the art.

[0345] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments. Example

[0346] Example 1: Sequence Design and Synthesis of dsRNA

[0347] In specific Examples 1-7 of the present application, each nucleotide sequence does not contain modifications not indicated in the examples. That is, each nucleotide sequence used in Examples 1-7 of the present application is a naked sequence or contains only the modifications indicated in the modification motif used.

[0348] 1.1 dsRNA design

[0349] With reference to the human FXI gene (Gene ID, 2160) mRNA sequence (NM_000128.4), multiple FXI dsRNAs were designed at different sites. All designed single dsRNAs had the lowest homology with all other non-target gene sequences after sequence similarity comparison using sequence similarity software. For details, please refer to the sequence listing at the end of the article. In addition, for modified dsRNA, if the first nucleotide at the 3' end of the sense strand is not A or U, a uracil ribonucleotide is used to replace the original nucleotide; if the first nucleotide at the 5' end of the antisense sequence is not A or U, an adenine ribonucleotide is used to replace the original nucleotide, and the original nucleic acid sequence is changed from n to n' for detection. For example, the nucleic acid sequences of E3, E4, E9, E14, E16, E24, E26, E51, E52, E69, and E70 are shown in the dsRNA numbered 3', 4', 9', 14', 16', 24', 26', 51', 52', 69', and 70' in the sequence listing at the end of the article, respectively.

[0350] In this application, we selected the ASO sequence of ISIS-416858 disclosed in the patent of ionis (patent number: CN 109797150 A), the top 4 sequences of FXI gene inhibition rate disclosed in patent number: CN113227376A (naked sequences named: RB2, RB4, RB6, RB7; modified sequences named: ERB2, ERB4, ERB6, ERB7, the naked sequences and modified sequences correspond to the sequences without ligand coupling in the molecules named L10-siFXIa1M1SP, L10-siFXIc1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP in the patent, respectively), and selected patent number: WO The top three sequences for FXI gene inhibition rates disclosed in patent 2022028457A1 (naked sequences named: TJ632, TJ645, and TJ772; modified sequences named: ETJ632, ETJ645, and ETJ772; the naked and modified sequences correspond to the ligand-free sequences in the patent molecules named TRD0632, TRD0645, and TRD0772, respectively) served as FXI gene positive control sequences. The naked sequences of the aforementioned dsRNA sequences and positive control sequences are shown in the sequence listing at the end of the article.

[0351] 1.2 Synthesis and purification of dsRNA and its conjugates

[0352] The dsRNA was synthesized using modified (e.g., 2'-methoxy or 2'-fluoro or thio modified) or unmodified ribonucleotides, and the synthesis was completed according to the theoretical yield of 1 μmol, using 1 μmol of a universal Frit vector ( All oligonucleotides were prepared on an LK-192X synthesizer (Jiangsu Lingkun Biotechnology Co., Ltd.) using CPG vectors (Ailiying) or the protected GANLNAC derivative L96. According to sequence requirements, all phosphoramidite monomers corresponding to the nucleoside were diluted in anhydrous acetonitrile at a ratio of 1:40 (g / mL) and coupled twice for 3 minutes. Deprotection was performed using 3% TCA, activation was performed using 0.3 M benzylthiotetrazolyl in acetonitrile, and capping and oxidation were performed using CAPA / CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated in a 65°C oven for 3 hours to remove the protecting groups. The tube was then cooled to room temperature and concentrated under vacuum for 30 minutes. The solution was then filtered through a 0.22 μm filter into a vial. Single-stranded fragments were purified using a semi-preparative reverse-phase instrument with an elution gradient of 7% to 30% (ACN:100 mM TEAA) over 10 minutes at a flow rate of 5 mL / min. After purification, the fragment was concentrated under vacuum and dried at room temperature. Finally, the sample was dissolved in water and purified on a GE Each solution was desalted on a Hi-Trap desalting column to elute the final oligonucleotide product. All identities and purity were confirmed using ESI-MS and IEX HPLC, respectively. Concentration was determined using a UV-visible microplate reader. Equimolar amounts of the sense and antisense strands were mixed and transferred to a new shipping tube. The mixture was heated at 95°C for 5 minutes, slowly annealed to room temperature, and finally dried using a vacuum concentrator at room temperature to yield the final dsRNA product.

