[go: up one dir, main page]

WO2025157271A1 - Nucleic acid molecule inhibiting f11 gene expression - Google Patents

Nucleic acid molecule inhibiting f11 gene expression

Info

Publication number
WO2025157271A1
WO2025157271A1 PCT/CN2025/074764 CN2025074764W WO2025157271A1 WO 2025157271 A1 WO2025157271 A1 WO 2025157271A1 CN 2025074764 W CN2025074764 W CN 2025074764W WO 2025157271 A1 WO2025157271 A1 WO 2025157271A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
nucleotides
seq
nucleic acid
antisense
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/074764
Other languages
French (fr)
Chinese (zh)
Inventor
张雪艳
石延君
王凌宇
董云霞
赵成龙
钟强
邵昱
刘建国
王传金
徐海亮
苏晓晔
李春雷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Original Assignee
CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd filed Critical CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Priority to CN202580000106.8A priority Critical patent/CN120712356A/en
Publication of WO2025157271A1 publication Critical patent/WO2025157271A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present application relates to the field of RNAi, and in particular to a nucleic acid molecule capable of inhibiting F11 gene expression through RNAi and its use.
  • RNA interference refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution.
  • dsRNA double-stranded RNA
  • dsRNA double-stranded RNA
  • RISC RNA interference
  • RISC can bind to mRNA in the cell that is complementary to the antisense strand of the dsRNA and cleave the mRNA, causing it to be degraded, preventing protein synthesis and resulting in gene "silencing.”
  • Thrombi are blood clots that restrict blood flow within blood vessels. They can occur in either the arterial or venous circulation and are the common pathological basis for most myocardial infarctions, ischemic strokes, and venous thromboembolism (VTE).
  • VTE venous thromboembolism
  • thrombin and FX or coagulation factor 10
  • Attempting to achieve anticoagulant effects by inhibiting either of these enzymes inevitably compromises hemostasis. This conflict limits the therapeutic efficacy of anticoagulants and means that patients at high risk of bleeding may not be suitable for anticoagulant therapy.
  • FXI coagulation factor XI
  • the present application provides a nucleic acid molecule that inhibits factor XI (FXI) gene expression in cells by RNAi, including compositions, preparations, and uses of the nucleic acid molecule.
  • the present application's examples provide sufficient data showing that preferred nucleic acid molecules of the present application include, but are not limited to, dsRNA molecules numbered 3, 3', 5, 8, 9, 9', 14, 14', 15, 19, 24, 24', 26, 26', 50, 70, 70', and 82, shRNA molecules whose structures and sequences include the aforementioned dsRNA molecules, precursor molecules of the shRNA molecules or dsRNA molecules, and modified shRNA molecules or dsRNA molecules.
  • Preferred modifications include, but are not limited to, modification E and modification E-1 described in detail in the present application's examples.
  • the present application relates to a nucleic acid molecule comprising or consisting of a sense sequence and an antisense sequence that are complementary to each other, and the antisense sequence comprises a polynucleotide sequence that is complementary to FXI gene mRNA, wherein:
  • complementary region 1 is formed by complementary base pairs in the antisense sequence and the FXI gene mRNA sequence
  • complementary region 2 is formed by complementary base pairs in the antisense sequence and the sense sequence;
  • complementary region 1 and complementary region 2 have at least 18, 19 or 20 identical base pairs
  • the complementary region 1 has a base pair number of 15 to 35 bp, and the complementary region 1 includes the following in the FXI gene mRNA sequence:
  • nucleotides 372 to 391 or 18 or 19 consecutive nucleotides therein nucleotides 411 to 430 or 18 or 19 consecutive nucleotides therein,
  • nucleotides 425 to 444 or 18 or 19 consecutive nucleotides therein nucleotides 465 to 484 or 18 or 19 consecutive nucleotides therein,
  • nucleotides 471 to 490 or 18 or 19 consecutive nucleotides therein nucleotides 518 to 537 or 18 or 19 consecutive nucleotides therein;
  • the site number of the nucleotide in the FXI gene mRNA sequence is the number of the corresponding nucleotide in the reference sequence SEQ ID NO: 244.
  • the “18 or 19 consecutive nucleotides” of a nucleotide sequence may be 19 consecutive nucleotides starting from the first nucleotide or the second nucleotide at the 5’ end of the nucleotide sequence and including the first nucleotide or the second nucleotide, which are consistent with the nucleotide sequence in the nucleotide sequence, or may be 18 consecutive nucleotides starting from the first nucleotide, the second nucleotide or the third nucleotide at the 5’ end of the nucleotide sequence and including the first nucleotide, the second nucleotide or the third nucleotide, which are consistent with the nucleotide sequence in the nucleotide sequence.
  • the sense sequence is equal to the antisense sequence. In some embodiments, the sense sequence and/or antisense sequence further comprise overhanging nucleotides outside the complementary region 2. In some embodiments, the sense sequence is longer than the antisense sequence. In some embodiments, the antisense sequence is longer than the sense sequence. In some embodiments, the sense sequence is 1, 2, 3, or 4 nucleotides longer than the antisense sequence. In some embodiments, the antisense sequence is 1, 2, 3, or 4 nucleotides longer than the sense sequence. In some embodiments, the sense sequence further comprises 1 or 2 nucleotides on the 5' end side and/or the 3' end side of the complementary region 2.
  • the antisense sequence further comprises 1 or 2 nucleotides on the 5' end side and/or the 3' end side of the complementary region 2. In some embodiments, the sense sequence and antisense sequence further comprise 1 or 2 nucleotides on the 5' end side and/or the 3' end side of the complementary region 2. In some embodiments, when the antisense sequence hybridizes to the sense sequence at maximum complementarity, the sense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the antisense sequence between the first and last nucleotides in complementary region 2.
  • the antisense sequence when the antisense sequence hybridizes to the sense sequence at maximum complementarity, the antisense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the sense sequence between the first and last nucleotides in complementary region 2.
  • the overhang nucleotides are two and are located in the antisense sequence, adjacent to the 5' end of complementary region 2, and the sense sequence does not include an overhang nucleotide.
  • the antisense sequence contains only 0, 1, or 2 nucleotides outside complementary region 1, and the sense sequence contains only 0, 1, or 2 nucleotides outside complementary region 2.
  • the antisense sequence when the antisense sequence hybridizes to the FXI gene mRNA sequence at maximum complementarity, the antisense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the FXI gene mRNA sequence between the first and last nucleotides in complementary region 1. In some embodiments, when the antisense sequence hybridizes with the FXI gene mRNA sequence at maximum complementarity, the FXI gene mRNA sequence does not contain nucleotides that are not complementary to the antisense sequence between the first nucleotide and the last nucleotide of complementary region 1. In some embodiments, the 3' end of complementary region 2 is an AU base pair.
  • complementary region 1 and complementary region 2 are uninterrupted, that is, continuous. In some embodiments, complementary region 1 and/or complementary region 2 are discontinuous, that is, interrupted by one or more bubbles (bubbles, non-complementary regions formed by non-complementary nucleotides in each of the two sequences forming the complementary region).
  • the number of base pairs in complementary region 1 is 15 to 35, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bp. In some embodiments, the number of base pairs in complementary region 2 is 15 to 35, such as 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bp. In some embodiments, complementary region 1 and complementary region 2 have 18, 19, 20, 21, 22, or 23 identical base pairs. In some embodiments, the length of the sense sequence and/or the antisense sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nt. In some embodiments, the sense sequence is 21nt in length and the antisense sequence is 23nt in length.
  • the nucleic acid molecule is composed of a sense sequence of 21nt in length and an antisense sequence of 23nt in length. In some embodiments, the nucleic acid molecule is composed of a sense sequence and an antisense sequence and a connecting chain (or shRNA loop), wherein the connecting chain connects the 3' end nucleotides of the sense sequence and the 5' end nucleotides of the antisense sequence.
  • the selection of the connecting chain is consistent in the art, for example, with reference to the literature Jensen, Stig The selection was performed as described in Gaard Rask et al. "Functional selection of shRNA loops from randomized retroviral libr aries.” PLoS one vol. 7, 8 (2012): e43095. doi: 10.1371/journal.pone.0043095, which is incorporated herein by reference in its entirety.
  • the sense sequence and/or antisense sequence further comprises 1 to 2 overhang nucleotides outside the complementary region 2.
  • the antisense sequence comprises only 0, 1, or 2 nucleotides outside complementary region 1, and the sense sequence comprises only 0, 1, or 2 nucleotides outside complementary region 2.
  • the 3' end of complementary region 2 is an AU base pair; in further embodiments, the 5' end A of the antisense strand is outside complementary region 1.
  • the FXI gene mRNA sequence complementary to the antisense strand is transcribed from a mammalian FXI gene coding region.
  • the mammal is a primate.
  • the primate is a human or a cynomolgus monkey.
  • the mRNA comprises the polynucleotide sequence shown in SEQ ID NO: 244.
  • the antisense sequence comprises the polynucleotide sequence shown in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201, or 213.
  • the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201 or 213, and additional 1, 2, 3 or 4 nucleotides.
  • the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 18 and 1 to 4 (e.g., 2 or 3) additional nucleotides
  • the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 137 and 1 to 4 (e.g., 2 or 3) additional nucleotides
  • the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 19 and 1 to 4 (e.g., 2 or 3) additional nucleotides
  • the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 138 and 1 to 4 (e.g., 2 or 3) additional nucleotides
  • the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 24 and another 1 to 4 (e.g., 2 or 3) nucleotides
  • the antisense sequence consists of a polynucleotide
  • the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 7 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 126.
  • the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 10 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 129.
  • the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 30 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 149.
  • the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 31 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 150.
  • the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 34 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 153; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 58 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 81 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 200; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 82 and another 1 to 4 (e.g., 2 or 3) nucleot
  • the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 24 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 143 and another 1 to 4 (e.g., 2 or 3) nucleotides;
  • the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 30 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 149 and another 1 to 4 (e.g., 2 or 3) nucleotides;
  • the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 31 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 150 and another 1 to 4 (e.g., 2 or 3) nucleotides;
  • the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 33 and the antisense sequence consists of the polynucleotide sequence shown in S
  • the present invention relates to a polynucleotide sequence comprising a polynucleotide sequence as shown in SEQ ID NO: 153 and 1 to 4 (e.g., 2 or 3) additional nucleotides;
  • the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 58 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 177 and 1 to 4 (e.g., 2 or 3) additional nucleotides;
  • the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 81 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 200 and 1 to 4 (e.g., 2 or 3) additional nucleotides;
  • the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 82 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 34
  • the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 153;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 33, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 152;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 3, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 122;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 4, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 123;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 7, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 126;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 10
  • the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 129;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 11
  • the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 130;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 12, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 131;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 17, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 136;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 18, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 137;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 19, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 138;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 24, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 143;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 30, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 149;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 31, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 150;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 81, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 200;
  • the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 82
  • the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 201; or
  • the sense sequence includes or is a polynucleotide sequence as shown in SEQ ID NO: 94
  • the antisense sequence includes or is a polynucleotide sequence as shown in SEQ ID NO: 213.
  • the nucleic acid molecule is a dsRNA or shRNA. In some embodiments, the ds RNA is an siRNA. In some embodiments, one or more nucleotides in the nucleic acid molecule are chemically modified. In some embodiments, the chemical modification makes the nucleic acid molecule more stable in a cell or in vivo environment. In some embodiments, the chemical modification prolongs the half-life of the nucleic acid molecule in vivo and/or in vitro. In some embodiments, the chemical modification comprises any one or more selected from the following, or consists of one or more modifications selected from the following:
  • Locked nucleic acid modification open ring or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification (2′-OM e), 2′-O-allyl modification, 2′-C-alkyl modification, 2′-C-allyl modification, 2′-fluoro modification (2′-F), 2′-deoxy modification (d), phosphorothioate modification (s), 2′-amino-modification, morpholino modification, phosphoramidate modification, methylphosphonate modification, tetrahydropyranyl modification, 1,5-anhydrohexitol modification, 5′-vinyl phosphate modification, and cyclohexenyl modification.
  • the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 14, and 16 of the antisense strand comprise a 2'-fluoro modification.
  • the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 14, and 16 of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification.
  • the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and/or the nucleotides at positions 2, 14, and 16 of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.
  • the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the 2nd, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification.
  • the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the 2nd, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification.
  • the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and/or the 2nd, 14th, and 16th nucleotides of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.
  • the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand comprise a 2'-fluoro modification.
  • the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification.
  • the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and/or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.
  • the nucleic acid molecule comprises a motif selected from any one of the following:
  • Sense sequence NmNmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmN
  • sense sequence NmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm
  • Ns from 5' to 3' of each sequence independently represent the nucleotides at the position of the sequence in which they are located, Nm represents a 2'-O-methyl modified ribonucleotide, and Nf represents a 2'-fluoro modified ribonucleotide; it should be understood that this scheme does not limit whether each nucleotide contains or does not contain other modifications besides the marked modifications.
  • the above embodiments further include at least one thiophosphate modification between the first and second nucleotides, and between the second and third nucleotides of the 5' sense sequence; and at least one thiophosphate modification between the first and second nucleotides, between the second and third nucleotides, between the first to last and the second to last, and between the second to last and the third nucleotides of the 5' antisense sequence.
  • the nucleic acid molecule comprises a motif selected from any one of the following (1)-(9):
  • the sense sequence is NmsNmsNmNmNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm
  • the sense sequence is NmsNmsNmNmNmNmNmfNmNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm
  • the sense sequence is NmsNmsNmNmNmNmNmfNmNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm
  • the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;
  • the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;
  • the nucleic acid molecule has a modification motif as any one of (1) to (6):
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • the sense sequence NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.
  • the nucleic acid molecule has the following modification motif:
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.
  • the nucleic acid molecule has the following modification motif:
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • the sense sequence NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm-L96
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.
  • the nucleic acid molecule has the following modification motif:
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • the sense sequence NmsNmsNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm-L96
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.
  • the naked sequence of the nucleic acid molecule specifically provided in this application is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has any of the modification patterns listed above.
  • the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or
  • the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe, and phosphorothioate;
  • Antisense sequence NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate; or
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.
  • the naked sequence of the nucleic acid molecule provided in the present application is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having any of the modification patterns listed above.
  • the naked sequence of the nucleic acid molecule provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having the modification pattern shown below:
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.
  • the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), and has the following modification pattern:
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe, and phosphorothioate;
  • Antisense sequence NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate; or
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.
  • the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.
  • the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm; or
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.
  • the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), and has the following modification pattern:
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;
  • Antisense sequence NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;
  • Antisense sequence NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate; or
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.
  • the present application also provides a precursor of the aforementioned nucleic acid molecule, which may be shRNA or dsRNA.
  • the third aspect of the present application further provides a nucleic acid delivery body comprising the nucleic acid molecule of the aforementioned first aspect, or the second nucleic acid molecule of the aforementioned second aspect.
  • the nucleic acid delivery body is a liposome, lipid nanoparticle or other polymer, endosome, exosome or vesicle.
  • the cell comprising the second nucleic acid molecule of the second aspect.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell, such as a stem cell, such as a hematopoietic stem cell, a mesenchymal stem cell, or the like.
  • the fourth aspect of the present application further provides uses of the nucleic acid of the first aspect, the second nucleic acid of the second aspect, the nucleic acid delivery body of the third aspect, and the viral particles, cells, and pharmaceutical compositions.
  • the present application provides the use of the nucleic acid molecule or salt thereof of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, or the cell for preparing a medicament for preventing or treating thromboembolic complications or coagulation disorders in a subject.
  • the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
  • the present application also provides a method for treating or preventing thromboembolic complications or coagulation disorders in a subject, the method comprising administering to a subject in need thereof an effective amount of the nucleic acid molecule of the first aspect or a salt thereof, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, the cell, or the pharmaceutical composition.
  • the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
  • a compound for treating or preventing thromboembolic complications or coagulation disorders in a subject comprising the nucleic acid molecule or salt thereof of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, the cell, or the pharmaceutical composition.
  • the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
  • the present application also provides use of the nucleic acid molecule or a salt thereof according to the first aspect or the second nucleic acid molecule according to the second aspect for inhibiting the expression of factor 11.
  • the present application also provides use of the nucleic acid molecule or a salt thereof of the first aspect or the second nucleic acid molecule of the second aspect for preparing a drug for inhibiting FXI expression in a subject.
  • the present application also provides a compound for inhibiting FXI expression in a subject, the compound comprising the nucleic acid molecule or a salt thereof of the first aspect, and a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent for inhibiting FXI expression in a subject
  • the compound comprising the nucleic acid molecule or a salt thereof of the first aspect
  • a pharmaceutically acceptable carrier or diluent a pharmaceutically acceptable carrier or diluent.
  • FIG1 shows the results of high-throughput screening of naked dsRNA molecules (5 nM) in HepG2 cells.
  • Figure 2 shows the relative content of FXI mRNA in HepG2 cells when 5nM and 1nM candidate dsRNA or Yangshen dsRNA naked nucleic acid molecules were applied, respectively.
  • Figure 3 shows the inhibition rate of FXI mRNA in HepG2 cells at a concentration of 1 nM by dsRNA modified with E.
  • Figure 4 shows the inhibition rate of FXI mRNA by GalNAc-coupled dsRNA at different concentrations.
  • FIG5 shows the results of target protein inhibition after in vivo administration of different GalNAc-conjugated dsRNAs.
  • FIG6 shows the comparative results after in vivo administration of different GalNAc
  • FIG7 shows the comparison results of IC50 of different modified dsRNAs at the cell level in vitro
  • FIG8 shows the comparative results after in vivo administration of dsRNA with different modified motifs
  • the present invention provides a nucleic acid molecule capable of initiating RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the coagulation factor FXI (or FX11) gene, a second nucleic acid molecule capable of transcribing the nucleic acid molecule, a delivery vehicle for the nucleic acid molecule and the second nucleic acid molecule, a virus or cell capable of transcribing the nucleic acid molecule, and uses of the nucleic acid molecule and the second nucleic acid molecule.
  • RISC RNA-induced silencing complex
