Nucleic acid molecules that inhibit F11 gene expression

Abstract

The present invention relates to a nucleic acid molecule that inhibits intracellular factor 11 (FXI) gene expression via RNAi, comprising mutually complementary sense sequences and antisense sequences or composed of mutually complementary sense sequences and antisense sequences, and the inhibition rate of said nucleic acid molecule against FXI is significantly superior to that of other small nucleic acid molecules that inhibit FXI expression via RNAi.

Description

Technology Field

[0001] The present application claims priority to patent application number 202410114595.7, filed with the National Intellectual Property Administration of China on January 26, 2024, with the invention title “Nucleic acid molecule inhibiting F11 gene expression,” and patent application number 202410568807.9, filed with the National Intellectual Property Administration of China on May 9, 2024, with the invention title “GalNAc derivative and oligonucleotide conjugate thereof.” The full text of the two prior applications is incorporated into the present application by means of citation.

[0002] The present application relates to the field of RNAi, specifically to a nucleic acid molecule capable of suppressing F11 gene expression via RNAi and its uses. Background Technology

[0003] RNA interference (RNAi) refers to the phenomenon in which homologous mRNA is efficiently and specifically degraded by double-stranded RNA (dsRNA), which is highly conserved during evolution. dsRNA is typically 19 to 30 bp in length and is one of the key tools in RNAi technology. In natural organisms, long dsRNA typically enters the cell, is specifically recognized by Dicer enzymes, and cleaved into small RNA fragments (i.e., siRNA) approximately 21 to 23 nucleotides long. These cleaved dsRNA fragments then unwind into single strands to form complexes (abbreviated as RISC) with specific proteins. Within the cell, RISC binds to mRNA complementary to the antisense strand of the dsRNA, cleaving and degrading the mRNA to render protein synthesis impossible, thereby causing a gene “silencing” phenomenon. In industrial production, there is a tendency to chemically synthesize and modify dsRNA to further enhance the stability and efficacy of dsRNA drugs.

[0004] A thrombus is a mass of blood coagulation that restricts blood flow within blood vessels; it can occur in arterial or venous circulation and is the common pathological basis for most myocardial infarctions, ischemic strokes, and venous thromboembolism (VTE). The primary targets of currently used anticoagulants are thrombin and FX (also known as blood coagulation factor 10), which are equally important in hemostasis and thrombus formation. Attempting to exert an anticoagulant effect by inhibiting both inevitably affects the hemostasis process. This contradiction limits the therapeutic intensity of anticoagulants, implying that anticoagulant therapy may not be suitable for patients at high risk of bleeding. There is a need for new anticoagulants that can achieve clinical anticoagulant effects while simultaneously reducing the risk of bleeding, and blood coagulation factor XI (FXI) is emerging as a new anticoagulant target. Compared to FX, FXI (also called blood coagulation factor FXI or F11) plays an auxiliary role in hemostasis but plays an essential role in thrombus formation, and spontaneous bleeding, central nervous system or gastrointestinal bleeding is rarely observed even in patients with severe FXI deficiency.

[0005] The present application provides a nucleic acid molecule that inhibits the expression of the intracellular factor XI (FXI) gene via RNAi, a composition comprising said nucleic acid molecule, a formulation, and uses thereof. According to sufficient data in the embodiments of the present application, the preferred nucleic acid molecule of the present application comprises, but is 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 having a structure and sequence comprising said dsRNA molecules, precursor molecules of said shRNA molecules or dsRNA molecules, and modified said shRNA molecules or dsRNA molecules. Here, the preferred modifications include, but are not limited to, modification E and modification E-1, which are described in detail in the embodiments of the present application.

[0006] Specifically, one aspect of the present application relates to a nucleic acid molecule comprising a mutually complementary sense sequence and an antisense sequence, or composed of a mutually complementary sense sequence and an antisense sequence, wherein the antisense sequence comprises a polynucleotide sequence complementary to the FXI gene mRNA, and wherein,

[0007] When the above antisense sequence and the above FXI gene mRNA are hybridized at a maximum complementarity rate, a complementary region 1 is formed with base pairs that are complementarily paired among the above antisense sequence and the FXI gene mRNA sequence;

[0008] When the above antisense sequence and the above sense sequence are hybridized at a maximum complementarity rate, a complementary region 2 is formed with base pairs that are complementarily paired among the antisense sequence and the above sense sequence;

[0009] Here, complementary region 1 and complementary region 2 have at least 18, 19, or 20 identical base pairs, and

[0010] The number of base pairs of the above complementary region 1 is 15 to 35 bp, and the above complementary region 1 comprises the following among the FXI gene mRNA sequences:

[0011] The 418th to 437th nucleotides or 18 or 19 consecutive nucleotides among them,

[0012] The 1141st to 1160th nucleotides or 18 or 19 consecutive nucleotides among them,

[0013] The 363rd to 382nd nucleotides or 18 or 19 consecutive nucleotides among them,

[0014] The 372nd to 391st nucleotides or 18 or 19 consecutive nucleotides among them,

[0015] The 411th to 430th nucleotides or 18 or 19 consecutive nucleotides among them,

[0016] The 421st to 440th nucleotides or 18 or 19 consecutive nucleotides among them,

[0017] The 422nd to 441st nucleotides or 18 or 19 consecutive nucleotides among them,

[0018] The 425th to 444th nucleotides or 18 or 19 consecutive nucleotides among them,

[0019] The 465th to 484th nucleotides or 18 or 19 consecutive nucleotides among them,

[0020] The 470th to 489th nucleotides or 18 or 19 consecutive nucleotides among them,

[0021] The 471st to 490th nucleotides or 18 or 19 consecutive nucleotides among them, or

[0022] The 518th to 537th nucleotides or 18 or 19 consecutive nucleotides among them; and

[0023] Here, the nucleotide position number in the FXI gene mRNA sequence is the number of the corresponding nucleotide in reference sequence number 244.

[0024] Here, “18 or 19 consecutive nucleotides among” in a set of nucleotide sequences may be 19 consecutive nucleotides starting from the first or second nucleotide at the 5' end of the nucleotide sequence, including the first nucleotide or the second nucleotide and matching the nucleotide arrangement of the nucleotide sequence, or 18 consecutive nucleotides starting from the first, second, or third nucleotide at the 5' end of the nucleotide sequence, including the first nucleotide, the second nucleotide, or the third nucleotide and matching the nucleotide arrangement of the nucleotide sequence.

[0025] In some embodiments, the sense sequence and the antisense sequence have the same length. In some embodiments, the sense sequence and / or the antisense sequence further comprise protruding terminal nucleotides in addition to 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 one side of the 5' end and / or one side of the 3' end of the complementary region 2. In some embodiments, the antisense sequence further comprises one or two nucleotides at one side of the 5' end and / or one side of the 3' end of the complementary region 2. In some embodiments, the sense sequence and the antisense sequence further comprise one or two nucleotides at one side of the 5' end and / or one side of the 3' end of the complementary region 2. In some embodiments, when the antisense sequence is hybridized with the sense sequence at a maximum complementarity rate, the sense sequence does not include a nucleotide between the first nucleotide and the last nucleotide of the complementary region 2 that is not paired complementarily with the antisense sequence nucleotide. In some embodiments, when the antisense sequence is hybridized with the sense sequence at a maximum complementarity rate, the antisense sequence does not contain nucleotides that are not paired complementarily with the sense sequence nucleotides between the first nucleotide and the last nucleotide of the complementarity region 2.In some embodiments, the protruding terminal nucleotides are two in number, located in the antisense sequence, adjacent to the 5' end of the complementary region 2, and the sense sequence does not contain protruding terminal nucleotides. In some embodiments, the antisense sequence contains only 0, 1, or 2 nucleotides in addition to the complementary region 1, and the sense sequence contains only 0, 1, or 2 nucleotides in addition to the complementary region 2. In some embodiments, when the antisense sequence is hybridized with the FXI gene mRNA sequence at a maximum complementarity rate, the antisense sequence does not contain nucleotides between the first and last nucleotides of the complementary region 1 that are not paired complementarily with the FXI gene mRNA sequence. In some embodiments, when the antisense sequence is hybridized with the FXI gene mRNA sequence at a maximum complementarity rate, the FXI gene mRNA sequence does not contain a nucleotide between the first and last nucleotides of the complementarity region 1 that is not paired complementarily with the antisense sequence. In some embodiments, the 3' end of the complementarity region 2 is an AU base pair. In some embodiments, the 3' end of the complementarity region 2 consists of an A derived from the antisense sequence and a U derived from the sense sequence (which may be denoted as T in this application). In some embodiments, the 3' end of the complementarity region 2 consists of an A derived from the sense sequence and a U derived from the antisense sequence (which may be denoted as T in this application). In some embodiments, the complementarity region 1 and the complementarity region 2 are continuous without interruption. In some embodiments, complementary region 1 and / or complementary region 2 are discontinuous, said discontinuity is interrupted by one or more bubbles (a non-complementary region formed by each non-complementary nucleotide of the two sequences forming the complementary region).

[0026] In some embodiments, the number of base pairs of the complementary region 1 is 15 to 35, for example 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 of the complementary region 2 is 15 to 35 bp, for example 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 complementary region 1 and the 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 30 nt. In some embodiments, the length of the sense sequence is 21 nt, and the length of the antisense sequence is 23 nt. In some embodiments, the nucleic acid molecule consists of a sense sequence of length 21 nt and an antisense sequence of length 23 nt. In some embodiments, the nucleic acid molecule is composed of a sense sequence, an antisense sequence, and a linking chain (or shRNA loop), wherein the linking chain links the 3' end nucleotide of the sense sequence and the 5' end nucleotide of the antisense sequence. The selection of the linking chain is consistent in the art and is selected, for example, in the manner described in the literature Jensen, Stig Mølgaard Rask et al. “Functional selection of shRNA loops from randomized retroviral libraries.” PloS one vol. 7,8 (2012): e43095. doi:10.1371 / journal.pone.0043095, the entirety of which is incorporated into the text by reference.

[0027] In some embodiments, the sense sequence and / or antisense sequence further comprise one to two protruding terminal nucleotides in addition to the complementary region 2.

[0028] In some embodiments, the antisense sequence comprises only 0, 1, or 2 nucleotides in addition to complementary region 1, and the sense sequence comprises only 0, 1, or 2 nucleotides in addition to complementary region 2.

[0029] In some embodiments, the 3' end of the complementary region 2 is an AU base pair; in additional embodiments, the 5' end A of the antisense strand is outside the complementary region 1.

[0030] 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 cyanomorph monkey. In some embodiments, the mRNA comprises a polynucleotide sequence represented by SEQ ID NO. 244. In some embodiments, the antisense sequence comprises a polynucleotide sequence represented by 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 a polynucleotide sequence represented by SEQ ID NOs 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 comprises a polynucleotide sequence represented by SEQ ID NOs 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 at one side of the 5' and / or 3' end of the polynucleotide sequence. In some embodiments, the antisense sequence is a polynucleotide sequence represented by SEQ ID NOs 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 represented by SEQ ID NOs 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 comprises a polynucleotide sequence represented by SEQ ID NOs 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 consists of a polynucleotide sequence represented by 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 one side of the 5' and / or 3' end of the polynucleotide sequence. In some embodiments, the antisense sequence is a polynucleotide sequence represented by 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82, or 94.