[0353] The structure of L96 used in the examples of this application is shown in Formula I below:

[0354] in, It represents a phosphodiester bond attached to the 3' end of the sense or antisense strand of an RNA molecule.

[0355] Example 2: High-throughput screening assay for FXI-dsRNA in vitro activity

[0356] 2.1 FXI dsRNA transfection into HepG2 cells

[0357] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator. After cell digestion, the cell density was adjusted to 4×10 5Cells were plated at 1 ml per well at 12-well plates. Prepare 100 ul of transfection complex: 45 μL of Opti-MEM and 5 μL of dsRNA (naked nucleic acid, unmodified) of different concentrations were mixed, 48 μL of Opti-MEM and 2 μL of RNAiMax transfection reagent were mixed, and the two mixtures were mixed and allowed to stand for 20 minutes to form a transfection complex. The above transfection complex was added to a 12-well plate and cultured in a 5% CO2, 37°C constant temperature incubator for 24 hours. Cells were harvested 24 hours after transfection, and RNA was extracted using the TRIZOL method.

[0358] 2.2 Real-time fluorescence quantitative PCR analysis

[0359] 24 hours after transfection, cells were lysed and total RNA was extracted using the Novagen FastPure Cell / Tissue Total RNA Isolation Kit V2 (refer to the Novagen RC112-01 instructions). Reverse transcription was performed into cDNA using Takara PrimeScript RT Master Mix RR036Q. qPCR primer sequences are listed in the primer section at the end of the article. PCR reactions were performed using a Bio-Rad CFX96 fluorescence quantitative PCR instrument, using the human GAPDH gene as the internal reference gene. Mock controls were used for normalization, assuming FXI mRNA expression in the mock group was equal to 1.

[0360] 2.3 Data Analysis

[0361] After the PCR reaction, relative quantitative analysis was performed using the reference gene as a standard and statistical analysis was performed using CFX96 software and GarphPad software. As shown in Table 1 and Figure 1, the results of high-throughput screening and validation of dsRNA (naked nucleic acid, unmodified) molecules in HepG2 cells are shown.

[0362] Table 1: Results of high-throughput screening of single-dose 5nM in HepG2 cells

[0363] Screening of FXI dsRNA in HepG2 cells identified 28 optimal sequences. dsRNA molecules with high efficacy and targeting both human and cynomolgus macaque FXI were retained as candidate sequences. We further compared several candidate Yangshen sequences with our candidate sequences. Table 2 and Figure 2 show the results of single-dose screening of candidate sequences and positive control sequences in HepG2 cells.

[0364] Table 2: Single-dose screening of unmodified dsRNA in HepG2 cells

[0365] The results showed that among the 7 candidate Yang Shen, TJ632 showed the best effect, and many of our candidate sequences showed superior or equivalent effects to Yang Shen TJ632, such as dsRNA No. 3, 5, 8, 9, 24, 26, 50, 70, 82, etc.

[0366] Example 3: Optimization of FXI-dsRNA

[0367] 3.1 Inhibitory activity detection

[0368] To further confirm the preferred dsRNA molecules, we used two different modification motifs on the above preferred sequences, and performed fluorination and methoxy modification combinations on different positions of the candidate sequences.

[0369] Specific modifications are selected from any of the following:

[0370] (1) Modification of E (dsRNA modified with E is referred to as En, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as E3 after modification with E):

[0371] Sense sequence: starting from the 5' end, the 7th, 9th, 10th, and 11th positions are 2'-fluoro-modified nucleotides, and the other positions are 2'-O-methyl-modified nucleotides, and the first and second phosphate bonds at the 5' end are thiolated to form phosphorothioate bonds; for example, with N representing any ribonucleotide, the modification of the sense sequence is: NmsNmsNmNmNmNmfNmNfNfNmNmNmNmNmNmNmNmsNmsNm;

[0372] Antisense sequence: the 2nd, 6th, 14th and 16th positions starting from the 5' end are 2'-fluoro-modified nucleotides (in other words, the 2nd, 6th, 14th and 16th positions starting from the 3' end of the complementary region between the antisense sequence and the sense chain are 2'-fluoro-modified nucleotides), the other positions are 2'-O-methyl-modified nucleotides, and the first and second phosphates at the 5' and 3' ends are thiolated to form a phosphorothioate bond; for example, if N represents any ribonucleotide, the modification of the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm.