  • dsRNA is double-stranded RNA. Since siRNA is a double-stranded RNA, the term “dsRNA” encompasses siRNA. dsRNA also includes double-stranded RNA that is longer than siRNA. The length greater than siRNA can refer to its sense strand being longer than siRNA, or its antisense strand being longer than siRNA, or its sense strand and antisense strand being longer than siRNA. After a double-stranded RNA that is generally longer than the siRNA sequence it contains enters the cell, it is broken down into siRNA by a type III endonuclease called Dicer.
  • Dicer a type III endonuclease
  • the lengths of the two chains of the dsRNA are each independently 15 to 30 nt (in this application, “nt” refers to nucleotides).
  • siRNA When "siRNA” is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in RISC unwind the siRNA double helix. When bound to a target mRNA complementary to the antisense strand in the siRNA, one or more endonucleases in RISC cleave the target, inducing gene silencing.
  • RISC RNA-induced silencing complex
  • each nucleotide in the dsRNA is a ribonucleotide.
  • a dsRNA may include one or more chemically modified nucleotides or may not include chemically modified nucleotides.
  • FXI coagulation factor FXI
  • Factor 11 The terms “FXI,” “coagulation factor FXI,” and “Factor 11” are used interchangeably and are also referred to in the art as FXI or PTA.
  • FXI may be mammalian-derived FXI.
  • FXI is primate-derived FXI.
  • FXI is human-derived FXI.
  • FXI is cynomolgus monkey-derived FXI.
  • FXI gene mRNA refers to mRNA encoding Factor 11 protein, which may be transcribed from FXI gene DNA and may be mature mRNA or pre-mRNA (Pre-mRNA), and thus may or may not contain introns.
  • the Factor 11 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the Factor 11 gene mRNA sequence of the present application is intended to include all mRNA sequences transcribed from Factor 11 gene mutants.
  • the human FXI gene mRNA sequence can be found, for example, in GenBank Accession No. GI:1732746318 (NM_000128.4).
  • the rhesus macaque FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 1622942384 (XM_015139652.2).
  • the cynomolgus macaque FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No.
  • GI: 2161917139 (XM_005556483.3).
  • the mouse FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 2293430447 (NM_028066.3).
  • the rat FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 2293332621 (NM_001411666.1).
  • Other examples of FXI gene mRNA sequences are readily available using publicly available databases, such as GenBANK.
  • the position numbering of the bases in the FXI gene mRNA sequence is the numbering of the corresponding bases in the reference sequence SEQ ID NO: 244” means that after the FXI gene mRNA sequence and the reference sequence have the same bases at as many positions as possible by introducing gaps or deleting nucleotides into the FXI gene mRNA sequence, the nucleotides of the reference sequence are numbered consecutively in sequence order starting from the first nucleotide at the 5’ end, and the positions of the nucleotides corresponding to each other are defined by the same numbering by aligning the FXI gene mRNA sequence and the reference sequence.
  • substantially complementary refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • Watson-Crick base pairing rules when A pairs with T or U, or C pairs with G or I, and vice versa, they are said to be complementary, paired, or matched; base pairings other than these are said to be non-complementary.
  • hybridization of nucleic acids refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner.
  • the complex can include two chains forming a double-stranded structure, three or more chains forming a multi-stranded complex, a single self-hybridizing chain, or a combination thereof.
  • hybridizing at maximum complementarity when referring to two polynucleotide sequences or two nucleic acid chains "hybridizing at maximum complementarity", it refers to a hybridization mode in which as many nucleotides as possible in the two chains are paired with each other by forming hydrogen bonds.
  • hybridizing at maximum complementarity one or more mismatches may be allowed to occur, and one or more bulges may be allowed to appear in one or more of the two chains.
  • the two chains “hybridized at maximum complementarity” may not have mismatches and bulges after hybridization.
  • complementary region refers to all base pairs paired by hydrogen bonds from the first base pair paired by hydrogen bonds at the 5' end to the last base pair paired by hydrogen bonds after hybridization of the two chains.
  • the complementary region formed by the two hybridized chains may be continuous or intermittent.
  • “complementary region 1” specifically refers to the complementary region consisting of the complementary base pairs in the antisense sequence and the FXI gene mRNA sequence when the antisense sequence hybridizes with the FXI gene mRNA at maximum complementarity
  • complementary region 2 refers to the complementary region consisting of the complementary base pairs in the antisense sequence and the sense sequence when the antisense sequence hybridizes with the sense sequence at maximum complementarity.
  • the antisense sequence portions of complementary regions 1 and 2 typically have partially or completely identical nucleotide compositions.
  • the antisense sequence and sense sequence are relative to a third sequence that is complementary to one of the sequences.
  • the antisense sequence and sense sequence in the "nucleic acid molecule that inhibits the expression of the factor 11 (FXI) gene in cells through RNAi" are relative to the third sequence, the FXI gene mRNA sequence.
  • the antisense sequence refers to the sequence in the nucleic acid molecule that has a complementary region with the FXI gene mRNA sequence
  • the sense sequence refers to the sequence in the nucleic acid molecule that has at least 10 consecutive identical nucleotides with the FXI gene mRNA sequence.
  • the 5' end of any complementary region in the present application refers to the position of the nucleotide or base pair in the complementary region that is closest to the 5' end of the sense strand or the third sequence
  • the 5' end of the complementary region refers to the side or end relatively close to the 5' end of the sense strand or the third sequence.
  • any complementary region in the present application refers to the position of the nucleotide or base pair in the complementary region that is closest to the 3' end of the sense strand or the third sequence
  • the 3' end of the complementary region refers to the side or end relatively close to the 3' end of the sense strand or the third sequence.
  • "close”, “near” or “far” refers to the number of nucleotides between the two nucleotide positions.
  • overhang nucleotides are relative to complementary region 2, and are nucleotides located outside complementary region 2 in the sense sequence and/or antisense sequence nucleotides after the sense sequence and antisense sequence hybridize at maximum complementarity. In some embodiments, the overhang nucleotides are located only in the sense sequence, in some embodiments, the overhang nucleotides are located only in the antisense sequence, and in some embodiments, the overhang nucleotides are located in both the sense sequence and the antisense sequence. In some embodiments, the overhang nucleotides are only present on the 5' end of complementary region 2.
  • the overhang nucleotides are only present on the 5' end of complementary region 2 of the sense strand. In some embodiments, the overhang nucleotides are only present on the 5' end of complementary region 2 of the antisense strand. In some embodiments, the overhang nucleotides are only present on the 3' end of complementary region 2 of the sense strand. In some embodiments, the overhang nucleotides are only present on the 3' end of complementary region 2 of the antisense strand. In some embodiments, the number of nucleotides in the overhang region on the same side (e.g., the 5' end or 3' end) of the sense sequence or antisense sequence is no more than two (i.e., one or two).
  • “on the 5' end side” and “on the 3' end side” are both used to describe the relative positional relationship between two sequences, two nucleotides, or one nucleotide and one sequence in the same polynucleotide sequence; wherein “5' end” refers to the end of the polynucleotide sequence containing a 5' free phosphate group or a 5' free hydroxyl group, and “3' end” refers to the end of the polynucleotide sequence containing a free 3'-hydroxyl group or a 3'-phosphate group, and the sequence or nucleotide on the 3' end side of a sequence in the nucleic acid chain is closer to the 3' end of the nucleic acid chain than the sequence.
  • complementary region 2 also contains a certain sequence or a certain nucleotide or nucleotides, which means that the "certain sequence or a certain nucleotide or nucleotides” are closer to the 5' end of the polynucleotide sequence (e.g., the antisense sequence or the sense sequence) with which they are co-located, relative to the sequence of "complementary region 2."
  • nucleotide in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, is also understood herein to refer to related structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used, unless the context clearly indicates otherwise.
  • nucleotide refers to a deoxyribonucleotide or a ribonucleotide.
  • a nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), a nucleotide isomer, or a nucleotide analog.
  • a nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety.
  • a nucleotide analog can be a naturally occurring nucleotide (e.g., inosine, pseudouridine, etc.) or a non-naturally occurring nucleotide.
  • Non-limiting examples of modifications on the sugar or base portion of a nucleotide include the addition (or removal) of an acetyl group, an amino group, a carboxyl group, a carboxymethyl group, a hydroxyl group, a methyl group, a phosphoryl group, and a thiol group, and the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-deazapurine).
  • Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino oligonucleotides.
  • the "nucleotides” of the present application do not include non-natural nucleotides with modified bases. In some embodiments, the “nucleotides” of the present application do not include nucleotides with modified bases.
  • “G”, “C”, “A”, “T” and “U” generally represent nucleotides with guanine, cytosine, adenine, thymine and uracil as bases, respectively. Unless otherwise specified, “G”, “C”, “A”, “T” and “U” represent nucleotides with no limitation on the modifications they contain, that is, they can be used to represent natural nucleotides or non-natural nucleotides.
  • the non-natural nucleotides may contain ribose and/or modified bases, as long as the bases therein can still be paired with their naturally paired bases (i.e., paired according to the Watson-Crick principle) through hydrogen bonding.
  • T refers to uridine or uracil.
  • nucleotide “nucleotide residue,” and “base” are used interchangeably in contexts related to nucleotide sequences. Base pairs are measured in bp, with one bp being one base pair. Nucleotides are measured in nt, with one nt being one nucleotide.
  • internucleotide linkage refers to the chemical bond (or linking group) between two adjacent nucleotides or between a nucleotide and a ligand. Unless otherwise specified, the chemical bond is a phosphate bond or a phosphoester bond. However, when an "s" is specifically indicated in a modification motif, i.e., the position of a phosphorothioate modification, only the internucleotide linkage at the position where "s" appears is a phosphorothioate bond, and all other unindicated internucleotide linkages are phosphate bonds. For example:
  • Sense strand NmNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm
  • internucleotide linkages in (1) can be either phosphate bonds or phosphorothioate bonds
  • Antisense strand vp-UmsAfsAmUmGdUmGdCmCmUmCmGmAmUfAmAfCmUmCmUmGmsGmsCm; because it has a specially marked "s", except for the following internucleotide linkages which are phosphorothioate bonds, the internucleotide linkages at other positions are phosphate bonds: the first and second internucleotide linkages from the 5' end of the sense strand, the internucleotide linkage between the 3' terminal nucleotide of the sense strand and ligand 1, the first and second internucleotide linkages from the 5' end of the antisense strand, and the first and second internucleotide linkages from the 3' end of the antisense strand.
  • the first internucleotide linkage refers to the chemical bond between the first and second nucleotides starting from one end (such as the 5' end or the 3' end);
  • the second internucleotide linkage refers to the chemical bond between the second and third nucleotides starting from one end (such as the 5' end or the 3' end), and so on;
  • the nth internucleotide linkage refers to the chemical bond between the nth and n+1th nucleotides or ligands starting from one end (such as the 5' end or the 3' end), and so on.
  • overhang As used herein, “overhang,” “overhang,” and “overhang sequence” are used interchangeably and refer to one or more unpaired nucleotides that extend beyond the duplex region at the end of a chain.
  • a nucleotide overhang is typically formed when the 3' end of one chain extends beyond the 5' end of the other chain, or when the 5' end of one chain extends beyond the 3' end of the other chain.
  • the length of the nucleotide overhang is typically between 1 and 6 nucleotides, between 1 and 5 nucleotides, between 1 and 4 nucleotides, between 1 and 3 nucleotides, between 2 and 6 nucleotides, between 2 and 5 nucleotides, or between 2 and 4 nucleotides.
  • the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a specific embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide.
  • the nucleotides in the overhang can be ribonucleotides or modified nucleotides as described herein.
  • the nucleotides in the overhang are 2'-modified nucleotides (e.g., 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides), deoxyribonucleotides, reverse nucleotides (e.g., reverse abasic nucleotides, reverse deoxyribonucleotides), or combinations thereof.
  • the nucleotides in the overhang are deoxyribonucleotides, such as deoxythymidine.
  • the nucleotides in the overhang are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, or combinations thereof.
  • the overhang comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotides.
  • the UU dinucleotides can comprise ribonucleotides or modified nucleotides, such as 2'-modified nucleotides.
  • the overhang comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotides.
  • the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (e.g., a polypyrimidine or polypurine sequence, such as UU, TT, AA, GG, etc.).
  • a polypyrimidine or polypurine sequence such as UU, TT, AA, GG, etc.
  • the nucleotide overhangs can be at the 5' end or the 3' end of one or both strands.
  • the RNA molecule comprises nucleotide overhangs at the 5' end and the 3' end of the antisense strand.
  • the RNA molecule comprises nucleotide overhangs at the 5' end and the 3' end of the sense strand.
  • the RNA molecule comprises nucleotide overhangs at the 5" end of the sense strand and the 5' end of the antisense strand.
  • the RNA molecule comprises nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.
  • RNA molecule may comprise a nucleotide overhang at one end of a double-stranded RNA molecule and a flat end at the other end.
  • "Flat end” means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the duplex region.
  • the RNA molecule comprises a nucleotide overhang at the 3' end of the sense strand and a flat end at the 5' end of the sense strand and the 3' end of the antisense strand.
  • the RNA molecule comprises a nucleotide overhang at the 3' end of the antisense strand and a flat end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNA molecule comprises a flat end at both ends of the double-stranded RNA molecule.
  • the sense strand and the antisense strand have the same length, and the length of the duplex region is the same as that of the sense strand and the antisense strand (i.e., the molecule is double-stranded over its entire length).
  • GalNAc or "N-acetylgalactosamine”: refers to 2-(acetylamino)-2-deoxy-D-galactopyranose.
  • the term “GalNAc” or “N-acetylgalactosamine” includes both the ⁇ form: 2-(acetylamino)-2-deoxy- ⁇ -D-galactopyranose and the ⁇ form: 2-(acetylamino)-2-deoxy- ⁇ -D-galactopyranose.
  • the GalNAc compound of the present application is the ⁇ form, i.e., 2-(acetylamino)-2-deoxy- ⁇ -D-galactopyranose.
  • VP modification or "E-VP” refers to a phosphate mimic modification located at the 5' end of the antisense strand, with the specific structure being:
  • Bx1 is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine;
  • the term "3' end” specifically refers to the position of the first nucleotide or base pair at the 3' end of a single nucleotide sequence or a double-stranded polynucleotide.
  • the term “5' end” specifically refers to the position of the first nucleotide or base pair at the 5' end of a single nucleotide sequence or a double-stranded polynucleotide.
  • nucleic acid molecule may be used to refer to any molecule having a nucleotide sequence composed of two or more nucleotides linked by a phosphate bond, or a modified phosphate bond (eg, a phosphorothioate bond).
  • nucleotide sequence refers to a polynucleotide chain composed of nucleotides arranged in sequence according to a certain order. This polynucleotide chain can constitute a nucleic acid molecule in this application, or it can constitute a section of a chain of a nucleic acid molecule.
  • nucleotide sequence can be represented by a specific polynucleotide sequence composed of various nucleotides (such as ATCG) (such as any one of SEQ ID NO: 1 to 100), or it can be represented as nucleotides from a certain position to a certain position in a certain sequence, such as “nucleotides from positions 363 to 382 in the FXI gene mRNA sequence".
  • nucleotide sequence can be RNA or DNA, or a hybrid molecule of RNA and DNA, and can also incorporate non-natural nucleotides or artificially modified nucleotides.
  • REL or "Relative expression level” appearing in the figures of this application refers to the relative expression level of mRNA.
  • N represents a ribonucleotide
  • dN represents a deoxyribonucleotide (DNA)
  • Nm represents a 2'-O-Me modified nucleotide, also known as a 2'-O-methyl modified nucleotide
  • Nf represents a 2'-F modified ribonucleotide or a 2'-fluoro modified ribonucleotide
  • s represents a phosphorothioate modification, i.e., a 5'-thio modified phosphate.
  • the term “about” refers to the typical error range for each value that is readily known to those skilled in the art. Reference to an "about” value or parameter herein includes (and describes) embodiments directed to that value or parameter itself. As used herein, when the term “about” precedes a numerical value, it means within a range of 10% above or below that numerical value. For example, “about 100” encompasses 90 and 110.
  • each nucleotide sequence does not contain modifications not indicated in the examples. That is, each nucleotide sequence used in Examples 1-7 of the present application is a naked sequence or contains only the modifications indicated in the modification motif used.
  • modified dsRNA if the first nucleotide at the 3' end of the sense strand is not A or U, a uracil ribonucleotide is used to replace the original nucleotide; if the first nucleotide at the 5' end of the antisense sequence is not A or U, an adenine ribonucleotide is used to replace the original nucleotide, and the original nucleic acid sequence is changed from n to n' for detection.
  • nucleic acid sequences of E3, E4, E9, E14, E16, E24, E26, E51, E52, E69, and E70 are shown in the dsRNA numbered 3', 4', 9', 14', 16', 24', 26', 51', 52', 69', and 70' in the sequence listing at the end of the article, respectively.
  • the human GAPDH gene was used as the internal reference gene, and the PCR reaction was performed using the CFX96 fluorescence quantitative PCR instrument from Bio-Rad, USA.
  • the mock group was used as the control for normalization, so that the expression level of FXI mRNA in the mock group was 1.
  • E Modification of E (dsRNA modified with E is referred to as En, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as E3 after modification with E):
  • Sense sequence starting from the 5' end, the 7th, 9th, 10th, and 11th positions are 2'-fluoro modified nucleotides, and the other positions are 2'-O-methyl modified nucleotides, and the first and second phosphate bonds at the 5' end are thiolated to form phosphorothioate bonds; for example, with N representing any ribonucleotide, the modification of the sense sequence is: NmsNmsNmNmNmNmNmNmfNmNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm;
  • Antisense sequence the 2nd, 6th, 14th and 16th positions starting from the 5’ are 2’-fluoro-modified nucleotides (in other words, the 2nd, 6th, 14th and 16th positions starting from the 3’ end of the complementary region between the antisense sequence and the sense chain are 2’-fluoro-modified nucleotides), other sites are 2’-O-methyl-modified nucleotides, and the first and second phosphates at the 5’ and 3’ ends are connected by thiolation to form a phosphorothioate bond; for example, if N represents any ribonucleotide, the modification of the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm.
  • E-1 Modification E-1 (dsRNA modified with E-1 is referred to as En-1, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as E3-1 after modification with E),
  • modification E-1 is that the sixth nucleotide from the 5' end of the antisense strand of the E-1 modified dsRNA is a 2'-O-methyl modified nucleotide instead of a 2'-fluoro modified nucleotide, i.e., for example:
  • the modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNm.
  • s represents a phosphorothioate bond
  • m represents a 2′-O-methyl modification
  • f represents a 2′-fluoro modification
  • the antisense compound ISIS-416858 adopts the structure and modification method disclosed in CN109797150A, namely the "5-10-5" structure, with 5 2'-O-methoxyethyl modified nucleotides on each flank, 10 deoxynucleotides in the middle, and all-thio and methylcytosine replacing cytosine.
  • the transfected cells were HepG2 cells, and the synthesis, transfection, and quantitative PCR detection steps were the same as in Example 2.
  • Table 3 and Figure 3 show the average target gene expression levels relative to the blank control group (the relative mRNA expression level of the blank control group was set to 1).
  • the candidate modified dsRNA is subjected to GalNAc coupling to form a GAL-dsRNA complex, that is, each dsRNA is subjected to G modification.
  • the G modification refers to conjugating L96 to the 3' end of the dsRNA sense sequence, including GE modification and GE-1 modification, wherein:
  • the modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm-L96;
  • GEn The dsRNA modified by GE is referred to as GEn, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as GE3 after GE modification;
  • the GE-1 is shown below:
  • the modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm-L96;
  • GEn-1 The dsRNA modified with GE-1 is referred to as GEn-1, where n is the number of the candidate dsRNA molecule.
  • dsRNA molecule No. 3 is referred to as GE3-1 after modification with GE-1.
  • the detection steps were the same as in Example 2, using the human GAPDH gene as the internal reference gene.
  • the detection primers used are listed in the primer section of the sequence listing at the end of the article.
  • Table 4 shows the average mRNA levels of the target gene expression levels in HepG2 cells treated with G-modified dsRNA relative to the untreated group (the relative mRNA expression level of the untreated group was set to 1). The statistical results are shown in Figure 4.
  • the IC50 of GalNAc-coupled dsRNA in HepG2 cells is lower than 0.1 nM, which once again proves the efficient inhibitory activity of the candidate sequence on the target gene.
  • the only difference between the GN-E05 modification and the LN-E05 modification is that the L96 ligand conjugated to the 3’ end of the sense chain is replaced with ligand 1, and ligand 1 is connected to the 3’ end of the sense chain through a phosphorothioate bond.
  • the wavy line indicates the connection to the 3' end of the sense strand of the dsRNA via a phosphorothioate bond.
  • the molecule of Formula II is a trivalent GalNac conjugate prepared from two compounds 6 and one compound 8 using conventional raw materials and synthetic methods in the art. The synthetic methods of compounds 6 and 8 are as follows:
  • Compound 8 is sequentially reacted with two or three compounds 6, and through conventional solid-phase synthesis cycles (deprotection, coupling, oxidation, and capping), a precursor (or intermediate) of ligand 1 attached to a solid support is obtained. Further solid-phase synthesis cycles are performed using the corresponding 2'-modified monomers. After completion of the reaction, the entire synthesized molecule is dissociated from the solid support by aminolysis, yielding the oligonucleotide sequence attached to ligand 1.
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm;
  • Antisense sequence vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmsNmsNm.
  • GN-E05 The modification of GN-E05 was the same as in Example 6. The only difference between it and the GN-E05VP modification was that GN-E05 did not have a vinyl phosphate modification at the 5' end of the antisense strand.
  • Example 5 The experimental protocol followed that of Example 5.
  • dsRNA No. 50 with various modifications was administered as a single subcutaneous injection at 1 mpk. Blood was collected on days 7, 14, 21, and 28 after administration, and plasma was assayed for FXI protein content. The residual percentages are relative to the pre-drug dose, as shown in Table 11 and Figure 8.
  • sequences used in the above examples of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of this application and do not limit the present invention in any way.
  • the nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When they represent RNA sequences, “T” represents uridine. Furthermore, in the context of RNA, unless otherwise specified, “T” and “U” both refer to uracil or uridine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