[0031] In some embodiments, the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 3, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 122; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 4, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 123; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 7, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 126; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 10, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 129; The sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 11, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 130; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 12, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 131; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 17, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 136; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 18, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 137; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 19, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 138;The sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 24, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 143; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 30, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 149; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 31, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 150; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 33, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 152; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 34, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 153; The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 58, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 177; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 81, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 200; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 82, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 201; or the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 94, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 213.

[0032] In some embodiments, the sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 3 and additional 1, 2, 3, or 4 nucleotides, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 122 and additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 4 and additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 123 and additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 7 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 126 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 10 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 129 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 11 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 130 and an additional 1 to 4 (e.g., 2 or 3) nucleotides;The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 12 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 131 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 17 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 136 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 18 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 137 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 19 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 138 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 24 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 143 and an additional 1 to 4 (e.g., 2 or 3) nucleotides;The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 30 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 149 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 31 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 150 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 33 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 152 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 34 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 153 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 58 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 177 and an additional 1 to 4 (e.g., 2 or 3) nucleotides;The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 81 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 200 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 82 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 201 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 94 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 213 and an additional 1 to 4 (e.g., 2 or 3) nucleotides.

[0033] In some embodiments, the sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 3 and additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 122; the sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 4 and additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 123; the sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 7 and additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 126; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 10 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 129; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 11 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 130; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 12 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 131; The sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 17 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 136;The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 18 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 137; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 19 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 138; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 24 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 143; The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 30 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 149; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 31 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 150; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 33 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 152; The sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 34 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 153;The sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 58 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 177; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 81 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 200; the sense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 82 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence comprises a polynucleotide sequence represented by SEQ ID NO. 201; The sense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 94 and an additional 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 213.;

[0034] In some embodiments, the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 3 and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 122 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 4 and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 123 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 7 and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 126 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence is a polynucleotide sequence represented by SEQ ID NO. 10, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 129 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 11, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 130 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 12, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 131 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence is a polynucleotide sequence represented by SEQ ID NO. 17, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 136 and consists of an additional 1 to 4 (e.g., 2 or 3) nucleotides;The sense sequence is a polynucleotide sequence represented by SEQ ID NO. 18, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 137 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 19, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 138 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 24, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 143 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence is a polynucleotide sequence represented by SEQ ID NO. 30, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 149 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 31, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 150 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 33, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 152 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; The sense sequence is a polynucleotide sequence represented by SEQ ID NO. 34, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 153 and an additional 1 to 4 (e.g., 2 or 3) nucleotides;The sense sequence is a polynucleotide sequence represented by SEQ ID NO. 58, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 177 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 81, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 200 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 82, and the antisense sequence is composed of a polynucleotide sequence represented by SEQ ID NO. 201 and an additional 1 to 4 (e.g., 2 or 3) nucleotides; Or, the sense sequence is a polynucleotide sequence represented by SEQ ID NO. 94, and the antisense sequence is a polynucleotide sequence represented by SEQ ID NO. 213 and consists of an additional 1 to 4 (e.g., 2 or 3) nucleotides.;

[0035] In some embodiments:

[0036] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 34, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 153;

[0037] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 58, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 177; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 33, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 152;

[0038] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 3, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 122;

[0039] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 4, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 123;

[0040] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 7, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 126;

[0041] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 10, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 129;

[0042] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 11, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 130;

[0043] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 12, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 131;

[0044] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 17, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 136;

[0045] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 18, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 137;

[0046] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 19, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 138;

[0047] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 24, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 143;

[0048] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 30, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 149;

[0049] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 31, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 150;

[0050] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 81, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 200;

[0051] The sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 82, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 201; or

[0052] The sense sequence above includes or is the polynucleotide sequence represented by SEQ ID NO. 94, and the antisense sequence above includes or is the polynucleotide sequence represented by SEQ ID NO. 213.

[0053] In some embodiments, the nucleic acid molecule is dsRNA or shRNA. In some embodiments, the dsRNA is siRNA. In some embodiments, one or more nucleotides of 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 extends 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 group consisting of:

[0054] Locked nucleic acid modification, ring-opening or unlocked nucleic acid modification, 2'-methoxyethyl modification, 2'-O-methyl modification (2'-OMe), 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, tetrahydropyran modification, 1,5-anhydrohexitol modification, 5'-vinylphosphate modification, and cyclohexenyl modification.

[0055] In some embodiments, the 7th, 9th to 12th 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 to 12th 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 group modification. In some embodiments, the 7th, 9th to 12th nucleotides of the sense strand of the nucleic acid molecule are modified by a 2'-fluoro group and / or the 2nd, 14th, and 16th nucleotides of the antisense strand are modified by a 2'-fluoro group, and the remaining nucleotides are modified by a 2'-O-methyl group, and the nucleic acid molecule does not additionally contain any modifications other than phosphorothioate.

[0056] In some embodiments, the 7th, 9th to 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 to 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 group modification. In some embodiments, the 7th, 9th to 11th nucleotides of the sense strand of the nucleic acid molecule are modified by a 2'-fluoro group and / or the 2nd, 14th, and 16th nucleotides of the antisense strand are modified by a 2'-fluoro group, and the remaining nucleotides are modified by a 2'-O-methyl group, and the nucleic acid molecule does not additionally contain any modifications other than phosphorothioate.

[0057] In some embodiments, the 7th, 9th to 11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the 7th, 9th to 11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl group modification. In some embodiments, the 7th, 9th to 11th nucleotides of the sense strand of the nucleic acid molecule are modified by a 2'-fluoro group and / or the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are modified by a 2'-fluoro group, and the remaining nucleotides are modified by a 2'-O-methyl group, and the nucleic acid molecule does not additionally contain any modifications other than phosphorothioate.

[0058] In some embodiments, the nucleic acid molecule comprises a motif selected from the group consisting of:

[0059] (1) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNmNmNm, Antisense sequence: NmNfNm

[0060] (2) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNm, Antisense sequence: NmNfNm

[0061] (3) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm, Antisense sequence: NmNfNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm;

[0062] Here, N from 5' to 3' of each sequence sequentially represents, respectively, the nucleotide at the position of the sequence, Nm represents a ribonucleotide modified by a 2'-O-methyl group, and Nf represents a ribonucleotide modified by a 2'-fluoro group; it should be understood that the present invention does not limit whether each nucleotide includes modifications other than those indicated.

[0063] In some embodiments, the embodiment further has a phosphorothioate modification at at least one of the positions numbered from the sense sequence 5' between the first and second nucleotides and between the second and third nucleotides; and has a phosphorothioate modification at at least one of the positions numbered from the antisense sequence 5' between the first and second nucleotides, between the second and third nucleotides, between the last from the first and second, and between the last from the second and third.

[0064] In some embodiments, the nucleic acid molecule comprises a motif selected from any one of (1) to (9) below:

[0065] (1) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0066] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0067] (2) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0068] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNm

[0069] (3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0070] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0071] (4) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm, Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm;

[0072] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0073] (5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm;

[0074] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0075] (6) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0076] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0077] (7) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms, Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0078] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0079] (8) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0080] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmNm, wherein each nucleotide does not include modifications other than 2'-F, 2'-OMe and phosphorothioate;

[0081] (9) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0082] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain a modification other than 2'-F, 2'-OMe and phosphorothioate.

[0083] Here, in all motifs of the present application, Nm represents a ribonucleotide modified by a 2'-O-methyl group, Nf represents a ribonucleotide modified by a 2'-fluoro group, and s represents a phosphorothioate modification.

[0084] In some embodiments, the nucleic acid molecule is linked to a ligand having organ targeting, e.g., a ligand having liver targeting. 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 3' end of the sense sequence. In some embodiments, the ASGPR ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched chain structure. In some embodiments, the GalNAc derivative comprises the following structure:

[0085]

[0086] In some embodiments, the GalNAc derivative is L96, and the structure of L96 is as follows:

[0087]

[0088] In some embodiments, the GalNAc derivative is ligand 1, and the structure of ligand 1 is as follows:

[0089] (X=S- or O-)(Equation II);

[0090] Here, in formulas I and II indicates that it is connected to the 3' end of the sense sequence or antisense sequence of the nucleic acid molecule; preferably, it is connected through a phosphodiester bond or a phosphorothioate diester bond.

[0091] Additionally, preferably, the nucleic acid molecule has any one of (1) to (6) modification motifs:

[0092] (1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96; Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm;

[0093] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96; antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not include a modification other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0094] (2) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0095] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNms-ligand 1, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm; wherein each nucleotide does not include a modification other than ligand 1, 2'-F, 2'-OMe and phosphorothioate;

[0096] (3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0097] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not include a modification other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0098] (4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0099] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not include a modification other than ligand 1, 2'-F, 2'-OMe and phosphorothioate;

[0100] (5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm;

[0101] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmNm; wherein each nucleotide does not include a modification other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0102] (6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm;

[0103] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not include a modification other than ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0104] In some embodiments, the nucleic acid molecule comprises a 5' phosphate or a 5' phosphate mimic modification, for example, a phosphate or a phosphate mimic at the 5' end on the antisense strand; preferably, the 5' phosphate mimic modification is 5'-VP.

[0105] In some embodiments, the nucleic acid molecule has the following modified motif:

[0106] GN-E20VP:

[0107] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1

[0108] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0109] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1

[0110] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0111] In some embodiments, the nucleic acid molecule has the following modified motif:

[0112] GN-E04VP:

[0113] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1

[0114] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0115] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1

[0116] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0117] In some embodiments, the nucleic acid molecule has the following modified motif:

[0118] GN-E05VP:

[0119] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1

[0120] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0121] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1

[0122] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0123] In some embodiments, the nucleic acid molecule has the following modified motif:

[0124] LN-E20VP:

[0125] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96

[0126] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0127] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96

[0128] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0129] In some embodiments, the nucleic acid molecule has the following modified motif:

[0130] LN-E04VP:

[0131] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0132] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0133] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0134] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0135] In some embodiments, the nucleic acid molecule has the following modified motif:

[0136] LN-E05VP:

[0137] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0138] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0139] In some other embodiments, sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

[0140] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0141] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 58 and antisense sequence: SEQ ID NO. 177 (siRNA ID NO. 50), and has any of the variant patterns listed above.

[0142] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 58 and antisense sequence: SEQ ID NO. 177 (siRNA ID NO. 50), and has the following modification pattern:

[0143] GN-E20:

[0144] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0145] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0146] GN-E20VP:

[0147] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0148] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0149] GN-E04:

[0150] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0151] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0152] GN-E04VP:

[0153] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0154] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0155] GN-E05:

[0156] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0157] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or

[0158] GN-E05VP:

[0159] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0160] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.