[0373] (2) Modification E-1 (dsRNA modified with E-1 is referred to as En-1, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as E3-1 after modification with E),

[0374] The only difference between modification E-1 and E1 modification is that the sixth nucleotide from the 5' end of the antisense strand of the E-1 modified dsRNA is a 2'-O-methyl modified nucleotide instead of a 2'-fluoro modified nucleotide, i.e., for example:

[0375] The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm;

[0376] The modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm.

[0377] In the sequence modifications of the present application, s represents a phosphorothioate bond, m represents a 2′-O-methyl modification, and f represents a 2′-fluoro modification.

[0378] In addition, the antisense compound ISIS-416858 adopts the structure and modification method disclosed in CN109797150A, namely the "5-10-5" structure, with 5 2'-O-methoxyethyl modified nucleotides on each flank, 10 deoxynucleotides in the middle, and all-thio and methylcytosine replacing cytosine.

[0379] The transfected cells were HepG2 cells, and the synthesis, transfection, and quantitative PCR detection steps were the same as in Example 2. Table 3 and Figure 3 show the average target gene expression levels relative to the blank control group (the relative mRNA expression level of the blank control group was set to 1).

[0380] Table 3: Single-dose (1 nM) screening of modified dsRNA in HepG2 cells

[0381] As can be seen, compared to the chemically modified positive control sequence, many of our candidate sequences, such as E8, E14, E19, E24, E26, and E50, still showed equivalent or superior efficacy to the best Yangshen ETJ632. This result further demonstrates that the selected sequences have the potential to effectively reduce FXI gene nucleic acid levels.

[0382] Example 4 In vitro effectiveness detection of GalNAc-dsRNA

[0383] The candidate modified dsRNA is subjected to GalNAc coupling to form a GAL-dsRNA complex, that is, each dsRNA is subjected to G modification.

[0384] The G modification refers to conjugating L96 to the 3' end of the dsRNA sense sequence, including GE modification and GE-1 modification, wherein:

[0385] The GE modification is shown below, where "-L96" indicates that L96 is conjugated to the 3' end of the dsRNA sense sequence:

[0386] The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96;

[0387] The modification of the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0388] The dsRNA modified by GE is referred to as GEn, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as GE3 after GE modification;

[0389] The GE-1 is shown below:

[0390] The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96;

[0391] The modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0392] Similarly, "-L96" indicates that L96 is conjugated to the 3' end of the dsRNA sense sequence through a phosphate bond. The dsRNA modified with GE-1 is referred to as GEn-1, where n is the number of the candidate dsRNA molecule. For example, dsRNA molecule No. 3 is referred to as GE3-1 after modification with GE-1.

[0393] We further validated the activity of the G-modified candidate complexes in HepG2 cells.

[0394] 4.1 Detection of mRNA expression levels in HepG2 cells

[0395] The detection steps were the same as in Example 2, using the human GAPDH gene as the internal reference gene. The detection primers used are listed in the primer section of the sequence listing at the end of the article. Table 4 shows the average mRNA levels of the target gene expression levels in HepG2 cells treated with G-modified dsRNA relative to the untreated group (the relative mRNA expression level of the untreated group was set to 1). The statistical results are shown in Figure 4.

[0396] Table 4 IC50 data of some GalNAc-coupled dsRNA in HepG2 cells

[0397] It can be seen that the IC50 of GalNAc-coupled dsRNA in HepG2 cells is lower than 0.1 nM, which once again proves the efficient inhibitory activity of the candidate sequence on the target gene.

[0398] Example 5 In vivo effectiveness test

[0399] The study used 6- to 8-week-old SPF-grade humanized FXI homozygous male mice (Shanghai Model Organism Laboratory Animal Co., Ltd.) and randomly divided them into groups of six. Each group received a single subcutaneous injection of different GE-modified dsRNAs, while the blank group received saline. Blood was collected from the back of the eye on days 7 and 13 after administration for serum analysis of FXI protein levels.

[0400] The specific dosage is shown in Table 5.