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

Description

一种抑制F11基因表达的核酸分子A nucleic acid molecule for inhibiting F11 gene expression

本申请要求2024年01月26日向中国国家知识产权局提交的专利申请号为202410114595.7，发明名称为“一种抑制F11基因表达的核酸分子”；以及2024年05月09日向中国国家知识产权局提交的专利申请号为202410568807.9，发明名称为“GalNAc衍生物及其寡聚核苷酸缀合物”的在先申请的优先权。该两件在先申请的全文通过引用的方式结合于本申请中。This application claims priority to prior applications, including patent application number 202410114595.7, filed with the State Intellectual Property Office of China on January 26, 2024, entitled "A Nucleic Acid Molecule for Inhibiting F11 Gene Expression," and patent application number 202410568807.9, filed with the State Intellectual Property Office of China on May 9, 2024, entitled "GalNAc Derivatives and Oligonucleotide Conjugates Thereof." The entire contents of both prior applications are incorporated herein by reference.

技术领域Technical Field

本申请涉及RNAi领域，具体涉及一种可通过RNAi抑制F11基因表达的核酸分子及其用途。The present application relates to the field of RNAi, and in particular to a nucleic acid molecule capable of inhibiting F11 gene expression through RNAi and its use.

背景技术Background Art

RNA干扰(RNAi)是指在进化过程中高度保守的、由双链RNA(dsRNA)诱发的同源mRNA高效特异性降解的现象。dsRNA长度通常为19～30bp是RNAi技术中重要的工具之一。自然机体内，通常较长的dsRNA进入细胞后可被Dicer酶特异性识别并切割成约21～23个核苷酸长的小分子RNA的片段(即siRNA)，切割产生的dsRNA片断解开变成单链，和某些蛋白质形成复合物(简称RISC)。RISC能结合到细胞内与dsRNA反义链互补的mRNA上，并切割该mRNA，使其被降解，造成蛋白质无法合成，产生基因“沉默”现象。工业生产中，人们更倾向于化学合成dsRNA并进行修饰，进一步提高dsRNA药物的稳定性和有效性。RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. dsRNA is typically 19 to 30 bp in length and is one of the important tools in RNAi technology. In natural organisms, after entering cells, longer dsRNA can be specifically recognized by the Dicer enzyme and cleaved into small RNA fragments (i.e., siRNA) of approximately 21 to 23 nucleotides in length. The resulting dsRNA fragments unwind into single strands and form complexes with certain proteins (referred to as RISC). RISC can bind to mRNA in the cell that is complementary to the antisense strand of the dsRNA and cleave the mRNA, causing it to be degraded, preventing protein synthesis and resulting in gene "silencing." In industrial production, people prefer to chemically synthesize dsRNA and modify it to further improve the stability and effectiveness of dsRNA drugs.

血栓是血管内限制血液流动的血凝块，可发生在动脉或静脉循环中，是大多数心肌梗死、缺血性脑卒中和静脉血栓栓塞症(VTE)的共同病理基础。目前常用抗凝药的主要靶点为凝血酶和FX(或称凝血因子10)，其在止血与血栓形成中同样重要，若试图通过抑制二者来发挥抗凝作用，则不可避免会影响到止血进程。这一矛盾限制了抗凝剂的治疗强度，意味着高出血风险患者可能不适合接受抗凝治疗。临床上需要出现新的抗凝剂，实现在抗凝的同时降低出血风险，而凝血因子XI(FXI)成为新的抗凝靶点。因为相较于FX，FXI(或称凝血因子FXI或F11)在止血中起辅助作用，但在血栓形成中起必要作用，即使是在FXI严重缺乏的患者中，很少观察到自发性出血、中枢神经系统或胃肠道出血。Thrombi are blood clots that restrict blood flow within blood vessels. They can occur in either the arterial or venous circulation and are the common pathological basis for most myocardial infarctions, ischemic strokes, and venous thromboembolism (VTE). Currently, the main targets of commonly used anticoagulants are thrombin and FX (or coagulation factor 10), which are equally important in hemostasis and thrombosis. Attempting to achieve anticoagulant effects by inhibiting either of these enzymes inevitably compromises hemostasis. This conflict limits the therapeutic efficacy of anticoagulants and means that patients at high risk of bleeding may not be suitable for anticoagulant therapy. New anticoagulants are clinically needed that can simultaneously reduce bleeding risk while providing anticoagulation, and coagulation factor XI (FXI) has emerged as a new anticoagulant target. Compared to FX, FXI (or coagulation factor FXI or F11) plays a supporting role in hemostasis but plays an essential role in thrombosis. Even in patients with severe FXI deficiency, spontaneous bleeding, central nervous system bleeding, or gastrointestinal bleeding is rarely observed.

申请概述Application Overview

本申请提供了一种通过RNAi抑制细胞中因子XI(FXI)基因表达的核酸分子，包括所述核酸分子的组合物、制剂、及其用途。本申请实施例有充分数据显示，本申请的优选核酸分子，包括但不限于编号为3、3’、5、8、9、9’、14、14’、15、19、24、24’、26、26’、50、70、70’、及82的dsRNA分子，结构及序列包含前述dsRNA分子的shRNA分子，所述shRNA分子或dsRNA分子的前体分子、以及修饰的所述shRNA分子或dsRNA分子。其中，优选的修饰包括但不限于本申请实施例中详细描述的修饰E及修饰E-1。The present application provides a nucleic acid molecule that inhibits factor XI (FXI) gene expression in cells by RNAi, including compositions, preparations, and uses of the nucleic acid molecule. The present application's examples provide sufficient data showing that preferred nucleic acid molecules of the present application include, but are not limited to, dsRNA molecules numbered 3, 3', 5, 8, 9, 9', 14, 14', 15, 19, 24, 24', 26, 26', 50, 70, 70', and 82, shRNA molecules whose structures and sequences include the aforementioned dsRNA molecules, precursor molecules of the shRNA molecules or dsRNA molecules, and modified shRNA molecules or dsRNA molecules. Preferred modifications include, but are not limited to, modification E and modification E-1 described in detail in the present application's examples.

具体地，本申请一方面涉及一种核酸分子，其包含彼此互补的正义序列和反义序列或由彼此互补的正义序列和反义序列组成，且所述反义序列包含与FXI基因mRNA互补的多核苷酸序列，其中，Specifically, the present application relates to a nucleic acid molecule comprising or consisting of a sense sequence and an antisense sequence that are complementary to each other, and the antisense sequence comprises a polynucleotide sequence that is complementary to FXI gene mRNA, wherein:

当所述反义序列与所述FXI基因mRNA以最大互补率杂交时，由所述反义序列和FXI基因mRNA序列中互补配对的碱基对组成互补区1；When the antisense sequence hybridizes with the FXI gene mRNA at a maximum complementarity rate, complementary region 1 is formed by complementary base pairs in the antisense sequence and the FXI gene mRNA sequence;

当所述反义序列与所述正义序列以最大互补率杂交时，由所述反义序列和所述正义序列中互补配对的碱基对组成互补区2；When the antisense sequence hybridizes with the sense sequence at a maximum complementarity rate, complementary region 2 is formed by complementary base pairs in the antisense sequence and the sense sequence;

其中互补区1与互补区2具有至少18、19或20个相同的碱基对，wherein complementary region 1 and complementary region 2 have at least 18, 19 or 20 identical base pairs,

所述互补区1的碱基对数量为15至35bp，并且所述互补区1包含FXI基因mRNA序列中：The complementary region 1 has a base pair number of 15 to 35 bp, and the complementary region 1 includes the following in the FXI gene mRNA sequence:

418至437位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 418 to 437 or 18 or 19 consecutive nucleotides therein,

1141至1160位的核苷酸或其中的连续18或19个核苷酸；Nucleotides 1141 to 1160 or 18 or 19 consecutive nucleotides therein;

363至382位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 363 to 382 or 18 or 19 consecutive nucleotides therein,

372至391位的核苷酸或其中的连续18或19个核苷酸、411至430位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 372 to 391 or 18 or 19 consecutive nucleotides therein, nucleotides 411 to 430 or 18 or 19 consecutive nucleotides therein,

421至440位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 421 to 440 or 18 or 19 consecutive nucleotides therein,

422至441位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 422 to 441 or 18 or 19 consecutive nucleotides therein,

425至444位的核苷酸或其中的连续18或19个核苷酸、465至484位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 425 to 444 or 18 or 19 consecutive nucleotides therein, nucleotides 465 to 484 or 18 or 19 consecutive nucleotides therein,

470至489位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 470 to 489 or 18 or 19 consecutive nucleotides therein,

471至490位的核苷酸或其中的连续18或19个核苷酸、518至537位的核苷酸或其中的连续18或19个核苷酸；Nucleotides 471 to 490 or 18 or 19 consecutive nucleotides therein, nucleotides 518 to 537 or 18 or 19 consecutive nucleotides therein;

其中FXI基因mRNA序列中核苷酸的位点编号为参比序列SEQ ID NO：244中对应核苷酸的编号。The site number of the nucleotide in the FXI gene mRNA sequence is the number of the corresponding nucleotide in the reference sequence SEQ ID NO: 244.

其中一段核苷酸序列“其中的连续18或19个核苷酸”，可以是该段核苷酸序列5’端从第一个核苷酸或第二个核苷酸开始且包含所述第一个核苷酸或第二个核苷酸的连续19个与所述核苷酸序列中核苷酸排序一致的核苷酸，或者也可以是该段核苷酸序列5’端从第一个核苷酸、第二个核苷酸或第三个核苷酸开始且包含所述第一个核苷酸、第二个核苷酸或第三个核苷酸的连续18个与所述核苷酸序列中核苷酸排序一致的核苷酸。The “18 or 19 consecutive nucleotides” of a nucleotide sequence may be 19 consecutive nucleotides starting from the first nucleotide or the second nucleotide at the 5’ end of the nucleotide sequence and including the first nucleotide or the second nucleotide, which are consistent with the nucleotide sequence in the nucleotide sequence, or may be 18 consecutive nucleotides starting from the first nucleotide, the second nucleotide or the third nucleotide at the 5’ end of the nucleotide sequence and including the first nucleotide, the second nucleotide or the third nucleotide, which are consistent with the nucleotide sequence in the nucleotide sequence.