[0161] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 58 and antisense sequence: SEQ ID NO. 177 (siRNA ID NO. 50), and has the following modification pattern:

[0162] GN-E20:

[0163] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0164] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than the ligands 1,2'-F,2'-OMe and phosphorothioate;

[0165] GN-E20VP:

[0166] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0167] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide is vp, ligand 1, 2'-F, 2'-OMe and does not contain modifications other than phosphorothioate;

[0168] GN-E04:

[0169] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0170] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than the ligands 1,2'-F,2'-OMe and phosphorothioate;

[0171] GN-E04VP:

[0172] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0173] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide is vp, ligand 1, 2'-F, 2'-OMe and does not contain modifications other than phosphorothioate;

[0174] GN-E05:

[0175] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0176] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate; or

[0177] GN-E05VP:

[0178] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0179] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0180] In some embodiments, the naked sequence of the nucleic acid molecule provided by the present application is sense sequence: SEQ ID NO. 34 and antisense sequence: SEQ ID NO. 153 (siRNA ID NO. 26'), and has any of the variant patterns listed above.

[0181] In some embodiments, the naked sequence of the nucleic acid molecule provided by the present application is sense sequence: SEQ ID NO. 34 and antisense sequence: SEQ ID NO. 153 (siRNA ID NO. 26'), and has the following modification pattern:

[0182] GN-E20:

[0183] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0184] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0185] GN-E20VP:

[0186] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0187] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0188] GN-E04:

[0189] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0190] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0191] GN-E04VP:

[0192] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0193] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0194] GN-E05:

[0195] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0196] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or

[0197] GN-E05VP:

[0198] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0199] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.

[0200] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 34 and antisense sequence: SEQ ID NO. 153 (siRNA ID NO. 26'), and has the following modification pattern:

[0201] GN-E20:

[0202] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0203] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than the ligands 1,2'-F,2'-OMe and phosphorothioate;

[0204] GN-E20VP:

[0205] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0206] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide is vp, ligand 1, 2'-F, 2'-OMe and does not contain modifications other than phosphorothioate;

[0207] GN-E04:

[0208] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0209] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than the ligands 1,2'-F,2'-OMe and phosphorothioate;

[0210] GN-E04VP:

[0211] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0212] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide is vp, ligand 1, 2'-F, 2'-OMe and does not contain modifications other than phosphorothioate;

[0213] GN-E05:

[0214] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0215] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate; or

[0216] GN-E05VP:

[0217] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1,

[0218] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe and phosphorothioate.

[0219] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 58 and antisense sequence: SEQ ID NO. 177 (siRNA ID NO. 50), and has the following modification pattern:

[0220] LN-E20:

[0221] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0222] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0223] LN-E20VP:

[0224] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0225] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0226] LN-E04:

[0227] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0228] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0229] LN-E04VP:

[0230] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0231] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0232] LN-E05:

[0233] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0234] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or

[0235] LN-E05VP:

[0236] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0237] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.

[0238] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 58 and antisense sequence: SEQ ID NO. 177 (siRNA ID NO. 50), and has the following modification pattern:

[0239] LN-E20:

[0240] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0241] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0242] LN-E20VP:

[0243] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0244] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0245] LN-E04:

[0246] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0247] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0248] LN-E04VP:

[0249] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0250] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0251] LN-E05:

[0252] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0253] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate; or

[0254] LN-E05VP:

[0255] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0256] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0257] In some embodiments, the naked sequence of the nucleic acid molecule provided by the present application is sense sequence: SEQ ID NO. 34 and antisense sequence: SEQ ID NO. 153 (siRNA ID NO. 26'), and has the following modification pattern:

[0258] LN-E20:

[0259] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0260] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

[0261] LN-E20VP:

[0262] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0263] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0264] LN-E04:

[0265] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0266] Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0267] LN-E04VP:

[0268] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0269] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0270] LN-E05:

[0271] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0272] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or

[0273] LN-E05VP:

[0274] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0275] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.

[0276] In some embodiments, the naked sequence of the nucleic acid molecule specifically provided by the present application is sense sequence: SEQ ID NO. 34 and antisense sequence: SEQ ID NO. 153 (siRNA ID NO. 26'), and has the following modification pattern:

[0277] LN-E20:

[0278] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0279] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0280] LN-E20VP:

[0281] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

[0282] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0283] LN-E04:

[0284] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0285] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

[0286] LN-E04VP:

[0287] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0288] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate;

[0289] LN-E05:

[0290] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0291] Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe, and phosphorothioate; or

[0292] LN-E05VP:

[0293] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

[0294] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, where each nucleotide does not contain modifications other than vp, L96, 2'-F, 2'-OMe and phosphorothioate.

[0295] In addition, the present application further provides a precursor of the aforementioned nucleic acid molecule, said precursor may be shRNA or dsRNA.

[0296] A second aspect of the present application provides a second nucleic acid molecule capable of being transcribed into a dsRNA or shRNA precursor within a cell, wherein the dsRNA or shRNA is the nucleic acid molecule of the first aspect described above. In some embodiments, the second nucleic acid molecule is a cyclic or linear nucleic acid molecule. In some embodiments, the second nucleic acid molecule is a cyclic or linear plasmid. In some embodiments, the nucleic acid molecule belongs to an artificially constructed viral genome and may be selected from, for example, a lentivirus vector or other retrovirus vector, an adenovirus vector, an AAV vector, a poxvirus vector, a baculovirus vector, or a herpes simplex virus vector, but is not limited thereto. In some embodiments, the nucleic acid molecule belongs to a cellular genome and is, for example, a nuclear genome, mitochondrial nucleic acid, or cytoplasmic free nucleic acid.

[0297] A third aspect of the present application further provides a nucleic acid carrier comprising the nucleic acid molecule of the first aspect described above, or the second nucleic acid molecule of the second aspect described above. In some embodiments, the nucleic acid carrier is a liposome, lipid nanoparticle or other polymer, endosome, exosome, or vesicle.

[0298] Further provide a virus particle comprising a second nucleic acid molecule of the second aspect described above. In some embodiments, the virus particle is an enveloped virus or a coated virus particle. In some embodiments, the virus particle is a pseudovirus particle. In some embodiments, the virus particle belongs to AAV, baculovirus, poxvirus, herpesvirus, alphavirus, lentivirus, or other retrovirus.

[0299] At the same time, a cell comprising the second nucleic acid molecule of the second aspect described above is further provided. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, e.g., a stem cell, e.g., a hematopoietic stem cell, a mesenchymal stem cell, etc.

[0300] The present application further provides a pharmaceutical composition comprising the nucleic acid molecule of the first aspect described above or a salt thereof, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition is used to treat or prevent thromboembolic complications or coagulation disorders in a subject, wherein the thromboembolic complications are preferably one or more selected from the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0301] A fourth aspect of the present application further provides the use of the nucleic acid of the first aspect described above, the second nucleic acid of the second aspect described above, the nucleic acid carrier of the third aspect described above, and the virus particle, the cell described above, and the pharmaceutical composition described above.

[0302] For example, the present application provides the use of the nucleic acid molecule or salt thereof of the first aspect described above, the second nucleic acid molecule of the second aspect described above, the nucleic acid carrier described above, the virus particle described above, or the cell described above in the manufacture of a drug for preventing or treating thromboembolic complications or coagulation disorders in a subject. In some embodiments, the thromboembolic complications are preferably one or more selected from the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0303] The present application further provides a method for treating or preventing thromboembolic complications or coagulation disorders in a subject, said method comprising the step of administering an effective amount of the nucleic acid molecule of the first aspect described above or a salt thereof, the second nucleic acid molecule of the second aspect described above, the nucleic acid carrier described above, the virus particle described above, the cell described above, or the pharmaceutical composition described above to a subject in need. In some embodiments, said thromboembolic complications are preferably one or more selected from the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0304] Further providing a compound for treating or preventing thromboembolic complications or coagulation disorders in a subject, comprising the nucleic acid molecule of the first aspect described above or a salt thereof, the second nucleic acid molecule of the second aspect described above, the nucleic acid carrier described above, the virus particle described above, the cell described above, or the pharmaceutical composition described above. In some embodiments, the thromboembolic complication is preferably one or more selected from the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0305] The present application further provides the use of the nucleic acid molecule of the first aspect described above or its salt, or the second nucleic acid molecule of the second aspect described above, for inhibiting the expression of factor 11.

[0306] The present application further provides the use of the nucleic acid molecule of the first aspect or its salt, or the second nucleic acid molecule of the second aspect, in the manufacture of a drug for inhibiting the expression of FXI in vivo in a subject.

[0307] The present application further provides a compound for inhibiting the expression of FXI in vivo in a subject, said compound comprising the nucleic acid molecule of the first aspect described above or a salt thereof, and a pharmaceutically acceptable carrier or diluent. Although preferred embodiments of the present application have been described in detail above, the present application is not limited thereto. Within the scope of the technical concept of the present application, various simple modifications may be made to the technical solution of the present application, including combining each technical feature in any other suitable manner, and such simple modifications and combinations should likewise be considered as disclosed in the present application and all fall within the scope of protection of the present application. The aspects and embodiments of the present application described in the text include aspects and embodiments that are “comprising,” “consisting of,” and “substantially composed of ……”. Brief explanation of the drawing

[0308] Figure 1 shows the results of high-throughput screening of dsRNA naked nucleic acid molecules (5 nM) in HepG2 cells. Figure 2 shows the relative content of FXI mRNA in HepG2 cells when candidate dsRNA or positive reference dsRNA naked nucleic acid molecules of 5 nM and 1 nM, respectively, are administered. Figure 3 shows the inhibition rate of dsRNA modified by modification E against FXI mRNA in HepG2 cells at a concentration of 1 nM. Figure 4 shows the inhibition rate of GalNAc-conjugated dsRNA against FXI mRNA at different concentrations. Figure 5 illustrates the results of inhibition of a target protein after in vivo administration of different dsRNAs conjugated with GalNAc. Figure 6 illustrates the comparative results after in vivo administration of different GalNAc. Figure 7 shows the results of a comparison of the in vitro cell-level IC50 of different modified dsRNAs. Figure 8 illustrates the comparative results after in vivo administration of dsRNA with different modification motifs. Specific details for implementing the invention

[0309] The present invention provides a nucleic acid molecule capable of inducing the cleavage of an RNA transcript of a blood coagulation factor FXI (or referred to as FX11) gene mediated by an RNA-induced silencing complex (RISC), a second nucleic acid molecule capable of transcribing said nucleic acid molecule, a carrier of said nucleic acid molecule and said second nucleic acid molecule, a virus or cell capable of transcribing said nucleic acid molecule, and uses of said nucleic acid molecule and said second nucleic acid molecule.

[0310] terminology

[0311] For the purpose of interpreting this specification, the following definitions apply, where appropriate, terms used in the singular form include the plural form, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the said technology belongs. All technical and patent disclosures cited herein are incorporated by reference in their entirety.