[0401] Table 5 3mpk single dose regimen

[0402] 5.1 FXI protein ELISA detection

[0403] After serum was diluted 1:500, the residual FXI content in the plasma of each group was detected using the Abcam ab108834 ELISA detection kit. For specific operating steps, please refer to the ab108834 ELISA test instructions. The absorbance value was measured at a wavelength of 450 nm using a microplate reader and analyzed using GraphPad software. The percentage inhibition rate is a relative result relative to the non-drug treatment group. The results are shown in Table 6. Figure 5 shows the relative expression level of FXI protein in the plasma of each group after normalization with the blank control group.

[0404] Table 6: Inhibition rate of plasma FXI protein after a single dose of 3 mpk

[0405] As can be seen, after a single dose of 3 mpk, the plasma FXI levels in mice treated with the candidate dsRNA molecules were significantly lower than those in the blank control group. On day 13, the inhibition rates of GE26, GE50, and GE70 exceeded 80%, with GE50 exhibiting the highest inhibition rate of 92.8%. Furthermore, the inhibition rates in all treatment groups were >50% on day 13. These results demonstrate that the multiple dsRNA molecules designed to target FXI effectively inhibit the expression and secretion of FXI protein in the liver, making them potential therapeutic agents targeting the FXI gene.

[0406] Example 6 In vivo efficacy evaluation experiment of different ligands

[0407] We further validated the effects of different ligand conjugates on candidate dsRNAs. Table 7 shows the dsRNA sequence information. These dsRNAs were modified with either LN-E05 or GN-E05. The LN-E05 modification was similar to the GE-1 modification described in Example 4. The specific modifications for GN-E05 are as follows:

[0408] The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1;

[0409] The modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0410] That is, the only difference between the GN-E05 modification and the LN-E05 modification is that the L96 ligand conjugated to the 3' end of the sense chain is replaced with ligand 1, and ligand 1 is linked to the 3' end of the sense chain through a phosphorothioate bond.

[0411] Table 7 Synthetic oligonucleotides

[0412] Groups were divided according to Table 7 and administered a single subcutaneous injection of 3 mpk. Blood was collected at various time points after administration, and plasma was used for FXI protein analysis. The residual percentage is relative to the pre-drug dose. The results are shown in Figure 6. Following a single 3 mpk administration, the inhibition rate of FXI protein expression by dsRNA linked to ligand 1 was significantly higher (p<0.05 using a paired T-test) than that by dsRNA linked to L96.

[0413] The structure of ligand 1 is shown in Formula II:

[0414] The wavy line indicates the connection to the 3' end of the sense strand of the dsRNA via a phosphorothioate bond. The molecule of Formula II is a trivalent GalNac conjugate prepared from two compounds 6 and one compound 8 using conventional raw materials and synthetic methods in the art. The synthetic methods of compounds 6 and 8 are as follows:

[0415] 1. Synthesis of Compound 6

[0416] ①Synthesis of compound 2:

[0417] 1.2 g of compound 1 and 12 mL of pyridine were added to a three-necked flask. After stirring and dissolving, 1.2 g of DMTrCl was added and stirred at room temperature for 1 hour under nitrogen protection. Methanol (0.6 mL) was slowly added to the reaction mixture to quench the excess DMTrCl. After stirring at room temperature for 15 minutes, 303.5 mg of NaHCO3 was added. After concentration, the crude product was obtained, DCM / H2O was added, stirred and dissolved, and washed with water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product of compound 2, which was directly used in the next step reaction.

[0418] ②Synthesis of compound 3:

[0419] Add ethanol (24 mL) to the crude compound 2, slowly heat to 50 ° C, stir and dissolve, then add hydrazine hydrate (0.75 mL), continue stirring at 50 ° C overnight, then cool to room temperature, and continue stirring at room temperature for half an hour, at which time a large amount of white solid gradually precipitates. Filter, and wash the filter residue with ethanol. The obtained filtrate is concentrated under reduced pressure, then redissolved in DCM / H2O, separated, and the organic phase is separated. It is washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The collected organic phase is concentrated under vacuum to give the crude compound 3, which is directly used in the next reaction.

[0420] ③Synthesis of compound 5:

[0421] To the crude compound 3, 0.9 g of compound 4, DCM (24 mL), 0.84 mL of Et3N, and 2.3 g of HBTU were added in sequence and stirred at room temperature under nitrogen for 2 hours. 9 mL of water was added to quench the reaction, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by reverse phase preparation (75% ACN-H2O) to give 1.4 g of compound 5, with a three-step yield of 44.2%. ESI-MS: m / z 1025.6 [M + OAc - ] - .