在一些实施方案中，所述正义序列与所述反义序列等长。在一些实施方案中，所述正义序列和/或反义序列在所述互补区2之外还包含突出端核苷酸。在一些实施方案中，所述正义序列长于所述反义序列。在一些实施方案中，所述反义序列长于所述正义序列。在一些实施方案中，所述正义序列比所述反义序列长1、2、3或4个核苷酸。在一些实施方案中，所述反义序列比所述正义序列长1、2、3或4个核苷酸。在一些实施方案中，所述正义序列在互补区2的5’端一侧和/或3’端一侧还包括1或2个核苷酸。在一些实施方案中，所述反义序列在互补区2的5’端一侧和/或3’端一侧还包括1或2个核苷酸。在一些实施方案中，所述正义序列和反义序列在互补区2的5’端一侧和/或3’端一侧还包括1或2个核苷酸。在一些实施方案中，当所述反义序列与所述正义序列以最大互补率杂交时，所述正义序列在互补区2的第1位核苷酸与最后一位核苷酸之间不包含未与反义序列核苷酸互补配对的核苷酸。在一些实施方案中，当所述反义序列与所述正义序列以最大互补率杂交时，所述反义序列在互补区2的第1位核苷酸与最后一位核苷酸之间不包含未与正义序列核苷酸互补配对的核苷酸。在一些实施方案中，所述突出端核苷酸为2个且位于反义序列中，与所述互补区2的5’末端相邻，所述正义序列不包含突出端核苷酸。在一些实施方案中，所述反义序列在互补区1以外的区域仅包含0、1或2个核苷酸，且所述正义序列在互补区2以外仅包含0、1或2个核苷酸。在一些实施方案中，当所述反义序列与FXI基因mRNA序列以最大互补率杂交时，所述反义序列在互补区1的第1位核苷酸与最后一位核苷酸之间不包含未与FXI基因mRNA序列互补配对的核苷酸。在一些实施方案中，当所述反义序列与FXI基因mRNA序列以最大互补率杂交时，所述FXI基因mRNA序列在互补区1的第1位核苷酸与最后一位核苷酸之间不包含未与反义序列互补配对的核苷酸。在一些实施方案中，所述互补区2的3’末端为AU碱基对。在一些实施方案中，所述互补区2的3’末端由来自反义序列的A与来自正义序列的U(在本申请中可以写作T)组成。在一些实施方案中，所述互补区2的3’末端由来自正义序列的A与来自反义序列的U(在本申请中可以写作T)组成。在一些实施方案中，互补区1与互补区2是不间断的，即连续的。在一些实施方案中，互补区1和/或互补区2不连续，所述不连续即被一个或多个气泡(Bubble，由形成互补区的两条序列中各自的非互补核苷酸形成的非互补区)隔断。In some embodiments, the sense sequence is equal to the antisense sequence. In some embodiments, the sense sequence and/or antisense sequence further comprise overhanging nucleotides outside the complementary region 2. In some embodiments, the sense sequence is longer than the antisense sequence. In some embodiments, the antisense sequence is longer than the sense sequence. In some embodiments, the sense sequence is 1, 2, 3, or 4 nucleotides longer than the antisense sequence. In some embodiments, the antisense sequence is 1, 2, 3, or 4 nucleotides longer than the sense sequence. In some embodiments, the sense sequence further comprises 1 or 2 nucleotides on the 5' end side and/or the 3' end side of the complementary region 2. In some embodiments, the antisense sequence further comprises 1 or 2 nucleotides on the 5' end side and/or the 3' end side of the complementary region 2. In some embodiments, the sense sequence and antisense sequence further comprise 1 or 2 nucleotides on the 5' end side and/or the 3' end side of the complementary region 2. In some embodiments, when the antisense sequence hybridizes to the sense sequence at maximum complementarity, the sense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the antisense sequence between the first and last nucleotides in complementary region 2. In some embodiments, when the antisense sequence hybridizes to the sense sequence at maximum complementarity, the antisense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the sense sequence between the first and last nucleotides in complementary region 2. In some embodiments, the overhang nucleotides are two and are located in the antisense sequence, adjacent to the 5' end of complementary region 2, and the sense sequence does not include an overhang nucleotide. In some embodiments, the antisense sequence contains only 0, 1, or 2 nucleotides outside complementary region 1, and the sense sequence contains only 0, 1, or 2 nucleotides outside complementary region 2. In some embodiments, when the antisense sequence hybridizes to the FXI gene mRNA sequence at maximum complementarity, the antisense sequence does not include a nucleotide that is not complementary paired with a nucleotide in the FXI gene mRNA sequence between the first and last nucleotides in complementary region 1. In some embodiments, when the antisense sequence hybridizes with the FXI gene mRNA sequence at maximum complementarity, the FXI gene mRNA sequence does not contain nucleotides that are not complementary to the antisense sequence between the first nucleotide and the last nucleotide of complementary region 1. In some embodiments, the 3' end of complementary region 2 is an AU base pair. In some embodiments, the 3' end of complementary region 2 consists of an A from the antisense sequence and a U (which may be written as T in this application) from the sense sequence. In some embodiments, the 3' end of complementary region 2 consists of an A from the sense sequence and a U (which may be written as T in this application) from the antisense sequence. In some embodiments, complementary region 1 and complementary region 2 are uninterrupted, that is, continuous. In some embodiments, complementary region 1 and/or complementary region 2 are discontinuous, that is, interrupted by one or more bubbles (bubbles, non-complementary regions formed by non-complementary nucleotides in each of the two sequences forming the complementary region).

在一些实施方案中，所述互补区1的碱基对数量为15至35个，例如15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35bp。在一些实施方案中，所述互补区2的碱基对数量为15至35bp，例如5、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35bp。在一些实施方案中，所述互补区1与互补区2具有18、19、20、21、22或23个相同的碱基对。在一些实施方案中，所述正义序列和/或所述反义序列的长度为15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30nt。在一些实施方案中，所述正义序列长度为21nt，所述反义序列长度为23nt。在一些实施方案中，所述核酸分子由长度为21nt的正义序列和长度为23nt的反义序列组成。在一些实施方案中，所述核酸分子由正义序列和反义序列以及连接链(或称shRNA loop)组成，其中所述连接链连接所述正义序列的3’端核苷酸和反义序列的5’端核苷酸。所述连接链的选择是本领域一致的，例如参照文献Jensen,Stig gaard Rask et al.“Functional selection of shRNA loops from randomized retroviral libr aries.”PloS one vol.7,8(2012):e43095.doi:10.1371/journal.pone.0043095描述的方式进行选择，其中所述文献通过引用全文并入本文。In some embodiments, the number of base pairs in complementary region 1 is 15 to 35, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bp. In some embodiments, the number of base pairs in complementary region 2 is 15 to 35, such as 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bp. In some embodiments, complementary region 1 and complementary region 2 have 18, 19, 20, 21, 22, or 23 identical base pairs. In some embodiments, the length of the sense sequence and/or the antisense sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nt. In some embodiments, the sense sequence is 21nt in length and the antisense sequence is 23nt in length. In some embodiments, the nucleic acid molecule is composed of a sense sequence of 21nt in length and an antisense sequence of 23nt in length. In some embodiments, the nucleic acid molecule is composed of a sense sequence and an antisense sequence and a connecting chain (or shRNA loop), wherein the connecting chain connects the 3' end nucleotides of the sense sequence and the 5' end nucleotides of the antisense sequence. The selection of the connecting chain is consistent in the art, for example, with reference to the literature Jensen, Stig The selection was performed as described in Gaard Rask et al. "Functional selection of shRNA loops from randomized retroviral libr aries." PLoS one vol. 7, 8 (2012): e43095. doi: 10.1371/journal.pone.0043095, which is incorporated herein by reference in its entirety.

在一些实施方案中，所述正义序列和/或反义序列在所述互补区2之外还包含1至2个突出端核苷酸。In some embodiments, the sense sequence and/or antisense sequence further comprises 1 to 2 overhang nucleotides outside the complementary region 2.

在一些实施方案中，所述反义序列在互补区1以外的区域仅包含0、1或2个核苷酸，正义序列在互补区2以外仅包含0、1或2个核苷酸。In some embodiments, the antisense sequence comprises only 0, 1, or 2 nucleotides outside complementary region 1, and the sense sequence comprises only 0, 1, or 2 nucleotides outside complementary region 2.

在一些实施方案中，其中互补区2的3’末端为AU碱基对；在进一步的实施方案中，反义链的5’末端A在互补区1外。In some embodiments, the 3' end of complementary region 2 is an AU base pair; in further embodiments, the 5' end A of the antisense strand is outside complementary region 1.

在一些实施方案中，与所述反义链互补的FXI基因mRNA序列转录自哺乳动物FXI基因编码区。在一些实施方案中所述哺乳动物为灵长类动物。在一些实施方案中，所述灵长类动物为人或食蟹猴。在一些实施方案中，所述mRNA包含如SEQ ID NO：244所示的多核苷酸序列。在一些实施方案中，所述反义序列包含如SEQ ID NO：122、123、126、129、130、131、136、137、138、143、149、150、152、153、177、200、201或213所示的多核苷酸序列。在一些实施方案中，所述反义序列由SEQ ID NO：122、123、126、129、130、131、136、137、138、143、149、150、152、153、177、200、201或213所示的多核苷酸序列以及另外的1、2、3或4核苷酸组成。在一些实施方案中，所述反义序列由SEQ ID NO：122、123、126、129、130、131、136、137、138、143、149、150、152、153、177、200、201或213所示的多核苷酸序列以及所述多核苷酸序列5’和/或3’端一侧的另外的1至2个核苷酸组成。在一些实施方案中，所述反义序列为如SEQ ID NO：122、123、126、129、130、131、136、137、138、143、149、150、152、153、177、200、201或213所示的多核苷酸序列。在一些实施方案中，所述正义序列包含如SEQ ID NO：3、4、7、10、11、12、17、18、19、24、30、31、33、34、58、81、82或94所示的多核苷酸序列。在一些实施方案中，所述反义序列由SEQ ID NO：3、4、7、10、11、12、17、18、19、24、30、31、33、34、58、81、82或94所示的多核苷酸序列以及另外的1至4个(例如2或3个)核苷酸组成。在一些实施方案中，所述反义序列由3、4、7、10、11、12、17、18、19、24、30、31、33、34、58、81、82或94所示的多核苷酸序列以及所述多核苷酸序列5’和/或3’端一侧的另外的1至2个核苷酸组成。在一些实施方案中，所述反义序列为如3、4、7、10、11、12、17、18、19、24、30、31、33、34、58、81、82或94所示的多核苷酸序列。In some embodiments, the FXI gene mRNA sequence complementary to the antisense strand is transcribed from a mammalian FXI gene coding region. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human or a cynomolgus monkey. In some embodiments, the mRNA comprises the polynucleotide sequence shown in SEQ ID NO: 244. In some embodiments, the antisense sequence comprises the polynucleotide sequence shown in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201, or 213. In some embodiments, the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201 or 213, and additional 1, 2, 3 or 4 nucleotides. In some embodiments, the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201 or 213, and additional 1 to 2 nucleotides on the 5' and/or 3' side of the polynucleotide sequence. In some embodiments, the antisense sequence is a polynucleotide sequence as shown in SEQ ID NO: 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 152, 153, 177, 200, 201, or 213. In some embodiments, the sense sequence comprises a polynucleotide sequence as shown in SEQ ID NO: 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82, or 94. In some embodiments, the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82 or 94, and an additional 1 to 4 (e.g., 2 or 3) nucleotides. In some embodiments, the antisense sequence consists of a polynucleotide sequence as shown in 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82 or 94, and an additional 1 to 2 nucleotides on the 5' and/or 3' side of the polynucleotide sequence. In some embodiments, the antisense sequence is a polynucleotide sequence as shown in 3, 4, 7, 10, 11, 12, 17, 18, 19, 24, 30, 31, 33, 34, 58, 81, 82 or 94.

在一些实施方案中，所述正义序列包含或为如SEQ ID NO：3所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：122所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：4所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：123所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：7所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：126所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：10所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：129所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：11所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：130所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：12所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：131所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：17所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：136所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：18所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：137所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：19所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：138所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：24所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：143所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：30所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：149所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：31所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：150所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：33所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：152所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：34所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：153所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：58所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：177所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：81所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：200所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：82所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：201所示的多核苷酸序列；或所述正义序列包含或为如SEQ ID NO：94所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：213所示的多核苷酸序列。In some embodiments, the sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 3, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 122; the sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 4, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 123; the sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 7, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 126; the sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 10, and the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 129; the sense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 11 acid sequence, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 130; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 12, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 131; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 17, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 136; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 18, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 137; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 19, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 138 sequence; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 24, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 143; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 30, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 149; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 31, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 150; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 33, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 152; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 34, And the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 153; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 81, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 200; the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 82, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 201; or the sense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 94, and the antisense sequence contains or is the polynucleotide sequence shown in SEQ ID NO: 213.

在一些实施方案中，所述正义序列由如SEQ ID NO：3所示的多核苷酸序列以及另外1、2、3或4核苷酸组成，且所述反义序列由如SEQ ID NO：122所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：4所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：123所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：7所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：126所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：10所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：129所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：11所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：130所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：12所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：131所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：17所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：136所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：18所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：137所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：19所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：138所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：24所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：143所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：30所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：149所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：31所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：150所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：33所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：152所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：34所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：153所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：58所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：177所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：81所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：200所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列由如SEQ ID NO：82所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：201所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；或所述正义序列由如SEQ ID NO：94所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列由如SEQ ID NO：213所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成。In some embodiments, the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 3 and another 1, 2, 3 or 4 nucleotides, and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 122 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 4 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 123 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 7 nucleotide sequence and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 126 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 10 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 129 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 11 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 130 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 12 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 131 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 17 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 136. the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 18 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 137 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 19 and 1 to 4 (e.g., 2 or 3) additional nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 138 and 1 to 4 (e.g., 2 or 3) additional nucleotides; The sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 24 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 143 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 30 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 149 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 31 The present invention relates to a polynucleotide sequence as shown in SEQ ID NO: 150 and another 1 to 4 (e.g., 2 or 3) nucleotides, wherein the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 150 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 33 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 152 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 34 and another 1 to 4 (e.g., 2 or 3) nucleotides, and The antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 153 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 58 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 177 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 81 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 200 nucleotide sequence and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 82 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 201 and another 1 to 4 (e.g., 2 or 3) nucleotides; or the sense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 94 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 213 and another 1 to 4 (e.g., 2 or 3) nucleotides.

在一些实施方案中，所述正义序列由如SEQ ID NO：3所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：122所示的多核苷酸序列所述正义序列由如SEQ ID NO：4所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：123所示的多核苷酸序列所述正义序列由如SEQ ID NO：7所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：126所示的多核苷酸序列所述正义序列由如SEQ ID NO：10所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：129所示的多核苷酸序列所述正义序列由如SEQ ID NO：11所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：130所示的多核苷酸序列所述正义序列由如SEQ ID NO：12所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：131所示的多核苷酸序列所述正义序列由如SEQ ID NO：17所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：136所示的多核苷酸序列所述正义序列由如SEQ ID NO：18所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：137所示的多核苷酸序列所述正义序列由如SEQ ID NO：19所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：138所示的多核苷酸序列所述正义序列由如SEQ ID NO：24所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：143所示的多核苷酸序列所述正义序列由如SEQ ID NO：30所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：149所示的多核苷酸序列所述正义序列由如SEQ ID NO：31所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：150所示的多核苷酸序列所述正义序列由如SEQ ID NO：33所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：152所示的多核苷酸序列所述正义序列由如SEQ ID NO：34所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：153所示的多核苷酸序列所述正义序列由如SEQ ID NO：58所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：177所示的多核苷酸序列所述正义序列由如SEQ ID NO：81所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：200所示的多核苷酸序列所述正义序列由如SEQ ID NO：82所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：201所示的多核苷酸序列或所述正义序列由如SEQ ID NO：94所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成，且所述反义序列为如SEQ ID NO：213所示的多核苷酸序列。In some embodiments, the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 3 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 122. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 4 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 123. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 7 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 126. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 10 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 129. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 11 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 130; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 12 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 131; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 17 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 136; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 18 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 137; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 19 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 138 The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 24 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 143. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 30 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 149. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 31 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 150. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 33 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 152. The sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 34 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 153; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 58 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 81 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 200; the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 82 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 201; or the sense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 94 and another 1 to 4 (e.g., 2 or 3) nucleotides, and the antisense sequence is the polynucleotide sequence shown in SEQ ID NO: 213.