[0312] As used in the text, “dsRNA” refers to double-stranded RNA, and since siRNA is RNA with a double-stranded structure, the term “dsRNA” includes siRNA. dsRNA further includes double-stranded RNA that is longer than siRNA, and the length being longer than siRNA may mean that its sense strand is longer than siRNA, its antisense strand is longer than siRNA, or both its sense strand and antisense strand are longer than siRNA. Generally, when double-stranded RNA longer than the included siRNA sequence enters the cell, it is degraded into siRNA by a type III endonuclease called Dicer. In some embodiments, the lengths of the two strands of the dsRNA are each independently 15 to 30 nt (in this application, “nt” is a nucleotide). After the “siRNA” is incorporated into the RNA-induced silencing complex (RISC), one or more helicases within the RISC unwind the siRNA double helix. When binding to a target mRNA complementary to the antisense strand of siRNA, one or more endonucleases within RISC cleave the target to induce gene silencing. Generally, most nucleotides on each strand of a dsRNA molecule are ribonucleotides, but this does not exclude the inclusion of one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or non-natural nucleotides, on one or both strands. In some embodiments, the dsRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used in this application, the dsRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0313] The terms “FXI,” “blood coagulation factor FXI,” and “factor 11” may be used interchangeably and are also referred to as FXI or PTA in the art. FXI may be FXI of mammalian origin. In some embodiments, FXI is FXI of primate origin. In some embodiments, FXI is FXI of human origin. In some embodiments, FXI is FXI of cynomolgus monkey origin. As used herein, “FXI gene mRNA” means mRNA encoding the factor 11 protein, which may be transcribed from FXI gene DNA and may be mature mRNA or mRNA precursor (Pre-mRNA), and may or may not contain introns. Since a small number of nucleotide mutations may exist in the factor 11 gene depending on the individual, unless specifically stated otherwise, the factor 11 gene mRNA sequence of this application means that all mRNA sequences transcribed from factor 11 gene mutants are included. The human FXI gene mRNA sequence can be found, for example, in Gene Bank Registry No. GI: ​​1732746318 (NM_000128.4). The rhesus monkey FXI gene mRNA sequence can be found, for example, in Gene Bank Registry No. GI: ​​1622942384 (XM_015139652.2). The cyanomolgus monkey FXI gene mRNA sequence can be found, for example, in Gene Bank Registry No. GI: ​​2161917139 (XM_005556483.3). The mouse FXI gene mRNA sequence can be found, for example, in Gene Bank Registry No. GI: ​​2293430447 (NM_028066.3). The rat FXI gene mRNA sequence can be found, for example, in gene bank registration number GI: 2293332621 (NM_001411666.1).Other examples of FXI gene mRNA sequences can be easily obtained using publicly available databases, such as GenBANK. Unless otherwise noted, FXI gene mRNA uses SEQ ID NO. 244 (i.e., NM_000128.4) as the reference sequence, meaning that each nucleotide position number of FXI gene mRNA corresponds to a nucleotide number in the 5' to 3' direction of the reference sequence SEQ ID NO. 244. As used in the text, the term “reference sequence” refers to a standard sequence used for homologous sequence alignment and means a sequence that can be used to define amino acid positions within a homologous polynucleotide or polynucleotide sequence. For example, “the base position number of the FXI gene mRNA sequence is the corresponding base number of reference sequence number 244” is defined by introducing a space into the FXI gene mRNA sequence or deleting nucleotides to ensure that the FXI gene mRNA sequence has the same bases as the reference sequence at as many positions as possible, then sequentially numbering the nucleotides of the reference sequence starting from the first nucleotide at the 5' end according to sequence order, and aligning the nucleotides corresponding to each other in the FXI gene mRNA sequence and the reference sequence to define the positions of the nucleotides with the same number.

[0314] As used in the text, “complementarity” of nucleic acids means that one nucleic acid has the ability to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairs. Percentage complementarity means the percentage of the total nucleotides of the shorter nucleic acid molecule in two nucleic acid molecules that can form hydrogen bonds (i.e., Watson-Crick base pairs) with the other nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, and 10 out of 10 represent about 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). “Complete complementarity” means that every consecutive residue of a nucleic acid sequence forms hydrogen bonds with the same number of consecutive residues of the second nucleic acid sequence. As used in the text, “substantially complementary” means at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the degree of complementarity in a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or means two nucleic acids that hybridize under strict conditions. In the case of a single base or a single nucleotide, if A and T or U, or C and G or I are paired according to the Watson-Crick base pairing principle, it is said to be complementary, paired, or matching, and vice versa; on the other hand, all other base pair matching is said to be non-complementary.

[0315] As used in the text, “hybridization” of nucleic acids refers to a reaction in which one or more polynucleotides react to form a complex, said complex being stabilized through hydrogen bonding between nucleotide residues. Hydrogen bonding may occur through Watson-Crick base pairs, Hoogsteen bonds, or any other sequence-specific method. The complex may comprise two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridized strand, or a combination thereof.

[0316] In this application, “hybridizing to maximum complementarity” of two polynucleotide sequences or two nucleic acid chains refers to a hybridization method in which as many nucleotides as possible form hydrogen bonds to pair with each other. “Hybridizing to maximum complementarity” may allow for one or more mismatches to appear, and may allow for one or more bulges to appear on one or more of the two strands. However, in some embodiments, “hybridizing to maximum complementarity” may not allow for mismatches or bulges to appear even after the two strands have been hybridized with each other. As used in the text, “complementary region” refers to all base pairs paired via hydrogen bonds from the first base pair paired via hydrogen bond starting from the 5' end to the last base pair paired via hydrogen bond after the two strands have been hybridized, and the complementary region formed by the two hybridized strands may be continuous or discontinuous. In particular, “Complementary Region 1” of the present application refers to a complementary region composed of base pairs that are complementarily paired between the antisense sequence and the FXI gene mRNA sequence when the antisense sequence is hybridized with the FXI gene mRNA at a maximum complementarity rate; and “Complementary Region 2” refers to a complementary region composed of base pairs that are complementarily paired between the antisense sequence and the sense sequence when the antisense sequence is hybridized with the sense sequence at a maximum complementarity rate. Since both Complementary Region 1 and Complementary Region 2 are associated with the antisense sequence, the antisense sequence portions of Complementary Region 1 and Complementary Region 2 generally have the same nucleotide composition, either partially or wholly.In the present application, the antisense sequence and the sense sequence are relative to a third sequence that is complementary to one of the sequences, and, for example, in “nucleic acid molecule that inhibits intracellular factor 11 (FXI) gene expression via RNAi,” the antisense sequence and the sense sequence are relative to the third sequence, the FXI gene mRNA sequence; that is, the antisense sequence refers to a sequence in the nucleic acid molecule having a region complementary to the FXI gene mRNA sequence, and the sense sequence refers to a sequence in the nucleic acid molecule having at least 10 consecutive identical nucleotides with the FXI gene mRNA sequence. Additionally, in the present application, the 5' end of any complementary region refers to the position of the nucleotide or base pair closest to the sense strand or the 5' end of the third sequence in its complementary region, and the 5' end of the complementary region refers to one side or one end that is relatively close to the sense strand or the 5' end of the third sequence. Likewise, in this application, the 3' end of any complementary region refers to the position of the nucleotide or base pair closest to the sense strand or the 3' end of the third sequence in its complementary region, and the 3' end of the complementary region refers to one side or one end that is relatively close to the sense strand or the 3' end of the third sequence. In this application, “close,” “proximate,” or “far” describing nucleotide positions on the same sequence or nucleotide chain means that the number of nucleotides between two nucleotide positions is less or more.

[0317] As used in the text, “protruding terminal nucleotide” refers to a nucleotide among the nucleotides of the sense sequence and / or antisense sequence located outside the complementary region 2, relative to the complementary region 2, after the sense sequence and the antisense sequence have been hybridized at a maximum complementary rate. In some embodiments, the protruding region nucleotide is located only in the sense sequence; in some embodiments, the protruding region nucleotide is located only in the antisense sequence; and in some embodiments, the protruding terminal nucleotide is located in both the sense sequence and the antisense sequence. In some embodiments, the protruding terminal nucleotide is present only on one side of the 5' end of the complementary region 2. In some embodiments, the protruding terminal nucleotide is present only on one side of the 5' end of the sense strand complementary region 2. In some embodiments, the protruding terminal nucleotide is present only on one side of the 5' end of the antisense strand complementary region 2. In some embodiments, the protruding terminal nucleotide is present only on one side of the 3' end of the complementary region 2. In some embodiments, the protruding terminal nucleotide is present only on one side of the 3' end of the sense strand complementary region 2. In some embodiments, the protruding terminal nucleotide is present only on one side of the 3' end of the antisense strand complementary region 2. In some embodiments, the number of protruding region nucleotides on the same side (e.g., the 5' end or the 3' end) of the sense sequence or antisense sequence does not exceed two (i.e., one or two).In this application, “one side of the 5’ end” and “one side of the 3’ end” are both used to describe the relative positional relationship between two sequences, two nucleotides, or one nucleotide and one sequence within the same polynucleotide sequence; wherein “5’ end” means one end of the polynucleotide sequence containing a 5’ free phosphate group or a 5’ free hydroxyl group, and “3’ end” means one end of the polynucleotide sequence containing a free 3’-hydroxyl group or a 3’-phosphate group, and a sequence or nucleotide located on one side of the 3’ end of a specific sequence within a nucleic acid chain is closer to the 3’ end of the nucleic acid chain than to the specific sequence. For example, a specific sequence or one or several nucleotides are further included in “one side of the 5' end of the complementary region 2,” which means that the “specific sequence or one or several nucleotides” are closer to the 5' end of a polynucleotide sequence (e.g., an antisense sequence or a sense sequence) that they have in common with respect to the sequence of the “complementary region 2.”

[0318] The term “nucleotide” means not only naturally occurring ribonucleotide or deoxyribonucleotide monomers, but also, unless otherwise clearly stated in the context, to be understood to mean related structural variants, including functionally equivalent derivatives and analogs, in the specific context regarding the use of said nucleotides. For example, “nucleotide” means deoxyribonucleotide or ribonucleotide. Nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs mean nucleotides having modified purine or pyrimidine bases or modified ribose moiety. Nucleotide analogs may be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base portions of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphate groups, and thiol groups, and cases where carbon and nitrogen atoms of the base are substituted by other atoms (e.g., 7-deazpurine). Nucleotide analogs further include dideoxynucleotides, 2'-O-methylnucleotides, lock nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino oligonucleotides. In some embodiments, the “nucleotide” of this application does not include base-modified non-natural nucleotides. In some embodiments, the “nucleotide” of this application does not include base-modified nucleotides.In this application, “G,” “C,” “A,” “T,” and “U” generally denote nucleotides having guanine, cytosine, adenine, thymine, and uracil as bases, respectively, and unless specifically stated otherwise, “G,” “C,” “A,” “T,” and “U” indicate that they do not limit the included modified nucleotides, i.e., they may be used to denote natural nucleotides or non-natural nucleotides, said non-natural nucleotides may include nucleotides with ribose and / or bases modified, provided that the base can still be paired complementarily via hydrogen bonding with its naturally paired (i.e., paired according to the Watson-Crick principle) base. However, in the context of RNA and RNA sequences, “T” means uridine or uracil unless specifically stated otherwise. It should be understood that in the context relating to nucleotide sequences in this application, “nucleotide,” “nucleotide residue,” and “base” may be used interchangeably. The number of base pairs is in units of bp, where one bp is one base pair. The number of nucleotides is in units of nt, where one nt is one nucleotide.