[0422] ④Synthesis of compound 6:

[0423] Dissolve 1.4 g of compound 5 in DCM (14 mL) and cool at 0±2°C. Add 524.7 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, followed by 81.3 mg of 1H-tetrazole. Stir the reaction mixture at 0±2°C for 15 minutes, then warm to room temperature and continue stirring for 2 hours. Cool the reaction mixture at 0±2°C and quench with 14 mL of 5% NaHCO3. Separate the organic phase, wash with saturated brine (1 x 14 mL) at 0±2°C, dry over anhydrous sodium sulfate, and filter. Concentrate the collected organic phase under vacuum, and the resulting crude product is redissolved in DCM / MTBE and carefully added dropwise to a vigorously stirred solution of heptane. A large amount of viscous oil will gradually precipitate from the walls and bottom of the flask. After standing for 10 minutes, decanter the supernatant. The crude product was chromatographed using EA:hept. = 10:1 to 2:1 eluent, concentrated and dried to give 560 mg of a white solid powder with a yield of 32% and a phosphorus purity of 98.19%. ESI-MS: m / z 1225.4 [M + OAc - ] - .

[0424] 2. Synthesis of Compound 8

[0425] ①Synthesis of compound 7:

[0426] 2 g of compound 5 was dissolved in DCM (20 mL), followed by the addition of 248.4 mg of succinic anhydride and 0.69 mL of Et3N. Finally, 25.3 mg of DMAP was added. After stirring at room temperature for 16 hours, HPLC analysis indicated a significant residual starting material. 310.4 mg of succinic anhydride and 0.69 mL of Et3N were added. Finally, 25.3 mg of DMAP was added, and stirring continued at room temperature for 36 hours. The reaction mixture was cooled to 0±2°C, ice water was added, followed by DCM, and finally 40 mL of 1% aqueous HOAc solution. After stirring for 10 minutes, the organic phase was separated and washed sequentially with 1% aqueous HOAc solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to yield the crude product. The crude product was chromatographed using a DCM:MeOH ratio of 70:1 to 20:1 eluent. After concentration and drying, 0.9 g of a white solid powder was obtained, yielding 40.7%. No succinic acid residue was detected, and the residual succinic anhydride was 0.26%. ESI-MS: m / z 1065.7[M - H] - .

[0427] ②Synthesis of compound 8:

[0428] To a three-necked flask, 300 mg of compound 7, 72.7 mg of DIPEA, 106 mg of HBTU, and 10 mL of acetonitrile were added. After stirring at 25°C for 10 minutes, 650 mg of solid support PS was added and stirring continued at 25°C for 24 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with acetonitrile, and the filter cake was collected and concentrated under vacuum to remove the solvent, yielding 850 mg of solid. 700 mg of the solid was added to a three-necked flask, followed by 1.74 g of acetic anhydride, 4.15 mg of DMAP, 103 mg of triethylamine, and 10 mL of pyridine, and stirred at 25°C for 4 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with acetonitrile, methanol, and acetonitrile, and the filter cake was collected and concentrated under vacuum to remove the solvent, yielding 750 mg of compound 8. The loading was determined to be 254.82 μmol / g.

[0429] 3. Preparation of oligonucleotide sequence linked to ligand 1

[0430] Compound 8 is sequentially reacted with two or three compounds 6, and through conventional solid-phase synthesis cycles (deprotection, coupling, oxidation, and capping), a precursor (or intermediate) of ligand 1 attached to a solid support is obtained. Further solid-phase synthesis cycles are performed using the corresponding 2'-modified monomers. After completion of the reaction, the entire synthesized molecule is dissociated from the solid support by aminolysis, yielding the oligonucleotide sequence attached to ligand 1.

[0431] Example 7 In vitro and in vivo efficacy evaluation experiments of different modified motifs

[0432] We further validated the effects of different modification motifs in dsRNAs with the same base sequence as double-strand number 50 and double-strand number 26. The specific molecules used are listed in Table 8. As can be seen, all molecules in Table 8 were linked to Ligand 1 via a phosphorothioate bond at the 3' end of the sense strand, while the 5' terminal nucleotide of the antisense strand was modified with 5'-vp.