在一些实施方案中，所述正义序列为如SEQ ID NO：3所示的多核苷酸序列且所述反义序列由如SEQ ID NO：122所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：4所示的多核苷酸序列且所述反义序列由如SEQ ID NO：123所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：7所示的多核苷酸序列且所述反义序列由如SEQ ID NO：126所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：10所示的多核苷酸序列且所述反义序列由如SEQ ID NO：129所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：11所示的多核苷酸序列且所述反义序列由如SEQ ID NO：130所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：12所示的多核苷酸序列且所述反义序列由如SEQ ID NO：131所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：17所示的多核苷酸序列且所述反义序列由如SEQ ID NO：136所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：18所示的多核苷酸序列且所述反义序列由如SEQ ID NO：137所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：19所示的多核苷酸序列且所述反义序列由如SEQ ID NO：138所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：24所示的多核苷酸序列且所述反义序列由如SEQ ID NO：143所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：30所示的多核苷酸序列且所述反义序列由如SEQ ID NO：149所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：31所示的多核苷酸序列且所述反义序列由如SEQ ID NO：150所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：33所示的多核苷酸序列且所述反义序列由如SEQ ID NO：152所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：34所示的多核苷酸序列且所述反义序列由如SEQ ID NO：153所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：58所示的多核苷酸序列且所述反义序列由如SEQ ID NO：177所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：81所示的多核苷酸序列且所述反义序列由如SEQ ID NO：200所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；所述正义序列为如SEQ ID NO：82所示的多核苷酸序列且所述反义序列由如SEQ ID NO：201所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成；或所述正义序列为如SEQ ID NO：94所示的多核苷酸序列且所述反义序列由如SEQ ID NO：213所示的多核苷酸序列以及另外1至4个(例如2或3个)核苷酸组成。In some embodiments, the sense sequence is a polynucleotide sequence as shown in SEQ ID NO: 3 and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 122 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence as shown in SEQ ID NO: 4 and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 123 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence as shown in SEQ ID NO: 7 and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 126 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence as shown in SEQ ID NO: 10 and the antisense sequence consists of a polynucleotide sequence as shown in SEQ ID NO: 129 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is a polynucleotide sequence as shown in SEQ ID NO: 11 and the antisense sequence The invention relates to a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 130 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 12 and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 131 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 17 and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 136 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 18 and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 137 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence of SEQ ID NO: 19 and the antisense sequence is the polynucleotide sequence of SEQ ID NO: 138 and 1 to 4 (e.g., 2 or 3) additional nucleotides. wherein the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 24 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 143 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 30 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 149 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 31 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 150 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 33 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 152 and another 1 to 4 (e.g., 2 or 3) nucleotides; the sense sequence is the polynucleotide sequence shown in SEQ ID NO: 34 and the antisense sequence consists of the polynucleotide sequence shown in SEQ ID NO: 153. The present invention relates to a polynucleotide sequence comprising a polynucleotide sequence as shown in SEQ ID NO: 153 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 58 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 177 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 81 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 200 and 1 to 4 (e.g., 2 or 3) additional nucleotides; the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 82 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 201 and 1 to 4 (e.g., 2 or 3) additional nucleotides; or the sense sequence is the polynucleotide sequence as shown in SEQ ID NO: 94 and the antisense sequence consists of the polynucleotide sequence as shown in SEQ ID NO: 213 and 1 to 4 (e.g., 2 or 3) additional nucleotides.

在一些实施方案中:In some embodiments:

所述正义序列包含或为如SEQ ID NO：34所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：153所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 34, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 153;

所述正义序列包含或为如SEQ ID NO：58所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：177所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：33所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：152所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 33, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 152;

所述正义序列包含或为如SEQ ID NO：3所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：122所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 3, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 122;

所述正义序列包含或为如SEQ ID NO：4所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：123所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 4, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 123;

所述正义序列包含或为如SEQ ID NO：7所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：126所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 7, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 126;

所述正义序列包含或为如SEQ ID NO：10所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：129所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 10, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 129;

所述正义序列包含或为如SEQ ID NO：11所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：130所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 11, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 130;

所述正义序列包含或为如SEQ ID NO：12所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：131所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 12, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 131;

所述正义序列包含或为如SEQ ID NO：17所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：136所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 17, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 136;

所述正义序列包含或为如SEQ ID NO：18所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：137所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 18, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 137;

所述正义序列包含或为如SEQ ID NO：19所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：138所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 19, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 138;

所述正义序列包含或为如SEQ ID NO：24所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：143所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 24, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 143;

所述正义序列包含或为如SEQ ID NO：30所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：149所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 30, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 149;

所述正义序列包含或为如SEQ ID NO：31所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：150所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 31, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 150;

所述正义序列包含或为如SEQ ID NO：81所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：200所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 81, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 200;

所述正义序列包含或为如SEQ ID NO：82所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：201所示的多核苷酸序列；或The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 82, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 201; or

所述正义序列包含或为如SEQ ID NO：94所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：213所示的多核苷酸序列。The sense sequence includes or is a polynucleotide sequence as shown in SEQ ID NO: 94, and the antisense sequence includes or is a polynucleotide sequence as shown in SEQ ID NO: 213.

在一些实施方案中，所述核酸分子为dsRNA或shRNA。在一些实施方案中，所述ds RNA为siRNA。在一些实施方案中，所述核酸分子中的一个或多个核苷酸经过化学修饰。在一些实施方案中，所述化学修饰使所述核酸分子在细胞或体内环境中更稳定。在一些实施方案中，所述化学修饰延长了所述核酸分子体内和/或体外半衰期。在一些实施方案中，所述化学修饰包含选自如下的任一种或多种，或由选自如下的一种或多种修饰组成：In some embodiments, the nucleic acid molecule is a dsRNA or shRNA. In some embodiments, the ds RNA is an siRNA. In some embodiments, one or more nucleotides in the nucleic acid molecule are chemically modified. In some embodiments, the chemical modification makes the nucleic acid molecule more stable in a cell or in vivo environment. In some embodiments, the chemical modification prolongs the half-life of the nucleic acid molecule in vivo and/or in vitro. In some embodiments, the chemical modification comprises any one or more selected from the following, or consists of one or more modifications selected from the following:

锁核酸修饰、开环或非锁核酸修饰、2′-甲氧基乙基修饰、2′-O-甲基修饰(2’-OM e)、2′-O-烯丙基修饰、2’-C-烷基修饰、2′-C-烯丙基修饰、2′-氟代修饰(2’-F)、2′-脱氧修饰(d)、硫代磷酸酯修饰(s)、2’-氨基-修饰、吗啉基修饰、氨基磷酸酯修饰、甲基膦酸酯修饰、四氢吡喃修饰、1,5-脱水己糖醇修饰、5’-乙烯基磷酸酯修饰、以及环己烯基修饰。Locked nucleic acid modification, open ring or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification (2′-OM e), 2′-O-allyl modification, 2′-C-alkyl modification, 2′-C-allyl modification, 2′-fluoro modification (2′-F), 2′-deoxy modification (d), phosphorothioate modification (s), 2′-amino-modification, morpholino modification, phosphoramidate modification, methylphosphonate modification, tetrahydropyranyl modification, 1,5-anhydrohexitol modification, 5′-vinyl phosphate modification, and cyclohexenyl modification.

在一些实施方案中，所述核酸分子正义链的7、9-12位核苷酸包含2′-氟代修饰和/或反义链2、14、16位核苷酸包含2′-氟代修饰。在一些实施方案中，所述核酸分子正义链的7、9-12位核苷酸包含2′-氟代修饰和/或反义链2、14、16位核苷酸包含2′-氟代修饰，并且其余核苷酸包含2′-O-甲基修饰。在一些实施方案中，所述核酸分子正义链的7、9-12位核苷酸由2′-氟代修饰和/或反义链2、14、16位核苷酸由2′-氟代修饰，并且其余核苷酸由2′-O-甲基修饰，并且所述核酸分子不进一步包含其他除硫代磷酸酯以外的修饰。In some embodiments, the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 14, and 16 of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 14, and 16 of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, the nucleotides at positions 7, 9-12 of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and/or the nucleotides at positions 2, 14, and 16 of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.

在一些实施方案中，所述核酸分子正义链的7、9-11位核苷酸包含2′-氟代修饰和/或反义链2、14、16位核苷酸包含2′-氟代修饰。在一些实施方案中，所述核酸分子正义链的7、9-11位核苷酸包含2′-氟代修饰和/或反义链2、14、16位核苷酸包含2′-氟代修饰，并且其余核苷酸包含2′-O-甲基修饰。在一些实施方案中，所述核酸分子正义链的7、9-11位核苷酸由2′-氟代修饰和/或反义链2、14、16位核苷酸由2′-氟代修饰，并且其余核苷酸由2′-O-甲基修饰，并且所述核酸分子不进一步包含其他除硫代磷酸酯以外的修饰。In some embodiments, the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the 2nd, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the 2nd, 14th, and 16th nucleotides of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, the 7th, 9th-11th nucleotides of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and/or the 2nd, 14th, and 16th nucleotides of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.

在一些实施方案中，所述核酸分子正义链的7、9-11位核苷酸包含2′-氟代修饰和/或反义链2、6、14、16位核苷酸包含2′-氟代修饰。在一些实施方案中，所述核酸分子正义链的7、9-11位核苷酸包含2′-氟代修饰和/或反义链2、6、14、16位核苷酸包含2′-氟代修饰，并且其余核苷酸包含2′-O-甲基修饰。在一些实施方案中，所述核酸分子正义链的7、9-11位核苷酸由2′-氟代修饰和/或反义链2、6、14、16位核苷酸由2′-氟代修饰，并且其余核苷酸由2′-O-甲基修饰，并且所述核酸分子不进一步包含其他除硫代磷酸酯以外的修饰。In some embodiments, the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand comprise a 2'-fluoro modification. In some embodiments, the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule comprise a 2'-fluoro modification and/or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand comprise a 2'-fluoro modification, and the remaining nucleotides comprise a 2'-O-methyl modification. In some embodiments, the nucleotides at positions 7, 9-11 of the sense strand of the nucleic acid molecule are modified by 2'-fluoro and/or the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are modified by 2'-fluoro, and the remaining nucleotides are modified by 2'-O-methyl, and the nucleic acid molecule does not further comprise other modifications other than phosphorothioate.

在一些实施方案中，所述核酸分子包含选自如下任一种基序：In some embodiments, the nucleic acid molecule comprises a motif selected from any one of the following:

(1)正义序列：NmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm，反义序列：NmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm(1) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm

(2)正义序列：NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm；和(2) sense sequence: NmNmNmNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm; and

(3)正义序列：NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm；(3) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

其中，各序列从5’至3’的N依次分别独立地表示其所在序列的所在位置的核苷酸，Nm表示经2’-O-甲基修饰的核糖核苷酸，Nf表示经2’-氟代修饰的核糖核苷酸；应当理解本方案并未限定各核苷酸是否包含或不包含除标注的修饰以外的其他修饰。Among them, the Ns from 5' to 3' of each sequence independently represent the nucleotides at the position of the sequence in which they are located, Nm represents a 2'-O-methyl modified ribonucleotide, and Nf represents a 2'-fluoro modified ribonucleotide; it should be understood that this scheme does not limit whether each nucleotide contains or does not contain other modifications besides the marked modifications.

在一些实施方案中，上述实施方案进一步在正义序列5’的第一个和第二核苷酸之间，以及第二个和第三个核苷酸之间包括至少一个硫代磷酸酯修饰；在反义序列5’的第一个和第二核苷酸之间，第二个和第三个核苷酸之间，倒数第一个和第二个之间，以及倒数第二个和第三个之间包括至少一个硫代磷酸酯修饰。In some embodiments, the above embodiments further include at least one thiophosphate modification between the first and second nucleotides, and between the second and third nucleotides of the 5' sense sequence; and at least one thiophosphate modification between the first and second nucleotides, between the second and third nucleotides, between the first to last and the second to last, and between the second to last and the third nucleotides of the 5' antisense sequence.

在一些实施方案中，所述核酸分子包含选自如下(1)-(9)中任一种基序：In some embodiments, the nucleic acid molecule comprises a motif selected from any one of the following (1)-(9):

(1)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNfNmNmNmNmNmNmNmNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

(2)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

(3)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, and the antisense sequence is NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe, and phosphorothioate;

(4)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;

(5)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;

(6)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, and the antisense sequence is NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;

(7)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(7) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;

(8)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(8) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, and the antisense sequence is NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2'-F, 2'-OMe and phosphorothioate;

(9)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(9) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中是：正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含2’-F、2’-OMe及硫代磷酸酯以外的修饰。In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, and the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than 2’-F, 2’-OMe and phosphorothioate.

其中，在本申请的所有基序中，Nm表示经2’-O-甲基修饰的核糖核苷酸，Nf表示经2’-氟代修饰的核糖核苷酸，s表示硫代磷酸酯修饰。In all motifs of the present application, Nm represents a 2'-O-methyl modified ribonucleotide, Nf represents a 2'-fluoro modified ribonucleotide, and s represents a phosphorothioate modification.

在一些实施方案中，所述核酸分子连接具有器官靶向性的配体，例如肝靶向性的配体。在一些实施方案中，所述核酸分子连接至少一个去唾液酸糖蛋白受体(ASGPR)配体。在一些实施方案中，所述器官靶向性配体连接于所述正义序列的5’末端或3’末端。在一些实施方案中，所述ASGPR配体是通过二价或三价支链结构连接的一个或多个GalNAc衍生物。在一些实施方案中，所述GalNAc衍生物包含以下结构：
In some embodiments, the nucleic acid molecule is linked to a ligand having organ targeting, such as a liver targeting ligand. In some embodiments, the nucleic acid molecule is linked to at least one asialoglycoprotein receptor (ASGPR) ligand. In some embodiments, the organ targeting ligand is linked to the 5' end or the 3' end of the sense sequence. In some embodiments, the ASGPR ligand is one or more GalNAc derivatives connected by a divalent or trivalent branched structure. In some embodiments, the GalNAc derivative comprises the following structure:

在一些实施方案中，所述GalNac衍生物为L96，所述L96的结构如下所示：
In some embodiments, the GalNac derivative is L96, and the structure of L96 is shown below:

在一些实施方案中，所述GalNac衍生物为配体1,所述配体1结构如下所示：In some embodiments, the GalNac derivative is ligand 1, and the structure of ligand 1 is shown below:

(X＝S-或O-)(式II)； (X＝ S- or O- ) (Formula II);

其中式I和II中的代表与所述核酸分子的正义序列或反义序列的3’端连接；优选地，通过磷酸二酯键或硫代磷酸二酯键连接。wherein the The terminator represents a link to the 3' end of the sense sequence or antisense sequence of the nucleic acid molecule; preferably, the link is via a phosphodiester bond or a phosphorothioate diester bond.

进一步优选地，所述核酸分子具有如(1)-(6)中任一的修饰基序：Further preferably, the nucleic acid molecule has a modification motif as any one of (1) to (6):

(1)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96；反义链：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96; Antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96；反义链：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

(2)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-Ligand 1; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than Ligand 1, 2'-F, 2'-OMe and phosphorothioate;

(3)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is: NmsNmsNmNmNmNmfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

(4)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-Ligand 1; the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than Ligand 1, 2'-F, 2'-OMe and phosphorothioate;

(5)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；In other embodiments, the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than L96, 2'-F, 2'-OMe and phosphorothioate;

(6)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰。In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1; the antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than ligand 1, 2’-F, 2’-OMe and phosphorothioate.

在一些实施方案中，所述核酸分子包含5’磷酸酯或5’磷酸酯模拟物修饰，如在反义链上的5’端磷酸酯或磷酸酯模拟物；优选地，所述5’磷酸酯模拟物为5’-VP修饰。In some embodiments, the nucleic acid molecule comprises a 5' phosphate or a 5' phosphate mimic modification, such as a 5' phosphate or a phosphate mimic on the antisense strand; preferably, the 5' phosphate mimic is a 5'-VP modification.

在一些实施方案中，所述核酸分子具有如下修饰基序：In some embodiments, the nucleic acid molecule has the following modification motif:

GN-E20VP：GN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNms-Ligand 1

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.

在一些实施方案中，所述核酸分子具有如下修饰基序：In some embodiments, the nucleic acid molecule has the following modification motif:

GN-E04VP：GN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-Ligand 1

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.

在一些实施方案中，所述核酸分子具有如下修饰基序：In some embodiments, the nucleic acid molecule has the following modification motif:

GN-E05VP：GN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-Ligand 1

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.

在一些实施方案中，所述核酸分子具有如下修饰基序：In some embodiments, the nucleic acid molecule has the following modification motif:

LN-E20VP：LN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96Justice sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNm-L96

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.

在一些实施方案中，所述核酸分子具有如下修饰基序：In some embodiments, the nucleic acid molecule has the following modification motif:

LN-E04VP：LN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96Justice sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.

在一些实施方案中，所述核酸分子具有如下修饰基序：In some embodiments, the nucleic acid molecule has the following modification motif:

LN-E05VP：LN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96Justice sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

在另一些实施方案中，正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96In other embodiments, the sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm; wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：58和反义序列：SEQ ID NO：177(siRNA编号50)，且具有如上列举的任一修饰模式。In some embodiments, the naked sequence of the nucleic acid molecule specifically provided in this application is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has any of the modification patterns listed above.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：58和反义序列：SEQ ID NO：177(siRNA编号50)，且具有如下修饰模式：In some embodiments, the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

GN-E20：GN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E20VP：GN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E04：GN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

GN-E04VP：GN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E05：GN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；或Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

GN-E05VP：GN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：58和反义序列：SEQ ID NO：177(siRNA编号50)，且具有如下修饰模式：In some embodiments, the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

GN-E20：GN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

GN-E20VP：GN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe, and phosphorothioate;

GN-E04：GN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

GN-E04VP：GN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

GN-E05：GN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；或Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate; or

GN-E05VP：GN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.

在一些实施方案中，本申请提供的核酸分子裸序列为正义序列：SEQ ID NO：34和反义序列：SEQ ID NO：153(siRNA编号26')，具有如上列举的任一修饰模式。In some embodiments, the naked sequence of the nucleic acid molecule provided in the present application is a sense sequence: SEQ ID NO: 34 and an antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having any of the modification patterns listed above.