[0319] As used in the text, “internucleotide bond” refers to a chemical bond (or linker) between two adjacent nucleotides or between a nucleotide and a ligand, and unless specifically stated otherwise, said chemical bond is a phosphoester bond or a phosphodiester bond; however, where an “s” is specifically indicated on a modification motif, i.e., a phosphorothioate modification site, the internucleotide connection is a phosphorothioate bond only at the site where the s is indicated, and all other unindicated internucleotide connections are phosphoester bonds, for example:

[0320] (1) Sense strand: NmNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNm, Antisense strand: NmNfNmNmNdNmNdNmNmNmNmNmNmNmNmNmNmNmNmNmNm;

[0321] In (1), the connection between each nucleotide may be a phosphoester bond or a phosphorothioate bond;

[0322] On the other hand, sense strand: CmsAmsGmAmGmUmUfAmUfCdGfAmGmGmCmAmCmAmUmUmAms-ligand 1,

[0323] In the case of antisense strand: vp-UmsAfsAmUmGdUmGdCmCmUmCmGmAmUfAmAfCmUmCmUmGmsGmsCm; except where the following internucleotide connections are phosphothioate bonds due to the specially indicated “s”, all internucleotide connections at other positions are phosphoester bonds: the first and second internucleotide connections from the 5’ end of the sense strand, the internucleotide connection between the 3’ end nucleotide of the sense strand and ligand 1, the first and second internucleotide connections from the 5’ end of the antisense strand, and the first and second internucleotide connections from the 3’ end of the antisense strand. Here, the first nucleotide linkage refers to a chemical bond between the first nucleotide and the second nucleotide starting from one end (e.g., the 5' end or the 3' end); the second nucleotide linkage refers to a chemical bond between the second and third nucleotides starting from one end (e.g., the 5' end or the 3' end), and continues in this manner; the nth nucleotide linkage refers to a 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 continues in this manner.

[0324] As used in the text, “protruding end,” “suspended end,” and “suspended sequence” may be used interchangeably and refer to one or more unpaired nucleotides extending beyond the double-stranded region at the ends of the chains. A nucleotide protruding end is generally formed when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other chain. The length of the nucleotide protruding end is generally 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 protruding end comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one specific embodiment, the nucleotide protruding end comprises 1 to 4 nucleotides. In one specific embodiment, the nucleotide protruding end comprises 2 nucleotides. In another specific embodiment, the nucleotide protruding end comprises a single nucleotide.

[0325] The nucleotide at the protruding end may be the ribonucleotide or modified nucleotide described in the text. In some embodiments, the nucleotide at the protruding end is a 2'-modified nucleotide (e.g., 2'-fluoro-modified nucleotide, 2'-O-methyl-modified nucleotide), a deoxyribonucleotide, a reverse nucleotide (e.g., reverse baseless nucleotide, reverse deoxyribonucleotide), or a combination thereof. For example, in one embodiment, the nucleotide at the protruding end is a deoxyribonucleotide, e.g., deoxythymidine. In another embodiment, the nucleotide at the protruding end is a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, or a combination thereof. In another embodiment, the protruding end comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In these embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide (e.g., a 2'-modified nucleotide). In other embodiments, the protruding end comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When the nucleotide protruding end is present on the antisense strand, the nucleotide within the protruding end may be complementary to the target gene sequence, thereby forming a mismatch with the target gene sequence, or may comprise some other sequence (e.g., a polypyrimidine or polypurine sequence, e.g., UU, TT, AA, GG, etc.).

[0326] Nucleotide protruding ends may be located at the 5' or 3' ends of one or both strands. For example, in one embodiment, an RNA molecule includes nucleotide protruding ends at the 5' and 3' ends of the antisense strand. In another embodiment, an RNA molecule includes nucleotide protruding ends at the 5' and 3' ends of the sense strand. In some embodiments, an RNA molecule includes nucleotide protruding ends at the 5'' ends of the sense strand and the 5' ends of the antisense strand. In other embodiments, an RNA molecule includes nucleotide protruding ends at the 3' ends of the sense strand and the 3' ends of the antisense strand.

[0327] The RNA molecule may include a nucleotide protruding end at one end of the double-stranded RNA molecule and a flat end at the other end. “Flat end” means that the sense strand and the antisense strand are fully base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the RNA molecule includes a nucleotide protruding end at the 3' end of the sense strand and flat ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNA molecule includes a nucleotide protruding end at the 3' end of the antisense strand and flat ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNA molecule includes flat ends at both ends of the double-stranded RNA molecule. In these embodiments, the sense strand and the antisense strand have the same length, and the length of the double-stranded region is the same as the sense strand and the antisense strand (i.e., the molecule is double-stranded over its entire length). As used in the text, “GalNAc” or “N-acetylgalactosamine” means 2-(acetylamino)-2-deoxy-D-galactopyranos. Unless specifically stated otherwise, the term “GalNAc” or “N-acetylgalactosamine” includes both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranos and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranos. Preferably, the GalNAc compound of the present application is the β form, i.e., 2-(acetylamino)-2-deoxy-β-D-galactopyranos.

[0328] As used in the text, “VP variant” or “E-VP” refers to a phosphate-mimicking variant located at the 5’ of the antisense strand, and its specific structure is as follows:

[0329]

[0330] Here:

[0331] Bx1 is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine;

[0332] T2 is a phosphoester or phosphorothioate nucleoside linker that links the above compound to an oligonucleotide 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.

[0333] As used in the text, “3’ end” specifically refers to the position of the first nucleotide or first base pair at the 3’ end of a single nucleotide sequence or a double-stranded polynucleotide. “5’ end” specifically refers to the position of the first nucleotide or first base pair at the 5’ end of a single nucleotide sequence or a double-stranded polynucleotide.

[0334] As used in this application, the term “nucleic acid molecule” may mean any molecule having a nucleotide sequence in which two or more nucleotides are connected through a phosphoester bond or a modified phosphoester bond (e.g., a phosphorothioate bond).

[0335] As used in the text, “nucleotide sequence” refers to a set of polynucleotide chains composed of nucleotides arranged sequentially in a specific order, and said polynucleotide chain may constitute a single nucleic acid molecule in this application or one end of a single strand of a specific nucleic acid molecule. Accordingly, “nucleotide sequence” may be represented as a set of precise polynucleotide sequences composed of various nucleotides (e.g., ATCG) (e.g., any one of SEQ ID NOs 1 to 100), or may be represented as nucleotides from a specific position to a specific position within a specific sequence, e.g., “nucleotides 363 to 382 in the FXI gene mRNA sequence.” In this application, unless specifically described otherwise, in a sequence or nucleic acid molecule comprising “nucleotides from a specific position to a specific position within a specific sequence,” said nucleotide arrangement order from said specific position to a specific position corresponds to the order of the aforementioned nucleotides within said specific sequence. If no restrictions are imposed, the “nucleotide sequence” may be RNA or DNA, or a hybrid molecule of RNA and DNA, and may include non-natural nucleotides or artificially modified nucleotides.

[0336] Additionally, the REL or Relative expression level shown in the attached drawings of this application refers to the mRNA relative expression level. 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 group modified nucleotide; Nf represents a 2'-F modified ribonucleotide or a 2'-fluoro modified ribonucleotide, and s represents a phosphorothioate modification, i.e., a 5'-thio modified phosphate, and where said modification is between two nucleotides or between a nucleotide and a ligand, it replaces the phosphoester bond between natural nucleotides, which can be referred to as a phosphorothioate bond. As used in the text, unless specifically stated otherwise, “phosphorothioate bond” and “phosphorothioate diester bond” may be used interchangeably in this application, and likewise “phosphoester bond” and “phosphodiester bond” may also be used interchangeably.

[0337] As used in the text, the term “about” refers to a general range of error for each value that is readily known to those skilled in the art. References to “about” values ​​or parameters include (and describe) embodiments of said values ​​or parameters themselves in the text. As used in the text, when the term “about” precedes a numerical value, it means within a range of 10% above or below said numerical value. For example, “about 100” includes 90 and 110.

[0338] As used in the text, unless otherwise specified, the singular forms “one,” “a kind,” and “the above” include the plural forms.

[0339] Unless otherwise defined in the text, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains.

[0340] It should be understood that this application includes various aspects, embodiments, and combinations of said aspects and / or forms of implementation described in the text. The foregoing description and the embodiments described below are intended to explain the scope of this application, not to limit it. Within the scope of the technical concept of this application, various simple modifications may be made to the technical solution of this application, including combining each technical feature in any other appropriate manner. Such simple modifications and combinations should likewise be considered as disclosed in this application and are all within the scope of protection of this application.

[0341] Unless otherwise stated, the practice of this application will utilize the ordinary techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. The aforementioned ordinary techniques are described in the existing technical literature.

[0342] It should be understood that this application includes various aspects, embodiments, and combinations of said aspects and / or forms of implementation described herein. The foregoing description and the embodiments described below are intended to explain the scope of this application, but are not intended to limit it. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Accordingly, those skilled in the art should recognize that the scope of this application includes said improvements and modifications to said aspects and embodiments.

[0343] Examples

[0344] Example 1: Sequence Design and Synthesis of dsRNA

[0345] In specific Examples 1 to 7 of the present application, each nucleotide does not include variations not specified in the present example. That is, each nucleotide sequence used in Examples 1 to 7 of the present application includes only the naked sequence or the variations indicated in the variation motif used.

[0346] 1.1 dsRNA Design

[0347] Multiple FXI dsRNAs were designed by selecting different regions based on the mRNA sequence of the human FXI gene (Gene ID, 2160) (NM_000128.4), and all designed individual dsRNAs had the lowest homology compared to all other non-target gene sequences using sequence similarity software, and specific details were referred to in the sequence table at the end of the text. In addition, for modified dsRNA, if the first nucleotide at the 3' end of the sense strand is not A or U, the original nucleotide is replaced with uracil ribonucleotide, and if the first nucleotide at the 5' end of the antisense sequence is not A or U, the original nucleotide is replaced with adenine ribonucleotide, 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, E70 are as indicated by dsRNAs with numbers 3', 4', 9', 14', 16', 24', 26', 51', 52', 69', and 70' in the sequence table at the end of the text, respectively.