[0433] Table 8: Synthetic oligonucleotides

[0434] The modified motifs in Table 8 are as follows:

[0435] GN-E20VP:

[0436] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1

[0437] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0438] GN-E04VP:

[0439] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

[0440] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0441] GN-E05VP:

[0442] Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

[0443] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

[0444] The modification of GN-E05 was the same as in Example 6. The only difference between it and the GN-E05 VP modification was that GN-E05 did not have a vinyl phosphate modification at the 5' end of the antisense strand.

[0445] 7.1 In vitro efficacy evaluation of different modified motifs

[0446] First, we verified the effects of the different modifications listed in Table 8 on drug efficacy in vitro using HepG2 cells. The experimental methods were similar to those in Example 4. Table 4 shows the average mRNA levels of each treated group relative to the target gene expression levels of the untreated group (the relative mRNA expression level of the untreated group was set to 1). The in vitro experiments were performed independently in different batches, and the statistical results are shown in Tables 9 and 10.

[0447] Table 9 IC50 data of 5VP modified dsRNA in HepG2 cells

[0448] Table 10 IC50 data of different motif-modified dsRNA in HepG2 cells

[0449] The in vitro results, shown in Table 9 and Figure 7, demonstrate that modification of 5-VP further enhances its efficacy. Furthermore, given the same ligand and base sequences, the modified motifs of GN-E05VP and GN-E20VP significantly outperformed those of GN-E04VP (shown in Table 10). Furthermore, the modified motif of GN-E20VP also significantly outperformed that of GN-E05VP.

[0450] 7.2 In vivo efficacy evaluation of different modified motifs

[0451] The experimental protocol followed that of Example 5. For each group, as shown in Table 8, dsRNA No. 50 with various modifications was administered as a single subcutaneous injection at 1 mpk. Blood was collected on days 7, 14, 21, and 28 after administration, and plasma was assayed for FXI protein content. The residual percentages are relative to the pre-drug dose, as shown in Table 11 and Figure 8.

[0452] Table 11: Residual rate of FXI protein in plasma after single administration of 1 mpk

[0453] Molecular results from modified sample 50 show that, at a single dose of 1 mpk, with identical dsRNA base sequences, the E05VP and E20VP modification motifs outperformed E04VP (P < 0.05), with E20VP further outperforming E05VP (P < 0.05). Furthermore, the results suggest that 5'-VP modification can further enhance drug efficacy.

[0454] We further compared the in vivo efficacy of molecule 26 with different modified motifs (as shown in Table 8). Blood samples were collected on days 7 and 14 after dosing, and plasma was used to measure FXI protein levels. The residual percentage is relative to the predrug concentration, as shown in Table 12.

[0455] Table 12: Residual rate of FXI protein in plasma after single administration of 1 mpk

[0456] Molecule 26 once again demonstrated that E05VP and E20VP were more effective than E04VP, with the E20VP motif being the most effective. Furthermore, modification of the 5'-VP motif further enhanced efficacy.

[0457] The sequences used in the above examples of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of this application and do not limit the present invention in any way. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When they represent RNA sequences, "T" represents uridine. Furthermore, in the context of RNA, unless otherwise specified, "T" and "U" both refer to uracil or uridine.

[0458] Sequence Listing:

Claims

1. A nucleic acid molecule that inhibits the expression of factor XI (FXI) gene in cells by RNAi, which comprises a sense sequence and an antisense sequence that are complementary to each other or consists of a sense sequence and an antisense sequence that are complementary to each other, and the antisense sequence contains a polynucleotide sequence complementary to the mRNA of the FXI gene, wherein, when the antisense sequence hybridizes with the mRNA of the FXI gene at the maximum complementary rate, a complementary region 1 is composed of the base pairs complementary paired in the antisense sequence and the mRNA sequence of the FXI gene; when the antisense sequence hybridizes with the sense sequence at the maximum complementary rate, a complementary region 2 is composed of the base pairs complementary paired in the antisense sequence and the sense sequence; wherein the complementary region 1 and the complementary region 2 have at least 18, 19 or 20 identical base pairs, the number of base pairs in the complementary region 1 is 18 to 30 bp, and the complementary region 1 contains in the mRNA sequence of the FXI gene: nucleotides at positions 418 to 437 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 1141 to 1160 or 18 or 19 consecutive nucleotides thereof; nucleotides at positions 363 to 382 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 372 to 391 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 411 to 430 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 421 to 440 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 422 to 441 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 425 to 444 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 465 to 484 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 470 to 489 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 471 to 490 or 18 or 19 consecutive nucleotides thereof, nucleotides at positions 518 to 537 or 18 or 19 consecutive nucleotides thereof, or wherein the site numbers of the nucleotides in the mRNA sequence of the FXI gene are the numbers corresponding to the nucleotides in the reference sequence SEQ ID NO:

244.