在一些实施方案中，本申请提供的核酸分子裸序列为正义序列：SEQ ID NO：34和反义序列：SEQ ID NO：153(siRNA编号26')，具有如下所示的修饰模式：In some embodiments, the naked sequence of the nucleic acid molecule provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having the modification pattern shown below:

GN-E20：GN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E20VP：GN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E04：GN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

GN-E04VP：GN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E05：GN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；或Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

GN-E05VP：GN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：34和反义序列：SEQ ID NO：153(siRNA编号26')，且具有如下修饰模式：In some embodiments, the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), and has the following modification pattern:

GN-E20：GN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

GN-E20VP：GN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe, and phosphorothioate;

GN-E04：GN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

GN-E04VP：GN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate;

GN-E05：GN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰；或Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2'-F, 2'-OMe, and phosphorothioate; or

GN-E05VP：GN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1，Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、配体1、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, ligand 1, 2’-F, 2’-OMe and thiophosphate.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：58和反义序列：SEQ ID NO：177(siRNA编号50)，且具有如下修饰模式：In some embodiments, the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

LN-E20：LN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

LN-E20VP：LN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

LN-E04：LN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

LN-E04VP：LN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

LN-E05：LN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；或Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

LN-E05VP：LN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：58和反义序列：SEQ ID NO：177(siRNA编号50)，且具有如下修饰模式：In some embodiments, the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 58 and the antisense sequence: SEQ ID NO: 177 (siRNA number 50), and has the following modification pattern:

LN-E20：LN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;

LN-E20VP：LN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;

LN-E04：LN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;

LN-E04VP：LN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;

LN-E05：LN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；或Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate; or

LN-E05VP：LN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.

在一些实施方案中，本申请提供的核酸分子裸序列为正义序列：SEQ ID NO：34和反义序列：SEQ ID NO：153(siRNA编号26')，具有如下所示的修饰模式：In some embodiments, the naked sequence of the nucleic acid molecule provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), having the modification pattern shown below:

LN-E20：LN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

LN-E20VP：LN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

LN-E04：LN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

LN-E04VP：LN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

LN-E05：LN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；或Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; or

LN-E05VP：LN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

在一些实施方案中，本申请具体提供的核酸分子裸序列为正义序列：SEQ ID NO：34和反义序列：SEQ ID NO：153(siRNA编号26')，且具有如下修饰模式：In some embodiments, the naked nucleic acid sequence specifically provided herein is the sense sequence: SEQ ID NO: 34 and the antisense sequence: SEQ ID NO: 153 (siRNA number 26'), and has the following modification pattern:

LN-E20：LN-E20:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;

LN-E20VP：LN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;

LN-E04：LN-E04:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than L96, 2’-F, 2’-OMe and phosphorothioate;

LN-E04VP：LN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate;

LN-E05：LN-E05:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰；或Antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe, and phosphorothioate; or

LN-E05VP：LN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96，Justification sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96,

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm，其中各核苷酸不包含vp、L96、2’-F、2’-OMe及硫代磷酸酯以外的修饰。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm, wherein each nucleotide does not contain modifications other than vp, L96, 2’-F, 2’-OMe and phosphorothioate.

此外，本申请还提供了前述核酸分子的前体，所述前体可以是shRNA或dsRNA。In addition, the present application also provides a precursor of the aforementioned nucleic acid molecule, which may be shRNA or dsRNA.

本申请的第二方面提供了第二核酸分子，所述第二核酸分子在细胞中可转录为dsRNA或shRNA前体，所述dsRNA或shRNA为前述第一方面的核酸分子。在一些实施方案中，所述第二核酸分子为环状或线性核酸分子。在一些实施方案中，所述第二核酸分子为换装或线性质粒。在一些实施方案中，所述核酸分子属于人工构建的病毒基因组，可选自包括但不限于例如慢病毒载体或其他逆转录病毒载体、腺病毒载体、AAV载体、痘病毒载体、杆状病毒载体、单纯疱疹病毒载体。在一些实施方案中，所述核酸分子属于细胞基因组，例如核基因组、线粒体核酸、或细胞质游离核酸。The second aspect of the present application provides a second nucleic acid molecule, which can be transcribed into a dsRNA or shRNA precursor in a cell, and the dsRNA or shRNA is the nucleic acid molecule of the aforementioned first aspect. In some embodiments, the second nucleic acid molecule is a circular or linear nucleic acid molecule. In some embodiments, the second nucleic acid molecule is a repackaged or linear plasmid. In some embodiments, the nucleic acid molecule belongs to an artificially constructed viral genome, which can be selected from, for example, lentiviral vectors or other retroviral vectors, adenoviral vectors, AAV vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors. In some embodiments, the nucleic acid molecule belongs to a cellular genome, such as a nuclear genome, mitochondrial nucleic acid, or cytoplasmic free nucleic acid.

本申请的第三方面，还提供了核酸递送体，其包含前述第一方面的核酸分子，或前述第二方面的第二核酸分子。在一些实施方案中，所述核酸递送体是脂质体、脂质纳米颗粒或其他聚合物、内吞体、外泌体或囊泡。The third aspect of the present application further provides a nucleic acid delivery body comprising the nucleic acid molecule of the aforementioned first aspect, or the second nucleic acid molecule of the aforementioned second aspect. In some embodiments, the nucleic acid delivery body is a liposome, lipid nanoparticle or other polymer, endosome, exosome or vesicle.

还提供了病毒颗粒，所述病毒颗粒包含前述第二方面的第二核酸分子。在一些实施方案中，所述病毒颗粒为囊膜病毒或披膜病毒颗粒。在一些实施方案中，所述病毒颗粒为假病毒颗粒。在一些实施方案中，所述病毒颗粒属于AAV、杆状病毒、痘病毒、疱疹病毒、甲病毒、慢病毒或其他逆转录病毒。Also provided are viral particles comprising the second nucleic acid molecule of the aforementioned second aspect. In some embodiments, the viral particles are enveloped viruses or togavirus particles. In some embodiments, the viral particles are pseudovirions. In some embodiments, the viral particles are AAV, baculovirus, poxvirus, herpes virus, alphavirus, lentivirus, or other retroviruses.

同时还提供了细胞，所述细胞包含前述第二方面的第二核酸分子。在一些实施方案中，所述细胞为原核细胞。在一些实施方案中，所述细胞为真核细胞，例如干细胞，例如造血干细胞、间充质干细胞等。Also provided is a cell comprising the second nucleic acid molecule of the second aspect. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, such as a stem cell, such as a hematopoietic stem cell, a mesenchymal stem cell, or the like.

本申请还提供了药物组合物，其包含前述第一方面的核酸分子或其盐，以及要用可接受的载体或稀释剂。在一些实施方案中，所述药物组合物用于治疗或预防受试者的血栓栓塞性并发症或凝血紊乱，所述血栓栓塞性并发症优选自深静脉血栓形成、肺栓塞、心肌梗塞和中风中的一种或多种。The present application also provides a pharmaceutical composition comprising the nucleic acid molecule or salt thereof of the first aspect and an acceptable carrier or diluent. In some embodiments, the pharmaceutical composition is used to treat or prevent a thromboembolic complication or coagulopathy in a subject, wherein the thromboembolic complication is preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

本申请的第四方面，还提供了前述第一方面的核酸、第二方面的第二核酸、第三方面的核酸递送体、以及前述病毒颗粒、前述细胞、及前述药物组合物的用途。The fourth aspect of the present application further provides uses of the nucleic acid of the first aspect, the second nucleic acid of the second aspect, the nucleic acid delivery body of the third aspect, and the viral particles, cells, and pharmaceutical compositions.

例如本申请提供了将前述第一方面的核酸分子或其盐、前述第二方面的第二核酸分子、前述核酸递送体、前述病毒颗粒、或前述细胞用于制备预防或治疗受试者的血栓栓塞性并发症或凝血紊乱的药物的用途。在一些实施方案中，所述血栓栓塞性并发症优选自深静脉血栓形成、肺栓塞、心肌梗塞和中风中的一种或多种。For example, the present application provides the use of the nucleic acid molecule or salt thereof of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, or the cell for preparing a medicament for preventing or treating thromboembolic complications or coagulation disorders in a subject. In some embodiments, the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

本申请还提供了治疗或预防受试者的血栓栓塞性并发症或凝血紊乱的方法，所述方法包含向有需要的受试者施用有效量的前述第一方面的核酸分子或其盐、前述第二方面的第二核酸分子、前述核酸递送体、前述病毒颗粒、前述细胞或前述药物组合物。在一些实施方案中，所述所述血栓栓塞性并发症优选自深静脉血栓形成、肺栓塞、心肌梗塞和中风中的一种或多种。The present application also provides a method for treating or preventing thromboembolic complications or coagulation disorders in a subject, the method comprising administering to a subject in need thereof an effective amount of the nucleic acid molecule of the first aspect or a salt thereof, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, the cell, or the pharmaceutical composition. In some embodiments, the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

还提供了用于治疗或预防受试者的血栓栓塞性并发症或凝血紊乱的化合物，其包含前述第一方面的核酸分子或其盐、前述第二方面的第二核酸分子、前述核酸递送体、前述病毒颗粒、前述细胞或前述药物组合物。在一些实施方案中，所述所述血栓栓塞性并发症优选自深静脉血栓形成、肺栓塞、心肌梗塞和中风中的一种或多种。Also provided is a compound for treating or preventing thromboembolic complications or coagulation disorders in a subject, comprising the nucleic acid molecule or salt thereof of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery vehicle, the viral particle, the cell, or the pharmaceutical composition. In some embodiments, the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

本申请还提供了将前述第一方面的核酸分子或其盐或前述第二方面的第二核酸分子用于抑制因子11的表达的用途。The present application also provides use of the nucleic acid molecule or a salt thereof according to the first aspect or the second nucleic acid molecule according to the second aspect for inhibiting the expression of factor 11.

本申请还提供了将前述第一方面的核酸分子或其盐或前述第二方面的第二核酸分子用于制备抑制受试者体内FXI表达的药物的用途。The present application also provides use of the nucleic acid molecule or a salt thereof of the first aspect or the second nucleic acid molecule of the second aspect for preparing a drug for inhibiting FXI expression in a subject.

本申请还提供了用于抑制受试者体内FXI表达的化合物，所述化合物包含前述第一方面的核酸分子或其盐，以及药学上可接受的载体或稀释剂。以上详细描述了本申请的优选实施方式，但是，本申请并不限于此。在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本申请所公开的内容，均属于本申请的保护范围。本文描述的本申请的方面和实施方式包括“包含”，“组成”和“基本上由……组成”的方面和实施方式。The present application also provides a compound for inhibiting FXI expression in a subject, the compound comprising the nucleic acid molecule or a salt thereof of the first aspect, and a pharmaceutically acceptable carrier or diluent. The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the scope of the technical concept of the present application, a variety of simple variations can be made to the technical solution of the present application, including combining the various technical features in any other suitable manner. These simple variations and combinations should also be regarded as the contents disclosed in the present application and fall within the scope of protection of the present application. The aspects and embodiments of the present application described herein include aspects and embodiments of "comprising", "consisting of" and "essentially consisting of..."

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1示出了dsRNA裸核酸分子(5nM)在HepG2细胞中的高通量筛选结果。FIG1 shows the results of high-throughput screening of naked dsRNA molecules (5 nM) in HepG2 cells.

图2示出了分别施用5nM及1nM候选dsRNA或阳参dsRNA裸核酸分子时，HepG2细胞中FXI mRNA的相对含量。Figure 2 shows the relative content of FXI mRNA in HepG2 cells when 5nM and 1nM candidate dsRNA or Yangshen dsRNA naked nucleic acid molecules were applied, respectively.

图3示出了经修饰E进行修饰的dsRNA在1nM浓度下对HepG2细胞中FXI mRNA的抑制率。Figure 3 shows the inhibition rate of FXI mRNA in HepG2 cells at a concentration of 1 nM by dsRNA modified with E.

图4示出了偶联GalNAc的dsRNA在不同浓度下对FXI mRNA的抑制率。Figure 4 shows the inhibition rate of FXI mRNA by GalNAc-coupled dsRNA at different concentrations.

图5示出了偶联GalNAc的不同dsRNA体内给药后靶蛋白的抑制结果。FIG5 shows the results of target protein inhibition after in vivo administration of different GalNAc-conjugated dsRNAs.

图6示出了不同GalNAc体内给药后的比较结果FIG6 shows the comparative results after in vivo administration of different GalNAc

图7示出了不同修饰的dsRNA体外细胞水平IC50的比较结果FIG7 shows the comparison results of IC50 of different modified dsRNAs at the cell level in vitro

图8示出了不同修饰基序dsRNA体内给药后的比较结果FIG8 shows the comparative results after in vivo administration of dsRNA with different modified motifs

申请详述Application Details

本发明提供一种可引发RNA-诱导沉默复合体(RISC)介导裂解凝血因子FXI(或称FX11)基因的RNA转录本的核酸分子、可转录所述核酸分子的第二核酸分子，所述核酸分子及所述第二核酸分子的递送体、可转录所述核酸分子的病毒或细胞、以及所述核酸分子及所述第二核酸分子的用途。The present invention provides a nucleic acid molecule capable of initiating RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the coagulation factor FXI (or FX11) gene, a second nucleic acid molecule capable of transcribing the nucleic acid molecule, a delivery vehicle for the nucleic acid molecule and the second nucleic acid molecule, a virus or cell capable of transcribing the nucleic acid molecule, and uses of the nucleic acid molecule and the second nucleic acid molecule.

术语the term

为了解释本说明书的目的，将应用以下定义，并且在适当时，单数形式使用的术语也将包括复数形式，反之亦然。除非另有定义，否则本文使用的所有技术和科学术语具有与所述技术所属领域的普通技术人员通常所理解的相同的含义。本文引用的所有技术和专利公开通过引用以其整体并入本文。For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the technology belongs. All technical and patent disclosures cited herein are incorporated herein by reference in their entirety.

如本文所用，“dsRNA”即双链RNA，由于siRNA为双链结构的RNA，因此术语“dsRNA”涵盖了siRNA。dsRNA还包含长度大于siRNA的双链RNA，所述长度大于siRNA可以指其正义链长于siRNA，或其反义链长于siRNA，或其正义链及反义链均长于siRNA。通常长度大于其包含的siRNA序列的双链RNA进入细胞中后，会被称为Dicer的第III型内切核酸酶分解成siRNA。在一些实施方案中，所述dsRNA两条链的长度各自独立地为15至30nt(在本申请中，“nt”即核苷酸)。当“siRNA”被纳入RNA-诱导沉默复合体(RISC)后，RISC中的一种或多种解螺旋酶解开siRNA双螺旋。当与siRNA中的反义链互补的标靶mRNA结合时，RISC内的一种或多种内切核酸酶会裂解标靶，诱发沉默基因沉默。通常，dsRNA分子的每个链的大多数核苷酸是核糖核苷酸，但其并不排除其中任一条或两条链中包含一个或多个非核糖核苷酸，例如脱氧核糖核苷酸和/或非天然核苷酸。在一些实施方案中，所述dsRNA分子中不包含非天然核苷酸。在一些实施方案中，所述dsRNA中的每一个核苷酸均为核糖核苷酸。如本申请所用，dsRNA可以包含一个或多个化学修饰的核苷酸或不包含化学修饰的核苷酸。As used herein, "dsRNA" is double-stranded RNA. Since siRNA is a double-stranded RNA, the term "dsRNA" encompasses siRNA. dsRNA also includes double-stranded RNA that is longer than siRNA. The length greater than siRNA can refer to its sense strand being longer than siRNA, or its antisense strand being longer than siRNA, or its sense strand and antisense strand being longer than siRNA. After a double-stranded RNA that is generally longer than the siRNA sequence it contains enters the cell, it is broken down into siRNA by a type III endonuclease called Dicer. In some embodiments, the lengths of the two chains of the dsRNA are each independently 15 to 30 nt (in this application, "nt" refers to nucleotides). When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in RISC unwind the siRNA double helix. When bound to a target mRNA complementary to the antisense strand in the siRNA, one or more endonucleases in RISC cleave the target, inducing gene silencing. Typically, the majority of nucleotides in each chain of a dsRNA molecule are ribonucleotides, but this does not exclude the inclusion of one or more non-ribonucleotides, such as deoxyribonucleotides and/or non-natural nucleotides, in any one or both chains. In some embodiments, the dsRNA molecule does not include non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, a dsRNA may include one or more chemically modified nucleotides or may not include chemically modified nucleotides.