[0348] In this application, the inventors [refer to] the ASO sequence of ISIS-416858 disclosed in Ionis patent (Patent No.: CN 109797150 A), the top 4 sequences of FXI gene repression rate disclosed in Patent No.: CN113227376A (Naked sequence names: RB2, RB4, RB6, RB7; Modified sequence names: ERB2, ERB4, ERB6, ERB7. The naked sequences and modified sequences correspond, respectively, to the sequences in the patent where the naming is not ligand-conjugated among the molecules L10-siFXIa1M1SP, L10-siFXIc1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP), and the top 3 sequences of FXI gene repression rate disclosed in Patent No.: WO 2022028457A1 (Naked sequence names: TJ632, TJ645, TJ772; modified sequence naming: ETJ632, ETJ645, ETJ772. The naked sequence and modified sequence correspond to the sequences in the patent where the ligand is not attached among the molecules TRD0632, TRD0645, and TRD0772, respectively) were selected as the FXI gene positive control sequences. The naked sequences of the aforementioned dsRNA sequences and positive control sequences are as shown in the sequence table at the end of the text.

[0349] 1.2 Synthesis and Purification of dsRNA and Its Conjugates

[0350] dsRNA was synthesized using modified (e.g., 2'-methoxy group, 2'-fluoro modification, or thio modification) or unmodified ribonucleotides, and all oligonucleotides were prepared on an LK-192X synthesizer (Jiangsu Lingkun Biotechnology Co., Ltd.) using a 1 μmol universal Frit carrier (1000 Å = 100 nm, Biocomma) or a CPG carrier of the protecting group-attached GalNAc derivative L96 (Asymchem). Depending on the sequence requirements, all phosphoramidite monomers of the corresponding nucleotides were diluted with anhydrous acetonitrile solvent at a 1:40 (g / mL) ratio, conjugated for 3 minutes, and conjugated a total of 2 times. Deprotection was performed using 3% TCA, and after activation with a 0.3M benzylthiotetrazole acetonitrile solution, capping and oxidation were performed with 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 water was added, and the protecting group was removed by heating in an oven at 65°C for 3 hours. Subsequently, it was cooled to room temperature and vacuum concentrated for 30 minutes. The solution was then filtered through a 0.22 μm filter membrane and placed in a sample bottle. Single-strand purification was performed using a semi-preparative reverse-phase purifier with an elution gradient of 7% to 30% (ACN: 100 mM TEAA), a time of 10 minutes, and a flow rate of 5 mL / min. After purification and preparation, the solution was vacuum concentrated and rotary dried at room temperature. Finally, after dissolving the samples in water, each solution was desalted on a GE Hi-Trap desalting column to elute the final oligonucleotide products. All characteristics and purity were verified using ESI-MS and IEX HPLC, respectively.After measuring the UV quantification concentration using a microplate reader, equimolar amounts of sense strands and antisense strands were mixed and placed in a new shipping tube, heated at 95°C for 5 minutes and slowly annealed to room temperature, and finally rotary dried at room temperature using a vacuum concentrator to obtain the final dsRNA product.

[0351] The L96 structure used in the embodiments of the present application is as shown by the following formula I:

[0352]

[0353] In the above formula, It is connected to the 3' end of the sense strand or antisense strand of an RNA molecule through a phosphodiester bond.

[0354] Example 2: High-throughput screening detection of FXI-dsRNA activity in vitro

[0355] 2.1 Transfection of HepG2 cells with FXI dsRNA

[0356] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator. After digestion, the cell density was adjusted to 4 × 10⁵ cells / ml, and 1 ml of cell suspension per well was inoculated into a 12-well plate. Preparation of 100 μL of transfection complex: 45 μL of Opti-MEM and 5 μL of dsRNA (naked nucleic acid, unmodified) of different concentrations were mixed, and 48 μL of Opti-MEM and 2 μL of RNAiMax transfection reagent were mixed and left for 5 minutes. Then, the two mixtures were combined and left for 20 minutes to form a transfection complex. The transfection complex was added to a 12-well plate and cultured continuously for 24 hours in a 5% CO2, 37°C incubator. Cells were harvested 24 hours after transfection, and RNA was extracted using the TRIZOL method.

[0357] 2.2 Real-time Fluorescence Quantitative PCR Analysis

[0358] Cells were lysed 24 hours after transfection, and total cell RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (refer to Vazyme RC112-01 instructions) with the Vazyme column extraction method. It was reverse transcribed into cDNA using Takara PrimeScript RT Master Mix RR036Q. The qPCR primer sequence information is as shown in the primer section of the sequence table at the end of the text. The human GAPDH gene was used as the internal reference gene, and PCR reactions were performed using a Bio-Rad CFX96 fluorescent quantitative PCR instrument. In the experiment, a mock group was used as a control, and normalization treatment was performed so that the FXI mRNA expression level of the mock group was 1.

[0359] 2.3 Data Analysis

[0360] After the PCR reaction was completed, relative quantitative analysis was performed using CFX96 software with the reference gene as a standard, and statistical analysis was performed using GraphPad software. As shown in Table 1 and Figure 1, the results of the high-throughput screening validation of dsRNA (naked nucleic acid, unmodified) molecules in HepG2 cells were presented.

[0361] Table 1: Results of high-throughput screening at a single dose of 5 nM in HepG2 cells

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] Through FXI dsRNA screening performed in HepG2 cells, 28 optimized sequences were discovered, and dsRNA molecules that were highly effective and simultaneously targeted human and cyanomolgus monkey FXI were used as candidate sequences. Additionally, the inventors compared several candidate positive control sequences with the inventors' candidate sequences, and Table 2 and Figure 2 show the screening results of the candidate sequences and positive control sequences under a single dose in HepG2 cells.

[0368] Table 2: Single-dose screening of unmodified dsRNA in HepG2 cells

[0369]

[0370]

[0371]

[0372] According to the results, among the 7 candidate positive references, TJ632 showed the best effect, and several candidate sequences, such as dsRNAs 3, 5, 8, 9, 24, 26, 50, 70, and 82, showed effects superior to or equivalent to the positive reference TJ632.

[0373] Example 3, Optimization of FXI-dsRNA

[0374] 3.1 Detection of Inhibitory Activity

[0375] To further identify the optimized dsRNA molecule, the inventors performed combinations of fluorination and methoxy group modifications at different positions of candidate sequences using two different modification motifs for each of the optimized sequences.

[0376] Specific variations are selected from any of the following:

[0377] (1) Modified by E (the dsRNA modified by E is denoted as En, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule number 3 is denoted as E3 after being modified by E):

[0378] Sense sequence: Starting from 5', the 7th, 9th, 10th, and 11th positions are 2'-fluoro-modified nucleotides, other positions are 2'-O-methyl-modified nucleotides, and the first and second phosphoester bonds at the 5' end form phosphothioate bonds through thiolation; for example, if N represents any ribonucleotide, the modification of the sense sequence is: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNmNm;

[0379] Antisense sequence: starting from 5', the 2nd, 6th, 14th, and 16th positions are 2'-fluoro-modified nucleotides (i.e., starting from the 3' end of the region complementary to the sense strand with the antisense sequence, the 2nd, 6th, 14th, and 16th positions are 2'-fluoro-modified nucleotides), other positions are 2'-O-methyl group-modified nucleotides, and the first and second phosphoester bonds at the 5' and 3' ends form phosphothioate bonds through thiolation; for example, if N represents any ribonucleotide, the modification of the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNmNm.

[0380] (2) Modified by E-1 (the dsRNA modified by E-1 is denoted as En-1, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule number 3 is denoted as E3-1 after being modified by E-1),

[0381] The only difference between E-1 and E1 modification is that the 6th nucleotide starting from the 5' end of the E-1 modified dsRNA antisense strand is a 2'-O-methyl group modified nucleotide rather than a 2'-fluoro modification, i.e., for example:

[0382] The variation of the sense sequence is: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNmsNm;

[0383] The antisense sequence variant is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNmsNm.

[0384] In the sequence modifications of the present application, s represents a phosphorothioate bond, m represents a 2'-O-methyl group modification, and f represents a 2'-fluoro modification.

[0385] In addition, the antisense compound ISIS-416858 used the structure and modification method disclosed in CN109797150A, namely the “5-10-5” structure, with five 2’-O-methoxyethyl group modified nucleotides on each side, ten deoxynucleotides in the middle, and a total thio-modified methylcytosine replacing cytosine.

[0386] 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 values ​​of the target gene expression levels of the relative blank control (the relative mRNA expression level of the blank control was set to 1).

[0387] Table 3: Single-dose (1 nM) screening of modified dsRNA in HepG2 cells

[0388]

[0389]

[0390] As can be seen from this, compared to chemically modified positive control sequences, several candidate sequences such as E8, E14, E19, E24, E26, and E50 still showed results equivalent to or superior to the best positive reference ETJ632. The above results once again demonstrated that the optimized sequences screened by the inventors have the potential to efficiently lower FXI gene nucleic acid levels.

[0391] Example 4 Detection of GalNAc-dsRNA In Vivo Efficacy

[0392] Candidate modified dsRNAs were conjugated to GalNAc to form a GAL-dsRNA complex, that is, G modification was performed for each dsRNA.

[0393] The above G modification refers to conjugating L96 to the 3' end of the dsRNA sense sequence, and includes GE modification and GE-1 modification, where:

[0394] The GE modifications are as follows, where “-L96” indicates that L96 is attached to the 3' end of the dsRNA sense sequence:

[0395] The variation of the sense sequence is: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96;

[0396] The antisense sequence variation is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0397] GE-modified dsRNA is denoted as GEn, where n is the number of the candidate dsRNA molecule; for example, dsRNA molecule number 3 is denoted as GE3 after GE modification;

[0398] GE-1 is as follows:

[0399] The variation of the sense sequence is: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96;

[0400] The antisense sequence variation is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNmNm;

[0401] Likewise, “-L96” indicates that L96 is attached to the 3' end of the dsRNA sense sequence via a phosphoester bond, and the dsRNA modified by GE-1 is denoted as GEn-1, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule number 3 is denoted as GE3-1 after being modified by GE-1.

[0402] The inventors further verified the activity of the G-modified candidate complex in HepG2 cells.

[0403] 4.1 Detection of mRNA Expression Levels in HepG2 Cells

[0404] The detection step is the same as in Example 2, using the human GAPDH gene as the internal reference gene, and the detection primers used are as indicated in the primer section of the sequence table at the end of the text. Table 4 shows the average mRNA levels in HepG2 cells treated with G-modified dsRNA 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 statistical results are shown in Figure 4.

[0405] Table 4 IC50 data of some GalNAc-conjugated dsRNAs in HepG2 cells

[0406]

[0407] As can be seen from this, the IC50 of all GalNAc-conjugated dsRNAs in HepG2 cells was less than 0.1 nM, once again demonstrating that the candidate sequences exhibit high inhibitory activity against target genes.