2. The nucleic acid molecule according to claim 1, wherein the number of base pairs in the complementary region 1 is 20, 21, 22 or 23 bp.

3. The nucleic acid molecule according to claim 1 or 2, wherein the number of base pairs in the complementary region 2 is 21 bp.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the complementary region 1 and the complementary region 2 have 20 or 21 identical base pairs.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein the length of the sense sequence is 21 nt and the length of the antisense sequence is 23 nt.

6. The nucleic acid molecule according to any one of claims 1 to 5, wherein the sense sequence and / or the antisense sequence further comprises 1 to 2 overhanging end nucleotides outside the complementary region 2.

7. The nucleic acid molecule according to claim 6, wherein the number of overhanging end nucleotides is 2 and they are located in the antisense sequence, adjacent to the 5' end of the complementary region 2, and the sense sequence does not contain overhanging end nucleotides.

8. The nucleic acid molecule according to any one of claims 1 to 7, wherein the antisense sequence contains only 0, 1 or 2 nucleotides in the region outside complementary region 1, and the sense sequence contains only 0, 1 or 2 nucleotides outside complementary region 2.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the 3'-end of complementary region 2 is an AU base pair.

10. The nucleic acid molecule according to any one of claims 1 to 9, wherein the FXI gene is a human FXI gene or a cynomolgus monkey FXI gene.

11. The nucleic acid molecule according to claim 10, wherein the mRNA contains the polynucleotide sequence shown in SEQ ID NO:

244.

12. The nucleic acid molecule according to any one of claims 1 to 11, wherein the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 153, 177, 152, 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 200, 201 or 213.

13. The nucleic acid molecule according to any one of claims 1 to 12, wherein: the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 34, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 153; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 33, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 152; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 3, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 122; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 4, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 123; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 7, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 126; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 10, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 129; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 11, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 130; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 12, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 131; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 17, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 136; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 18, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 137; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 138; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 24, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 143; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 30, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 149; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 31, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 150; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 81, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 200; The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 82, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 201; or The sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 94, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO:

213.

14. The nucleic acid molecule according to any one of claims 1 to 13, which is dsRNA or shRNA.

15. The nucleic acid molecule according to claim 14, which is siRNA.

16. The nucleic acid molecule according to any one of claims 1 to 15, wherein one or more nucleotides are chemically modified.

17. The nucleic acid molecule according to any one of claims 1 to 16, wherein the chemical modification is selected from any one or more of the following: Locked nucleic acid modification, open-ring or non-locked nucleic acid modification, 2'-methoxyethyl modification, 2'-O-methyl modification, 2'-O-allyl modification, 2'-C-alkyl modification, 2'-C-allyl modification, 2'-fluoro modification, 2'-deoxy modification, phosphorothioate bond modification, 2'-amino modification, morpholino modification, aminophosphonate modification, methylphosphonate modification, tetrahydropyran modification, 1,5-anhydrohexitol modification, 5'-vinyl phosphate modification, and cyclohexenyl modification; Preferably, the 7th, 9th - 12th nucleotides of the sense strand of the nucleic acid molecule comprise 2'-fluoro modification and / or the 2nd, 14th, 16th nucleotides of the antisense strand comprise 2'-fluoro modification; more preferably, the remaining nucleotides comprise 2'-O-methyl modification; Preferably, the 7th, 9th - 11th nucleotides of the sense strand of the nucleic acid molecule comprise 2'-fluoro modification and / or the 2nd, 14th, 16th nucleotides of the antisense strand comprise 2'-fluoro modification; more preferably, the remaining nucleotides comprise 2'-O-methyl modification; Preferably, the 7th, 9th - 11th nucleotides of the sense strand of the nucleic acid molecule comprise 2'-fluoro modification, and the 2nd, 6th, 14th, 16th nucleotides of the antisense strand comprise 2'-fluoro modification; more preferably, the remaining nucleotides comprise 2'-O-methyl modification; Preferably, the nucleic acid molecule comprises any one of the following motifs: (1) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm, (2) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm, and (3) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm, Among them, Nm represents a ribonucleotide modified with 2'-O-methyl, and Nf represents a ribonucleotide modified with 2'-fluoro; More preferably, between the first and second nucleotides at the 5' end of the sense sequence, between the second and third nucleotides, between the penultimate and the last nucleotide, and between the antepenultimate and the penultimate nucleotide, there is at least one phosphorothioate modification; between the first and second nucleotides at the 5' end of the antisense sequence, between the second and third nucleotides, between the penultimate and the last nucleotide, and between the antepenultimate and the penultimate nucleotide, there is at least one phosphorothioate modification.