术语“FXI”、“凝血因子FXI”、及“因子11”可互换使用，在本领域中又称为FXI或PTA。FXI可以为哺乳动物来源的FXI。在一些实施方案中，FXI为灵长类动物来源的FXI。在一些实施方案中，FXI为人源的FXI。在一些实施方案中，FXI为食蟹猴来源的FXI。如本文所用，“FXI基因mRNA”是指编码因子11蛋白的mRNA，其可由FXI基因DNA转录而成，可以为成熟mRNA或mRNA前体(Pre-mRNA)，因此其可以包含内含子或不包含内含子。由于因子11基因在不同个体中可能存在少数核苷酸的突变，如非特别说明，本申请的因子11基因mRNA序列旨在包含全部由因子11基因突变体转录获得的mRNA序列。人FXI基因mRNA序列可以例如在基因库登录号GI:1732746318(NM_000128.4)中找到。恒河猴FXI基因mRNA序列可以例如在基因库登录号GI:1622942384(XM_015139652.2)中找到。食蟹猴FXI基因mRNA序列可以例如在基因库登录号GI:2161917139(XM_005556483.3)中找到。小鼠FXI基因mRNA序列可以例如在基因库登录号GI:2293430447(NM_028066.3)中找到。大鼠FXI基因mRNA序列可以例如在基因库登录号GI:2293332621(NM_001411666.1)中找到。使用公共可获得的数据库，例如基因库(GenBANK)容易获得FXI基因mRNA序列的其他实例。如非特别说明，FXI基因mRNA以SEQ ID NO：244(即NM_000128.4)为参比序列，即FXI基因mRNA中的各核苷酸位点编号为对应于参比序列SEQ ID NO：244中的5’至3’方向的核苷酸编号。如本文所用，术语“参比序列”即用于同源序列比对的标准序列，可用于定义同源多核苷酸或多核苷酸序列中核苷酸位置的序列。例如“FXI基因mRNA序列中碱基的位点编号为参比序列SEQ ID NO：244中对应碱基的编号”是指通过向所述FXI基因mRNA序列中引入空位或删除核苷酸等方式使所述FXI基因mRNA序列与参比序列在尽可能多的位置上具有相同碱基后，所述参比序列的核苷酸从5’端的第1位核苷酸开始按序列顺序依次连续编号，所述FXI基因mRNA序列与所述参比序列通过比对相互对应的核苷酸以相同编号定义该核苷酸的位置。The terms "FXI," "coagulation factor FXI," and "Factor 11" are used interchangeably and are also referred to in the art as FXI or PTA. FXI may be mammalian-derived FXI. In some embodiments, FXI is primate-derived FXI. In some embodiments, FXI is human-derived FXI. In some embodiments, FXI is cynomolgus monkey-derived FXI. As used herein, "FXI gene mRNA" refers to mRNA encoding Factor 11 protein, which may be transcribed from FXI gene DNA and may be mature mRNA or pre-mRNA (Pre-mRNA), and thus may or may not contain introns. Because the Factor 11 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the Factor 11 gene mRNA sequence of the present application is intended to include all mRNA sequences transcribed from Factor 11 gene mutants. The human FXI gene mRNA sequence can be found, for example, in GenBank Accession No. GI:1732746318 (NM_000128.4). The rhesus macaque FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 1622942384 (XM_015139652.2). The cynomolgus macaque FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 2161917139 (XM_005556483.3). The mouse FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 2293430447 (NM_028066.3). The rat FXI gene mRNA sequence can be found, for example, in Gene Bank Accession No. GI: 2293332621 (NM_001411666.1). Other examples of FXI gene mRNA sequences are readily available using publicly available databases, such as GenBANK. Unless otherwise specified, the FXI gene mRNA uses SEQ ID NO: 244 (i.e., NM_000128.4) as the reference sequence, that is, each nucleotide position number in the FXI gene mRNA is the nucleotide number corresponding to the 5’ to 3’ direction in the reference sequence SEQ ID NO: 244. As used herein, the term “reference sequence” is a standard sequence used for homologous sequence alignment, which can be used to define the sequence of nucleotide positions in homologous polynucleotides or polynucleotide sequences. For example, “the position numbering of the bases in the FXI gene mRNA sequence is the numbering of the corresponding bases in the reference sequence SEQ ID NO: 244” means that after the FXI gene mRNA sequence and the reference sequence have the same bases at as many positions as possible by introducing gaps or deleting nucleotides into the FXI gene mRNA sequence, the nucleotides of the reference sequence are numbered consecutively in sequence order starting from the first nucleotide at the 5’ end, and the positions of the nucleotides corresponding to each other are defined by the same numbering by aligning the FXI gene mRNA sequence and the reference sequence.

如本文所用，核酸的“互补”是指一条核酸具有通过传统的Watson-Crick碱基配对与另一条核酸形成氢键的能力。百分比互补性表示两条核酸分子中，较短一条的核酸分子中可与另一核酸分子形成氢键(即，Watson-Crick碱基配对)的核苷酸占所述较短一条的核苷酸中全部核苷酸的百分比(例如，10个中的约5、6、7、8、9、10个分别为约50％，60％，70％，80％，90％和100％互补)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数量的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250或更多个核苷酸的区域内，至少约70％，75％，80％，85％，90％，95％，96％，97％，98％，99％或100％中的任何一个的互补程度，或指在严格条件下杂交的两条核酸。对于单个碱基或单个核苷酸，按照Watson-Crick碱基配对原则，A与T或U、C与G或I配对时，被称为互补、配对或匹配，反之亦然；而除此以外的碱基配对都称为不互补。As used herein, "complementarity" of nucleic acids refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of nucleotides in the shorter of two nucleic acid molecules that can form hydrogen bonds (i.e., Watson-Crick base pairing) with the other nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, and 10 out of 10 are approximately 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For single bases or single nucleotides, according to the Watson-Crick base pairing rules, when A pairs with T or U, or C pairs with G or I, and vice versa, they are said to be complementary, paired, or matched; base pairings other than these are said to be non-complementary.

如本文所用，核酸的“杂交”是指一个或多个多核苷酸反应形成复合物的反应，该复合物通过核苷酸残基之间的氢键来稳定。氢键可以通过沃森克里克碱基配对，Hoogstein结合或任何其他序列特定方式发生。所述复合物可包括形成双链结构的两条链、形成多链复合物的三条或更多条链、单个自杂交链或其组合。As used herein, "hybridization" of nucleic acids refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can include two chains forming a double-stranded structure, three or more chains forming a multi-stranded complex, a single self-hybridizing chain, or a combination thereof.

在本申请中，当提及两条多核苷酸序列或两条核酸链“以最大互补率杂交”，则是指在使两条链中尽可能多的核苷酸彼此通过形成氢键配对的杂交方式。在“以最大互补率杂交”时，可以允许出现一个或多个错配，也可以允许两条链中的一条或多条链中出现一个或多个凸起(bulge)。但是，在一些实施方案中，“以最大互补率杂交”的两条链在彼此杂交后也可以不出现错配和凸起。如本文所用，“互补区”是指两条链杂交后，从5’端开始第一个通过氢键配对的碱基对开始至最后一个通过氢键配对的碱基对为止的全部通过氢键配对的碱基对，两条杂交链形成的互补区可以是连续的亦可以是间隔的。而本申请的“互补区1”特指当反义序列与FXI基因mRNA以最大互补率杂交时，由反义序列和FXI基因mRNA序列中互补配对的碱基对组成的互补区；而“互补区2”则是指当反义序列与正义序列以最大互补率杂交时，由所述反义序列和所述正义序列中互补配对的碱基对组成的互补区。由于互补区1与互补区2均涉及反义序列，因此互补区1和互补区2的反义序列部分通常具有部分或全部相同的核苷酸组成。本申请中，反义序列和正义序列是相对于与其中的一条序列互补的第三序列而言的，例如“通过RNAi抑制细胞中因子11(FXI)基因表达的核酸分子”中的反义序列和正义序列是相对于第三序列FXI基因mRNA序列来说的，即反义序列是指所述核酸分子中与FXI基因mRNA序列存在互补区的序列，正义序列是指所述核酸分子中与FXI基因mRNA序列具有至少10个以上连续相同核苷酸的序列。此外，本申请中任意互补区的5’末端是指其互补区中最接近于正义链或所述第三序列5’末端的核苷酸或碱基对的位置，互补区的5’端则是指其中相对靠近正义链或所述第三序列5’末端的一侧或一端。同理，本申请的任意互补区的3’末端是指其互补区中最接近于正义链或所述第三序列3’末端的核苷酸或碱基对的位置，互补区的3’端则是指其中相对靠近正义链或所述第三序列3’末端的一侧或一端。在本申请中，当描述同一条序列或核苷链上核苷酸位置的“接近”、“近”或“远”是指两个核苷酸位置间隔核苷酸数量的少或多。In this application, when referring to two polynucleotide sequences or two nucleic acid chains "hybridizing at maximum complementarity", it refers to a hybridization mode in which as many nucleotides as possible in the two chains are paired with each other by forming hydrogen bonds. When "hybridizing at maximum complementarity", one or more mismatches may be allowed to occur, and one or more bulges may be allowed to appear in one or more of the two chains. However, in some embodiments, the two chains "hybridized at maximum complementarity" may not have mismatches and bulges after hybridization. As used herein, "complementary region" refers to all base pairs paired by hydrogen bonds from the first base pair paired by hydrogen bonds at the 5' end to the last base pair paired by hydrogen bonds after hybridization of the two chains. The complementary region formed by the two hybridized chains may be continuous or intermittent. In this application, "complementary region 1" specifically refers to the complementary region consisting of the complementary base pairs in the antisense sequence and the FXI gene mRNA sequence when the antisense sequence hybridizes with the FXI gene mRNA at maximum complementarity; and "complementary region 2" refers to the complementary region consisting of the complementary base pairs in the antisense sequence and the sense sequence when the antisense sequence hybridizes with the sense sequence at maximum complementarity. Since both complementary regions 1 and 2 involve antisense sequences, the antisense sequence portions of complementary regions 1 and 2 typically have partially or completely identical nucleotide compositions. In this application, the antisense sequence and sense sequence are relative to a third sequence that is complementary to one of the sequences. For example, the antisense sequence and sense sequence in the "nucleic acid molecule that inhibits the expression of the factor 11 (FXI) gene in cells through RNAi" are relative to the third sequence, the FXI gene mRNA sequence. That is, the antisense sequence refers to the sequence in the nucleic acid molecule that has a complementary region with the FXI gene mRNA sequence, and the sense sequence refers to the sequence in the nucleic acid molecule that has at least 10 consecutive identical nucleotides with the FXI gene mRNA sequence. In addition, the 5' end of any complementary region in the present application refers to the position of the nucleotide or base pair in the complementary region that is closest to the 5' end of the sense strand or the third sequence, and the 5' end of the complementary region refers to the side or end relatively close to the 5' end of the sense strand or the third sequence. Similarly, the 3' end of any complementary region in the present application refers to the position of the nucleotide or base pair in the complementary region that is closest to the 3' end of the sense strand or the third sequence, and the 3' end of the complementary region refers to the side or end relatively close to the 3' end of the sense strand or the third sequence. In the present application, when describing the position of nucleotides on the same sequence or nucleoside chain, "close", "near" or "far" refers to the number of nucleotides between the two nucleotide positions.

如本文所用，“突出端核苷酸”是相对于互补区2而言的，当正义序列与反义序列以最大互补率杂交后，所述正义序列和/或反义序列核苷酸中位于互补区2以外核苷酸。在一些实施方案中，所述突出区核苷酸仅位于正义序列，在一些实施方案中，所述突出区核苷酸仅位于反义序列，在一些实施方案中所述突出端核苷酸位于正义序列及反义序列。在一些实施方案中，所述突出端核苷酸仅存在于互补区2的5’端一侧。在一些实施方案中，所述突出端核苷酸仅存在于正义链互补区2的5’端一侧。在一些实施方案中，所述突出端核苷酸仅存在于反义链互补区2的5’端一侧。在一些实施方案中，所述突出端核苷酸仅存在于互补区2的3’端一侧。在一些实施方案中，所述突出端核苷酸仅存在于正义链互补区2的3’端一侧。在一些实施方案中，所述突出端核苷酸仅存在于反义链互补区2的3’端一侧。在一些实施方案中，所述突出区核苷酸在正义序列或反义序列的同一侧(例如5’端或3’端)的数量不超过两个(即为1个或者2个)。在本申请中，“5’端一侧”及“3’端一侧”均用以描述同一条多核苷酸序列中两段序列，两个核苷酸或一个核苷酸与一段序列之间的相对位置关系；其中，“5’端”指所述多核苷酸序列包含5’游离磷酸基团或5’游离羟基的一端，“3’端”指所述多核苷酸序列包含游离3’-羟基或3’-磷酸基团的一端，而核酸链中某序列3’端一侧的序列或核苷酸比所述某序列更靠近所述核酸链的3’端。。而例如“在互补区2的5’端一侧”还包含某序列或者某个或某几个核苷酸，则指所述“某序列或者某个或某几个核苷酸”相对于“互补区2”的序列，更靠近其共同所在的多核苷酸序列(例如反义序列或正义序列)的5’端。As used herein, "overhang nucleotides" are relative to complementary region 2, and are nucleotides located outside complementary region 2 in the sense sequence and/or antisense sequence nucleotides after the sense sequence and antisense sequence hybridize at maximum complementarity. In some embodiments, the overhang nucleotides are located only in the sense sequence, in some embodiments, the overhang nucleotides are located only in the antisense sequence, and in some embodiments, the overhang nucleotides are located in both the sense sequence and the antisense sequence. In some embodiments, the overhang nucleotides are only present on the 5' end of complementary region 2. In some embodiments, the overhang nucleotides are only present on the 5' end of complementary region 2 of the sense strand. In some embodiments, the overhang nucleotides are only present on the 5' end of complementary region 2 of the antisense strand. In some embodiments, the overhang nucleotides are only present on the 3' end of complementary region 2 of the sense strand. In some embodiments, the overhang nucleotides are only present on the 3' end of complementary region 2 of the antisense strand. In some embodiments, the number of nucleotides in the overhang region on the same side (e.g., the 5' end or 3' end) of the sense sequence or antisense sequence is no more than two (i.e., one or two). In this application, "on the 5' end side" and "on the 3' end side" are both used to describe the relative positional relationship between two sequences, two nucleotides, or one nucleotide and one sequence in the same polynucleotide sequence; wherein "5' end" refers to the end of the polynucleotide sequence containing a 5' free phosphate group or a 5' free hydroxyl group, and "3' end" refers to the end of the polynucleotide sequence containing a free 3'-hydroxyl group or a 3'-phosphate group, and the sequence or nucleotide on the 3' end side of a sequence in the nucleic acid chain is closer to the 3' end of the nucleic acid chain than the sequence. For example, "on the 5' end side of complementary region 2" also contains a certain sequence or a certain nucleotide or nucleotides, which means that the "certain sequence or a certain nucleotide or nucleotides" are closer to the 5' end of the polynucleotide sequence (e.g., the antisense sequence or the sense sequence) with which they are co-located, relative to the sequence of "complementary region 2."

术语“核苷酸”，除了是指天然存在的核糖核苷酸或脱氧核糖核苷酸单体以外，本文中还应当理解为是指其相关的结构变体，包括衍生物和类似物，其关于使用该核苷酸的具体上下文在功能上是等效的，除非上下文明确另外指明。例如，“核苷酸”指脱氧核糖核苷酸或核糖核苷酸。核苷酸可以是标准核苷酸(即，腺苷、鸟苷、胞苷、胸苷和尿苷)，核苷酸异构体或核苷酸类似物。核苷酸类似物指具有修饰的嘌呤或嘧啶碱基或者修饰的核糖部分的核苷酸。核苷酸类似物可以是天然存在的核苷酸(例如肌苷、假尿苷等)或非天然存在的核苷酸。核苷酸的糖或碱基部分上的修饰的非限制性实例包括乙酰基、氨基、羧基、羧甲基、羟基、甲基、磷酰基和硫醇基的添加(或去除)，以及碱基的碳和氮原子被其它原子的取代(例如7-脱氮嘌呤)。核苷酸类似物还包括双脱氧核苷酸、2'-O-甲基核苷酸、锁核酸(LNA)、肽核酸(PNA)和吗啉代寡核苷酸(morpholino)。在一些实施方案中，本申请的“核苷酸”不包含碱基经过修饰的非天然核苷酸。在一些实施方案中，本申请的“核苷酸”不包含碱基经过修饰的核苷酸。在本申请中，“G”、“C”、“A”、“T”和“U”通常分别代表以鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶为碱基的核苷酸，如非特别说明，“G”、“C”、“A”、“T”和“U”表示不限定其包含的修饰的核苷酸，即其可用于表示天然核苷酸或非天然核苷酸，所述非天然核苷酸可以包含核糖和/或碱基经修饰的核苷酸，只要其中的碱基依然可与其天然配对(即依据Watson-Crick原则配对)的碱基通过氢键互补配对即可。但在RNA的语境下以及在RNA序列中，如非特别说明“T”代指尿苷或尿嘧啶。应当理解，本申请中凡涉及核苷酸序列的语境，“核苷酸”、“核苷酸残基”和“碱基”可互换使用。碱基对的个数以bp为单位，一个bp则为一个碱基对。核苷酸个数则以nt为单位，一个nt为一个核苷酸。The term "nucleotide", in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, is also understood herein to refer to related structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used, unless the context clearly indicates otherwise. For example, "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. A nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), a nucleotide isomer, or a nucleotide analog. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog can be a naturally occurring nucleotide (e.g., inosine, pseudouridine, etc.) or a non-naturally occurring nucleotide. Non-limiting examples of modifications on the sugar or base portion of a nucleotide include the addition (or removal) of an acetyl group, an amino group, a carboxyl group, a carboxymethyl group, a hydroxyl group, a methyl group, a phosphoryl group, and a thiol group, and the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino oligonucleotides. In some embodiments, the "nucleotides" of the present application do not include non-natural nucleotides with modified bases. In some embodiments, the "nucleotides" of the present application do not include nucleotides with modified bases. In the present application, "G", "C", "A", "T" and "U" generally represent nucleotides with guanine, cytosine, adenine, thymine and uracil as bases, respectively. Unless otherwise specified, "G", "C", "A", "T" and "U" represent nucleotides with no limitation on the modifications they contain, that is, they can be used to represent natural nucleotides or non-natural nucleotides. The non-natural nucleotides may contain ribose and/or modified bases, as long as the bases therein can still be paired with their naturally paired bases (i.e., paired according to the Watson-Crick principle) through hydrogen bonding. However, in the context of RNA and in RNA sequences, unless otherwise specified, "T" refers to uridine or uracil. It should be understood that throughout this application, "nucleotide," "nucleotide residue," and "base" are used interchangeably in contexts related to nucleotide sequences. Base pairs are measured in bp, with one bp being one base pair. Nucleotides are measured in nt, with one nt being one nucleotide.