[0408] Example 5: Detection of in vivo efficacy

[0409] The experiment was conducted using 6 to 8-week-old SPF-grade humanized FXI homozygous male mice (Shanghai Model Organisms Center, Inc.), randomly divided into groups of 6 mice each, and administered via a single subcutaneous injection. Each group was injected with a different dsRNA modified by GE, while the blank group was injected with physiological saline. Blood was collected on days 7 and 13 after administration, and serum was collected by bleeding from the posterior eye and used to detect FXI protein content.

[0410] The specific dosage is as shown in Table 5.

[0411] Table 5. Single Dose Plan for 3mpk

[0412]

[0413] 5.1 FXI Protein ELISA Detection

[0414] After diluting the serum to 1:500, the residual FXI content in the plasma of each group was detected using the ab108834ELISA detection kit from Abcam, and the specific operation steps were referred to in the ab108834ELISA detection instructions. The absorbance value was detected at a wavelength of 450 nm using a microplate reader and analyzed with GraphPad software. The percentage of inhibition rate is a relative result to the untreated group, and the results are as shown in Table 6. Figure 5 shows the relative expression levels of FXI protein in the plasma of each group, normalized to a blank control.

[0415] Table 6: Plasma FXI protein inhibition rate after a single dose of 3mpk

[0416]

[0417] As can be seen from this, following a single administration of 3mpk, the in vivo plasma FXI content in mice of the candidate dsRNA molecule administration group was found to be significantly lower compared to the blank control group. On day 13, the inhibition rates of GE26, GE50, and GE70 were greater than 80%, with GE50 showing the highest inhibition rate at 92.8%. At the same time, the inhibition rates of all administration groups were >50% on day 13. The above results demonstrate that the various dsRNA molecules designed by the inventors for FXI can effectively inhibit the expression and secretion of FXI protein in the liver, proving them to be potential therapeutic drugs targeting the FXI gene.

[0418] Example 6 In vivo efficacy evaluation experiment of different ligands

[0419] The inventors further verified the effects of different ligand conjugates on candidate dsRNAs. Table 7 contains the dsRNA sequence information. This uses the LN-E05 variant or the GN-E05 variant, where the LN-E05 variant is identical to the GE-1 variant of Example 4, and the GN-E05 variant is specifically as follows:

[0420] The modification of the sense sequence is: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1;

[0421] The antisense sequence variation is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNmNm;

[0422] The difference between the GN-E05 variant and the LN-E05 variant is that the L96 ligand attached to the 3' end of the sense strand is replaced by ligand 1, and ligand 1 is connected to the 3' end of the sense strand through a phosphorothioate bond.

[0423] Table 7 Synthesized Oligonucleotides

[0424]

[0425] Groups were divided according to Table 7, and each group was administered a single subcutaneous injection of 3 mpk. Blood samples were collected at different time points after administration, and plasma was used to detect FXI protein content. The percentage of retention rate is a relative result compared to before drug administration. As shown in Figure 6, the results indicated that with the single 3 mpk administration, the dsRNA of linkage ligand 1 showed a significantly higher inhibition rate of FXI protein than that of linkage L96 dsRNA (using paired T-test, p<0.05).

[0426] The structure of ligand 1 is represented by Formula II, and:

[0427]

[0428] Here, the wavy line indicates connection to the 3' end of the dsRNA sense strand 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 synthesis methods in the art. Here, the synthesis methods for compounds 6 and 8 are as follows:

[0429] 1. Synthesis of Compound 6

[0430]

[0431] ① Synthesis of Compound 2:

[0432] 1.2 g of Compound 1 and 12 mL of pyridine were added to a three-necked flask. After stirring to dissolve, 1.2 g of DMTrCl was added, and the mixture was stirred at room temperature for 1 hour under the protection of nitrogen gas. Methanol (0.6 mL) was slowly added to the reaction mixture to quench the excess DMTrCl, and after stirring at room temperature for 15 minutes, 303.5 mg of NaHCO3 was added. After concentration, a crude product was obtained, DCM / H2O was added and stirred to dissolve, the mixture was washed with water, the organic phase was dried with anhydrous sodium sulfate, and then filtered. The collected organic phase was concentrated under vacuum to obtain a crude product of Compound 2, which was used directly in the next step of the reaction.

[0433] ② Synthesis of Compound 3:

[0434] Ethanol (24 mL) was added to the crude product of Compound 2, the temperature was slowly raised to 50°C and stirred to dissolve it, then hydrazine hydrate (0.75 mL) was added, and the mixture was stirred overnight at 50°C, then cooled to room temperature and stirred continuously for 30 minutes at room temperature, during which a large amount of white solid gradually precipitated. The mixture was filtered, and the filtration residue was washed with ethanol. The obtained filtrate was concentrated under reduced pressure, redissolved in DCM / H2O, separated, and the organic phase was separated. The organic phase was washed with saturated saline solution, dried with anhydrous sodium sulfate, and then filtered. The collected organic phase was concentrated under vacuum to obtain the crude product of Compound 3, which was used directly in the next step of the reaction.

[0435] ③ Synthesis of Compound 5:

[0436] 0.9 g of Compound 4, 24 mL of DCM, 0.84 mL of Et3N, and 2.3 g of HBTU were sequentially added to the crude product of Compound 3, and the mixture was stirred at room temperature for 2 hours under nitrogen protection. The reaction was quenched by adding 9 mL of water, the organic phase was separated, washed with saturated saline, dried with anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified through a preparative reverse phase (75% ACN-H2O) to obtain 1.4 g of Compound 5, with a three-step yield of 44.2%. ESI-MS: m / z 1025.6 [M+OAc-]-.

[0437] ④ Synthesis of Compound 6:

[0438] 1.4 g of compound 5 was dissolved in DCM (14 mL) and cooled by leaving it at 0 ± 2 °C. 524.7 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite was added, followed by the addition of 81.3 mg of 1H-tetrazole. The reaction solution was first stirred at 0 ± 2 °C for 15 minutes, then heated to room temperature and stirred continuously for 2 hours. The reaction solution was cooled at 0 ± 2 °C, 14 mL of 5% NaHCO3 was added to quench the reaction, the organic phase was separated, washed with saturated saline (1 × 14 mL) at 0 ± 2 °C, dried with anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under reduced pressure, and the obtained crude product was redissolved in DCM / MTBE and carefully added dropwise to an n-heptane (hept.) solution being vigorously stirred. During this process, a large amount of viscous oily material gradually precipitated on the flask walls and bottom. After standing for 10 minutes, the supernatant was decanted. The crude product was chromatographed with an EA:hept. = 10:1 to 2:1 eluent, concentrated, and dried to obtain 560 mg of white solid powder. The yield was 32%, and the phosphorus spectral purity was 98.19%. ESI-MS: m / z 1225.4 [M+OAc-]-.

[0439] 2. Synthesis of Compound 8

[0440]

[0441] ① Synthesis of Compound 7:

[0442] 2 g of compound 5 was dissolved in DCM (20 mL), and 248.4 mg of succinic anhydride and 0.69 mL of Et3N were added sequentially. Finally, 25.3 mg of DMAP was added and stirred at room temperature for 16 hours. After monitoring that a large amount of raw material remained by HPLC, 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 solution was cooled to 0 ± 2°C, ice water was added to the reaction, additional DCM was added, and finally 40 mL of 1% HOAc aqueous solution was added and stirred for 10 minutes. The organic phase was separated, washed sequentially with 1% HOAc aqueous solution and water, dried with anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product. The crude product was chromatographed with a DCM:MeOH eluent of 70:1 to 20:1, concentrated, and dried to obtain 0.9 g of a white solid powder with a yield of 40.7%. Detection results showed no residue of succinic acid, and a residue of succinic anhydride of 0.26%. ESI-MS: m / z 1065.7[MH]-.

[0443] ② Synthesis of Compound 8:

[0444] 300 mg of compound 7, 72.7 mg of DIPEA, 106 mg of HBTU, and 10 mL of acetonitrile were added to a three-necked flask and stirred at 25°C for 10 minutes, after which 650 mg of solid-phase support PS was added and stirring continued at 25°C for 24 hours. After the reaction was complete, the mixture was filtered, the filter cake was washed with acetonitrile, the filter cake was collected, and the solvent was removed by concentrating under vacuum to obtain 850 mg of solid. 700 mg of this was taken and added to a three-necked flask, and 1.74 g of acetic anhydride, 4.15 mg of DMAP, 103 mg of triethylamine, and 10 mL of pyridine were further added, and the mixture was stirred at 25°C for 4 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed sequentially with acetonitrile, methanol, and acetonitrile, and the filter cake was collected and concentrated under vacuum to remove the solvent, thereby obtaining 750 mg of compound 8. The measured loading was 254.82 μmol / g.

[0445] 3. Preparation of the oligonucleotide sequence linked to ligand 1

[0446] A precursor (or intermediate) of ligand 1 linked to a solid-phase support could be obtained by sequentially reacting compound 8 with two compounds 6 or sequentially reacting three compounds 6 through the conventional solid-phase synthesis cycle steps (deprotection, coupling, oxidation, and capping). Additionally, a solid-phase synthesis cycle reaction was performed using the corresponding 2'-modified monomer. After the reaction was complete, the entire synthesized molecule was separated from the solid-phase support by ammonia digestion to obtain the oligonucleotide sequence linked to ligand 1.

[0447] Example 7 In vitro and in vivo efficacy evaluation experiment of different modified motifs

[0448] The inventors further verified the effects of different modification motifs in dsRNAs with base sequences of double strand number 50 and double strand number 26. The specific molecules used are shown specifically in Table 8. As can be seen from this, all molecules in Table 8 are connected to ligand 1 via a phorprothioate bond at the 3' end of the sense strand, whereas a 5'-vp modification was used at the 5' end nucleotide of the antisense strand.

[0449] Table 8: Synthesized Oligonucleotides

[0450]

[0451] Each variant motif in Table 8 is as follows:

[0452] GN-E20VP:

[0453] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1

[0454] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0455] GN-E04VP:

[0456] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1

[0457] Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;

[0458] GN-E05VP:

[0459] Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1

[0460] Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.

[0461] The modification of GN-E05 is the same as in Example 6, and the difference from the modification of GN-E05VP is that GN-E05 does not have a vinyl phosphate modification at the 5' end of the antisense strand.

[0462] 7.1 In Vitro Efficacy Evaluation of Different Modified Motifs

[0463] First, the inventors verified the effect of the different modification methods of Table 8 on pharmacological efficacy at the in vitro HepG2 cell level, referring to Example 4 for the experimental method. Table 4 shows the average mRNA levels in each treatment group relative to the target gene expression levels in the untreated group (the relative mRNA expression level of the untreated group was set to 1). The in vitro test was an independent replication result from different batches, and the statistical results are shown in Tables 9 and 10.

[0464] Table 9 IC50 data of 5VP modified dsRNA in HepG2 cells

[0465]

[0466] Table 10 IC50 data of different motif-modified dsRNAs in HepG2 cells

[0467]

[0468] The in vitro results are shown in Table 9 and Figure 7, and it was found that modifications of 5-VP help further enhance the efficacy. At the same time, when both the ligand and base sequence are identical, the modified motifs of GN-E05VP and GN-E20VP are significantly superior to GN-E04VP (see Table 10), and additionally, the modified motif of GN-E20VP was found to be significantly superior to GN-E05VP.