18. The nucleic acid molecule according to claim 17, wherein the nucleic acid molecule comprises any one of the following motifs: (1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; and (6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (7) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (8) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (9) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; Among them, Nm represents a ribonucleotide modified with 2'-O-methyl, Nf represents a ribonucleotide modified with 2'-fluoro, and s represents a phosphorothioate modification.

19. The nucleic acid molecule according to any one of claims 1 to 18, which is linked to at least one asialoglycoprotein receptor (ASGPR) ligand.

20. The nucleic acid molecule according to claim 19, wherein the ASGPR ligand is linked to the 5'-end or 3'-end of the sense sequence.

21. The nucleic acid molecule according to claim 19 or 20, wherein the ASGPR ligand is one or more GalNAc derivatives linked by a divalent or trivalent branched structure.

22. The nucleic acid molecule according to claim 21, wherein the GalNac derivative is L96 or ligand 1, and the structure of L96 is shown as follows: The structure of the ligand 1 is shown as follows: Wherein in Formula I and Formula II Respectively represent linkage to the 3'-end of the sense sequence or antisense sequence of the nucleic acid molecule; preferably, linked to the 3'-hydroxyl group through a phosphodiester bond or phosphorothioate bond; More preferably, the nucleic acid molecule has any of the following modification motifs: (1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

23. The nucleic acid molecule according to claims 1-22, wherein the nucleic acid molecule comprises a phosphate or phosphate mimetic modification at the 5'-end of the antisense strand; preferably, the 5'-phosphate mimetic is 5'-VP modification; More preferably, the nucleic acid molecule has any of the following modification motifs: GN-E20VP: Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms - Ligand 1; Antisense sequence: vp - NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; GN - E04VP: Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms - Ligand 1; Antisense sequence: vp - NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; GN - E05VP: Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms - Ligand 1; Antisense sequence: vp - NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

24. A second nucleic acid molecule which can be transcribed into a dsRNA or shRNA precursor in a cell, wherein the dsRNA or shRNA is the nucleic acid molecule according to any one of claims 1 - 23.

25. A viral particle which comprises the polynucleotide sequence of the second nucleic acid molecule according to claim 24.

26. A cell which comprises the polynucleotide sequence of the second nucleic acid molecule according to claim 24.

27. Use of the nucleic acid molecule according to any one of claims 1 to 23 or a salt thereof for inhibiting the expression of factor 11.

28. Use of the nucleic acid molecule according to any one of claims 1 to 23 or a salt thereof for preparing a medicament for inhibiting the expression of FXI in a subject.

29. A pharmaceutical composition which comprises the nucleic acid molecule according to any one of claims 1 to 23 or a salt thereof, the second nucleic acid molecule according to claim 24, the viral particle according to claim 25, or the cell according to claim 26.

30. Use of the nucleic acid molecule according to any one of claims 1 to 23 or a salt thereof, the second nucleic acid molecule according to claim 24, the viral particle according to claim 25, or the cell according to claim 26 or the pharmaceutical composition according to claim 29 for preparing a medicament for preventing or treating thromboembolic complications or coagulation disorders in a subject, wherein the thromboembolic complications are preferably one or more selected from deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

31. A method for treating or preventing thromboembolic complications or coagulation disorders in a subject, wherein the thromboembolic complications are preferably one or more selected from deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke, and the method comprises administering to a subject in need thereof an effective amount of a nucleic acid molecule or a salt thereof according to any one of claims 1 to 23, a second nucleic acid molecule according to claim 24, a viral particle according to claim 25, a cell according to claim 26, or a pharmaceutical composition according to claim 29.

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