[0469] 7.2 In vivo evaluation of different modified motifs

[0470] Refer to Example 5 for the experimental plan, and dsRNA 50 with different modifications was administered to each group in Table 8 via a single subcutaneous injection of 1 mpk. Blood samples were collected on days 7, 14, 21, and 28 after administration, and plasma was used to detect FXI protein content. The percentage of retention rate is a relative result to the time point before drug administration, and the results are shown in Table 11 and Figure 8.

[0471] Table 11: Plasma FXI protein retention rate after a single 1 mpk dose

[0472]

[0473] According to the results of the modified molecule 50, when the dsRNA base sequences were identical in a single 1 mpk dose, the E05VP and E20VP modified motifs were superior to E04VP (P<0.05), and E20VP was found to be additionally superior to E05VP (P<0.05). At the same time, the results showed that the 5'-VP modification helped to further enhance the drug's efficacy.

[0474] Additionally, the inventors compared the efficacy of different modified motifs of molecule 26 in vivo (as shown in Table 8). Blood samples were collected on days 7 and 14 after administration, respectively, and plasma was used to detect FXI protein content. The percentage of retention rate is a relative result to the time point before drug administration, and the results are shown in Table 12.

[0475] Table 12: Plasma FXI protein retention rate after a single 1 mpk dose

[0476]

[0477] It was once again proven that the efficacy of E05VP and E20VP in molecule 26 is superior to that of E04VP, and the efficacy of the E20VP motif was found to be the best among them. In addition, modifications of 5'-VP were found to help further enhance the efficacy.

[0478] The sequences used in the above embodiments of the present application are shown in the sequence table below. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application and do not constitute any limitation on the forms of the present application. The nucleic acid sequences in the following sequence table may represent DNA sequences or RNA sequences, and in the case of RNA sequences, “T” represents uridine. Also, in the context of RNA, unless specifically stated otherwise, “T” and “U” both mean uracil or uridine.

[0479] Ranking Table:

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

Claims

Claim 1 A nucleic acid molecule that inhibits intracellular factor 11 (FXI) gene expression via RNAi, comprising or composed of mutually complementary sense sequences and antisense sequences, wherein the antisense sequence comprises a polynucleotide sequence complementary to the FXI gene mRNA, wherein, when the antisense sequence and the FXI gene mRNA are hybridized at a maximum complementation rate, a complementary region 1 is formed by base pairs that are complementarily paired between the antisense sequence and the FXI gene mRNA sequence; and when the antisense sequence and the sense sequence are hybridized at a maximum complementation rate, a complementary region 2 is formed by base pairs that are complementarily paired between the antisense sequence and the sense sequence.Herein, complementary region 1 and complementary region 2 have at least 18, 19, or 20 identical base pairs, the number of base pairs of complementary region 1 is 18 to 30 bp, and complementary region 1 comprises the following in the FXI gene mRNA sequence: 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, 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, or nucleotides at positions 518 to 537 or 18 or 19 consecutive nucleotides thereof; and, wherein the nucleotide position number in the FXI gene mRNA sequence is the corresponding nucleotide number in reference sequence number 244, a nucleic acid molecule.; Claim 2 A 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. Claim 3 A nucleic acid molecule according to claim 1 or 2, wherein the number of base pairs in the complementary region 2 is 21 bp. Claim 4 A 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. Claim 5 A 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. Claim 6 A nucleic acid molecule according to any one of claims 1 to 5, wherein the sense sequence and / or antisense sequence further comprises 1 to 2 protruding terminal nucleotides in addition to the complementary region 2. Claim 7 A nucleic acid molecule according to claim 6, wherein the protruding terminal nucleotides are two, located in the antisense sequence, adjacent to the 5' end of the complementary region 2, and the sense sequence does not contain protruding terminal nucleotides. Claim 8 A nucleic acid molecule according to any one of claims 1 to 7, wherein the antisense sequence comprises only 0, 1, or 2 nucleotides in addition to complementary region 1, and the sense sequence comprises only 0, 1, or 2 nucleotides in addition to complementary region 2. Claim 9 A nucleic acid molecule according to any one of claims 1 to 8, wherein the 3' end of the complementary region 2 is an AU base pair. Claim 10 In any one of claims 1 to 9, the FXI gene is a nucleic acid molecule that is a human FXI gene or a cyanomorphic monkey FXI gene. Claim 11 In claim 10, the mRNA is a nucleic acid molecule comprising a polynucleotide sequence represented by SEQ ID NO.

244. Claim 12 A nucleic acid molecule according to any one of claims 1 to 11, wherein the antisense sequence comprises or is itself a polynucleotide sequence represented by SEQ ID NOs 153, 177, 152, 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 200, 201, or 213. Claim 13 In any one of claims 1 to 12, the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 34, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 153; the sense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 58, and the antisense sequence comprises or is the polynucleotide sequence represented by SEQ ID NO. 177; The sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 33, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 152; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 3, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 122; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 4, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 123; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 7, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 126; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 10, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 129; and the sense The sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 11, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 130; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 12, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 131;The sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 17, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 136; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 18, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 137; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 19, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 138; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 24, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 143; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 30, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 149; and the A nucleic acid molecule, wherein the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 31, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 150; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 81, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 200; the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 82, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 201; or the sense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO. 94, and the antisense sequence comprises or is itself the polynucleotide sequence represented by SEQ ID NO.

213. Claim 14 A nucleic acid molecule that is dsRNA or shRNA in any one of paragraphs 1 to 13. Claim 15 In paragraph 14, a nucleic acid molecule that is siRNA. Claim 16 A nucleic acid molecule according to any one of claims 1 to 15, wherein one or more nucleotides are chemically modified. Claim 17 In any one of claims 1 to 16, the chemical modification is one or more selected from the group consisting of: locking nucleic acid modification, ring-opening or unlocking 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 modification, 2'-amino modification, morpholino modification, phosphoramidate modification, methylphosphonate modification, tetrahydropyran modification, 1,5-anhydrohexitol modification, 5'-vinylphosphate modification, and cyclohexenyl modification; preferably, the 7th, 9th to 12th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and / or the 2nd, 14th of the antisense strand The 16th nucleotide comprises a 2'-fluoro modification, and additionally preferably, the remaining nucleotides comprise a 2'-O-methyl group modification; preferably, the 7th, 9th to 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; additionally preferably, the remaining nucleotides comprise a 2'-O-methyl group modification; preferably, the 7th, 9th to 11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification, and the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification; additionally preferably, the remaining nucleotides comprise a 2'-O-methyl group modification;Preferably, the nucleic acid molecule comprises a motif selected from the group consisting of: (1) sense sequence: NmNmNmNmNmNmNfNmNfNfNfNfNfNm NmNmNmNmNmNmNmNfNmNfNfNmNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm, where Nm represents a ribonucleotide modified by a 2'-O-methyl group and Nf represents a ribonucleotide modified by a 2'-fluoro group; additionally preferably, having a phosphorothioate modification at at least one of the positions numbered from 5' between the first and second nucleotides, between the second and third nucleotides, between the last and the first and second, and between the last and the second and third; A nucleic acid molecule having a phosphorothioate modification at at least one of the positions numbered from the antisense sequence 5', between the first and second nucleotides, between the second and third nucleotides, between the last and the first and second, and between the last and the second and third. Claim 18 In claim 17, the nucleic acid molecule comprises a motif selected from the group consisting of: (1) sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNm NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;(4) sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;(5) sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; and (6) sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; (7) sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm; (8) sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;(9) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms, Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; where Nm represents a ribonucleotide modified by a 2'-O-methyl group, Nf represents a ribonucleotide modified by a 2'-fluoro group, and s represents a phosphorothioate modification, nucleic acid molecule.; Claim 19 A nucleic acid molecule connected to at least one asialoglycoprotein receptor (ASGPR) ligand in any one of claims 1 to 18. Claim 20 In claim 19, the above ASGPR ligand is a nucleic acid molecule connected to the 5' end or 3' end of the sense sequence. Claim 21 In claim 19 or 20, the nucleic acid molecule wherein the ASGPR ligand is one or more GalNAc derivatives connected through a divalent or trivalent branched chain structure. Claim 22 In claim 21, the GalNAc derivative is L96 or ligand 1, and the structure of L96 is as follows: (Formula I); The structure of the above ligand 1 is as follows: (X=S- or O-)(Equation II); wherein, in the above formulas, in Equation I and Equation II each indicates being connected to the 3' end of the sense sequence or antisense sequence of the nucleic acid molecule; preferably, being connected to the 3' hydroxyl group via a phosphodiester bond or a phosphorothioate diester bond; additionally preferably, the nucleic acid molecule is a nucleic acid molecule having any one of the following modified motifs: (1) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm;(2) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;(3) Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;(4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;(5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNm-L96; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNfNmNmNmNmNmNmsNmsNm;(6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNmsNm. Claim 23 In any one of claims 1 to 22, the nucleic acid molecule comprises a phosphate or phosphate mimic modification at the 5' end of the antisense strand; Preferably, the 5' phosphate mimic modification is 5'-VP; additionally preferably, the nucleic acid molecule is a nucleic acid molecule having any one of the following modification motifs: GN-E20VP:Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; GN-E04VP:Sense sequence: NmsNmsNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1; Antisense sequence: vp-NmsNfsNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;GN-E05VP:Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNms-ligand 1;Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNmsNm. Claim 24 A second nucleic acid molecule that can be transcribed into a dsRNA or shRNA precursor within a cell, wherein the dsRNA or shRNA is a nucleic acid molecule according to any one of claims 1 to 23. Claim 25 A virus particle comprising the polynucleotide sequence of the second nucleic acid molecule according to paragraph 24. Claim 26 A cell comprising the polynucleotide sequence of the second nucleic acid molecule according to paragraph 24. Claim 27 Use of a nucleic acid molecule or a salt thereof according to any one of claims 1 to 23 for inhibiting the expression of factor 11. Claim 28 The use of a nucleic acid molecule or a salt thereof according to any one of claims 1 to 23 in the manufacture of a drug that inhibits FXI expression in the body of a subject. Claim 29 A pharmaceutical composition comprising 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 virus particle according to claim 25, or a cell according to claim 26. Claim 30 A use of a nucleic acid molecule or salt thereof according to any one of claims 1 to 23, a second nucleic acid molecule according to claim 24, a virus particle according to claim 25, a cell according to claim 26, or a pharmaceutical composition according to claim 29 in the manufacture of a drug for preventing or treating thromboembolic complications or coagulation disorders in a subject, wherein the thromboembolic complications are preferably one or more selected from the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Claim 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 the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke, and the method comprises the step of administering 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 virus particle according to claim 25, a cell according to claim 26, or a pharmaceutical composition according to claim 29 to a subject in need.

Images