如本文所用“核苷酸间连接”是指相邻两个核苷酸间或核苷酸与配体键的化学键(或连接基团)，如非特别说明，所述化学键为磷酸酯键或磷酸酯键；然而当一个修饰基序中特别标明“s”，即硫代磷酸酯修饰的位置时，则仅在s出现位置处的核苷酸间连接为硫代磷酸酯键，其他未标明的核苷酸间连接均为磷酸酯键，例如：As used herein, "internucleotide linkage" refers to the chemical bond (or linking group) between two adjacent nucleotides or between a nucleotide and a ligand. Unless otherwise specified, the chemical bond is a phosphate bond or a phosphoester bond. However, when an "s" is specifically indicated in a modification motif, i.e., the position of a phosphorothioate modification, only the internucleotide linkage at the position where "s" appears is a phosphorothioate bond, and all other unindicated internucleotide linkages are phosphate bonds. For example:

(1)有义链：NmNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNm，反义链：NmNfNmNmNdNmNdNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm；(1) Sense strand: NmNmNmNmNmNmNfNmNfNdNfNmNmNmNmNmNmNmNmNmNm, antisense strand: NmNfNmNmNdNmNdNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm;

则(1)中的各核苷酸间连接可以是磷酸酯键也可以是硫代磷酸酯键；Then the internucleotide linkages in (1) can be either phosphate bonds or phosphorothioate bonds;

而如果是:有义链：CmsAmsGmAmGmUmUfAmUfCdGfAmGmGmCmAmCmAmUmUmAms-配体1，And if it is: sense strand: CmsAmsGmAmGmUmUfAmUfCdGfAmGmGmCmAmCmAmUmUmAms-ligand 1,

反义链：vp-UmsAfsAmUmGdUmGdCmCmUmCmGmAmUfAmAfCmUmCmUmGmsGmsCm；由于其具有特别标明的“s”,则除以下核苷酸间连接为硫代磷酸酯键之外，其他位置的核苷酸间连接均为磷酸酯键：有义链5’端起的第1个核苷酸间连接和第2个核苷酸间连接，有义链3’末端核苷酸与配体1之间的核苷酸间连接，反义链5’末端起的第1个核苷酸间连接和第2个核苷酸间连接，以及反义链3’末端起的第1个核苷酸间连接和第2个核苷酸间连接。其中，第1个核苷酸间连接是指从一个末端(例如5’末端或3’末端)开始的第一个和第二个核苷酸之间的化学键；第2个核苷酸间连接是指从一个末端(例如5’末端或3’末端)开始的第二个和第三个核苷酸之间的化学键，依次类推；第n个核苷酸间连接是指从一个末端(例如5’末端或3’末端)开始的第n个和第n+1个核苷酸或配体之间的化学键，依次类推。Antisense strand: vp-UmsAfsAmUmGdUmGdCmCmUmCmGmAmUfAmAfCmUmCmUmGmsGmsCm; because it has a specially marked "s", except for the following internucleotide linkages which are phosphorothioate bonds, the internucleotide linkages at other positions are phosphate bonds: the first and second internucleotide linkages from the 5' end of the sense strand, the internucleotide linkage between the 3' terminal nucleotide of the sense strand and ligand 1, the first and second internucleotide linkages from the 5' end of the antisense strand, and the first and second internucleotide linkages from the 3' end of the antisense strand. Among them, the first internucleotide linkage refers to the chemical bond between the first and second nucleotides starting from one end (such as the 5' end or the 3' end); the second internucleotide linkage refers to the chemical bond between the second and third nucleotides starting from one end (such as the 5' end or the 3' end), and so on; the nth internucleotide linkage refers to the chemical bond between the nth and n+1th nucleotides or ligands starting from one end (such as the 5' end or the 3' end), and so on.

如本文所用，“突出端”、“悬垂端”及“悬垂序列”可互换使用，是指延伸超过在链末端的双链体区的未配对的一个或多个核苷酸。当一条链的3′端延伸超过另一条链的5′端时或者当一条链的5′端延伸超过另一条链的3′端时，典型地形成核苷酸突出端。核苷酸突出端的长度通常是在1与6个核苷酸之间、1与5个核苷酸之间、1与4个核苷酸之间、1与3个核苷酸之间、2与6个核苷酸之间、2与5个核苷酸之间、或2与4个核苷酸之间。在一些实施例中，核苷酸突出端包含1、2、3、4、5或6个核苷酸。在一个特定的实施例中，核苷酸突出端包含1至4个核苷酸。在某些实施例中，核苷酸突出端包含2个核苷酸。在某些其他实施例中，核苷酸突出端包含单个核苷酸。As used herein, "overhang," "overhang," and "overhang sequence" are used interchangeably and refer to one or more unpaired nucleotides that extend beyond the duplex region at the end of a chain. A nucleotide overhang is typically formed when the 3' end of one chain extends beyond the 5' end of the other chain, or when the 5' end of one chain extends beyond the 3' end of the other chain. The length of the nucleotide overhang is typically between 1 and 6 nucleotides, between 1 and 5 nucleotides, between 1 and 4 nucleotides, between 1 and 3 nucleotides, between 2 and 6 nucleotides, between 2 and 5 nucleotides, or between 2 and 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a specific embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide.

突出端中的核苷酸可以是如本文所述的核糖核苷酸或经修饰的核苷酸。在一些实施例中，突出端中的核苷酸是2′-经修饰的核苷酸(例如2'-氟修饰的核苷酸，2'-O-甲基修饰的核苷酸)、脱氧核糖核苷酸、反向核苷酸(例如反向无碱基核苷酸，反向脱氧核糖核苷酸)或其组合。例如，在一个实施例中，突出端中的核苷酸是脱氧核糖核苷酸，例如脱氧胸苷。在另一个实施例中，突出端中的核苷酸是2'-O-甲基修饰的核苷酸、2'-氟修饰的核苷酸、2'-甲氧基乙基修饰的核苷酸或其组合。在其他实施例中，突出端包含5'-尿苷-尿苷-3'(5'-UU-3')二核苷酸。在这类实施例中，UU二核苷酸可以包含核糖核苷酸或经修饰的核苷酸，例如2'-修饰的核苷酸。在其他实施例中，突出端包含5'-脱氧胸苷-脱氧胸-3'(5'-dTdT-3')二核苷酸。当核苷酸突出端存在于反义链中时，突出端中的核苷酸可以与靶基因序列互补，形成与靶基因序列的错配或包含一些其他序列(例如聚嘧啶或聚嘌呤序列，如UU、TT、AA、GG等)。The nucleotides in the overhang can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides in the overhang are 2'-modified nucleotides (e.g., 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides), deoxyribonucleotides, reverse nucleotides (e.g., reverse abasic nucleotides, reverse deoxyribonucleotides), or combinations thereof. For example, in one embodiment, the nucleotides in the overhang are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides in the overhang are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, or combinations thereof. In other embodiments, the overhang comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotides. In such embodiments, the UU dinucleotides can comprise ribonucleotides or modified nucleotides, such as 2'-modified nucleotides. In other embodiments, the overhang comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotides. When a nucleotide overhang is present in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (e.g., a polypyrimidine or polypurine sequence, such as UU, TT, AA, GG, etc.).

核苷酸突出端可以在一条或两条链的5'端或3'端。例如，在一个实施例中，RNA分子在反义链的5'端和3'端包含核苷酸突出端。在另一个实施例中，RNA分子在正义链的5'端和3′端包含核苷酸突出端。在一些实施例中，RNA分子在正义链的5”端和反义链的5′端包含核苷酸突出端。在其他实施例中，RNA分子在正义链的3′端和反义链的3′端包含核苷酸突出端。The nucleotide overhangs can be at the 5' end or the 3' end of one or both strands. For example, in one embodiment, the RNA molecule comprises nucleotide overhangs at the 5' end and the 3' end of the antisense strand. In another embodiment, the RNA molecule comprises nucleotide overhangs at the 5' end and the 3' end of the sense strand. In some embodiments, the RNA molecule comprises nucleotide overhangs at the 5" end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNA molecule comprises nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.

RNA分子可在双链RNA分子的一端包含核苷酸突出端，而在另一端包含平端。“平端”意味着正义链与反义链在分子末端完全地碱基配对，并且不存在延伸超出双链体区的未配对核苷酸。在一些实施例中，RNA分子在正义链的3′端包含核苷酸突出端，而在正义链的5′端和反义链的3′端包含平端。在其他实施例中，RNA分子在反义链的3′端包含核苷酸突出端，而在反义链的5′端和正义链的3′端包含平端。在某些实施例中，RNA分子在双链RNA分子的两端包含平端。在这类实施例中，正义链和反义链具有相同的长度，并且双链体区的长度与正义链和反义链相同(即该分子在其整个长度上是双链的)。如本文所用，“GalNAc”或“N-乙酰半乳糖胺”：是指2-(乙酰基氨基)-2-脱氧-D-吡喃半乳糖。如非特别说明，术语“GalNAc”或“N-乙酰基半乳糖胺”包括β形式：2-(乙酰基氨基)-2-脱氧-β-D-吡喃半乳糖与α形式：2-(乙酰基氨基)-2-脱氧-α-D-吡喃半乳糖两者。优选地，本申请的GalNAc化合物为β形式，即2-(乙酰基氨基)-2-脱氧-β-D-吡喃半乳糖。An RNA molecule may comprise a nucleotide overhang at one end of a double-stranded RNA molecule and a flat end at the other end. "Flat end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the duplex region. In some embodiments, the RNA molecule comprises a nucleotide overhang at the 3' end of the sense strand and a flat end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNA molecule comprises a nucleotide overhang at the 3' end of the antisense strand and a flat end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNA molecule comprises a flat end at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and the antisense strand have the same length, and the length of the duplex region is the same as that of the sense strand and the antisense strand (i.e., the molecule is double-stranded over its entire length). As used herein, "GalNAc" or "N-acetylgalactosamine": refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term "GalNAc" or "N-acetylgalactosamine" includes both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Preferably, the GalNAc compound of the present application is the β form, i.e., 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

如本文所用，“VP修饰”或“E-VP”是指位于反义链上5'的磷酸酯模拟修饰，具体结构为：
As used herein, "VP modification" or "E-VP" refers to a phosphate mimic modification located at the 5' end of the antisense strand, with the specific structure being:

其中：in:

Bx1为尿嘧啶、胸腺嘧啶、胞嘧啶、5-甲基胞嘧啶、腺嘌呤或鸟嘌呤；Bx1 is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine;

T2是将上述化合物连接到寡聚核酸链的磷酸酯或硫代磷酸酯核苷间连接基团；以及G为卤素、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或OCH2-N(H)-C(＝NH)NH2。 T2 is a phosphate or phosphorothioate internucleoside linker that connects the above compound to the oligonucleic acid chain; and G is a halogen, OCH3 , OCF3 , OCH2CH3 , OCH2CF3, OCH2 - CH =CH2, O( CH2 ) 2 - OCH3 , O( CH2 ) 2 -O( CH2 ) 2 - N ( CH3 ) 2 , OCH2C (=O) -N (H) CH3 , OCH2C (=O)-N(H)-( CH2 ) 2 -N( CH3 ) 2 , or OCH2 -N(H)-C(=NH) NH2 .

如本文所用，“3’末端”特指单核苷酸序列或双链多核苷酸3’端的第一个核苷酸或第一个碱基对的位置。“5’末端”则特指单核苷酸序列或双链多核苷酸5’端的第一个核苷酸或第一个碱基对的位置。As used herein, the term "3' end" specifically refers to the position of the first nucleotide or base pair at the 3' end of a single nucleotide sequence or a double-stranded polynucleotide. The term "5' end" specifically refers to the position of the first nucleotide or base pair at the 5' end of a single nucleotide sequence or a double-stranded polynucleotide.

如本申请所用，术语“核酸分子”可用于指代任何具有由2个以上核苷酸通过磷酸酯键，或经过修饰的磷酸酯键(例如硫代磷酸脂键)连接而成的核苷酸序列的分子。As used herein, the term "nucleic acid molecule" may be used to refer to any molecule having a nucleotide sequence composed of two or more nucleotides linked by a phosphate bond, or a modified phosphate bond (eg, a phosphorothioate bond).

如本文所用，术语“核苷酸序列”是指一段由核苷酸依据一定顺序依次排列组成的一段多核苷酸链，该段多核苷酸链本申请可以组成一个核酸分子，也可以构成某一个核酸分子的一条链中的一段。因此，“核苷酸序列”可以以一段确切的由各种核苷酸(例如ATCG)组成的多核苷酸序列(例如SEQ ID NO：1至100中的任一条)表示，也可以表示为某序列中的某位至某位的核苷酸，例如“FXI基因mRNA序列中”“363至382位的核苷酸”。在本申请中，如非特别说明，包含“某序列中的某位至某位的核苷酸”的序列或核酸分子中，所述某位至某位的核苷酸的排列顺序与所述某序列中的前述核苷酸的顺序一致。在不加限定的情况下，“核苷酸序列”可以是RNA或DNA，或RNA与DNA的杂合分子，还可以掺入非天然核苷酸或经人工修饰的核苷酸。As used herein, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in sequence according to a certain order. This polynucleotide chain can constitute a nucleic acid molecule in this application, or it can constitute a section of a chain of a nucleic acid molecule. Therefore, a "nucleotide sequence" can be represented by a specific polynucleotide sequence composed of various nucleotides (such as ATCG) (such as any one of SEQ ID NO: 1 to 100), or it can be represented as nucleotides from a certain position to a certain position in a certain sequence, such as "nucleotides from positions 363 to 382 in the FXI gene mRNA sequence". In this application, unless otherwise specified, in a sequence or nucleic acid molecule comprising "nucleotides from a certain position to a certain position in a certain sequence", the arrangement order of the nucleotides from a certain position to a certain position is consistent with the order of the aforementioned nucleotides in the certain sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid molecule of RNA and DNA, and can also incorporate non-natural nucleotides or artificially modified nucleotides.

此外，本申请附图中出现的REL或Relative expression level是指mRNA相对表达水平。在序列语境下出现的N代表核糖核苷酸，dN代表脱氧核糖核苷酸(DNA)，Nm代表2'-O-Me修饰的核苷酸或称为2’-O-甲基修饰的核苷酸；Nf表示2'-F修饰的核糖核苷酸或2’-氟代修饰的核糖核苷酸；s代表硫代磷酸脂修饰，即5'-硫代修饰的磷酸脂，当该修饰在两个核苷酸之间或核苷酸与配体之间，其替代天然核苷酸间的磷酸酯键，可称硫代磷酸酯键。如本文所用，如非特别说明，本申请中“硫代磷酸酯键”与“硫代磷酸二酯键”可互换使用；同样“磷酸酯键”与“磷酸二酯键”可互换使用。In addition, the term "REL" or "Relative expression level" appearing in the figures of this application refers to the relative expression level of mRNA. In the context of sequences, N represents a ribonucleotide, dN represents a deoxyribonucleotide (DNA), Nm represents a 2'-O-Me modified nucleotide, also known as a 2'-O-methyl 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. When this modification occurs between two nucleotides or between a nucleotide and a ligand, it replaces the phosphate bond between natural nucleotides and is referred to as a phosphorothioate bond. As used herein, unless otherwise specified, "phosphorothioate bond" and "phosphothiodiester bond" are used interchangeably; similarly, "phosphate bond" and "phosphodiester bond" are used interchangeably.

如本文所用，术语“约”是指本技术领域的技术人员容易知道的各个值的通常误差范围。提及“约”值或参数在本文中包括(及描述)针对该值或参数本身的实施方案。如本文所使用的，当术语“约”在数值之前时，表示该数值上或下10％的范围内。例如，“约100”涵盖90和110。As used herein, the term "about" refers to the typical error range for each value that is readily known to those skilled in the art. Reference to an "about" value or parameter herein includes (and describes) embodiments directed to that value or parameter itself. As used herein, when the term "about" precedes a numerical value, it means within a range of 10% above or below that numerical value. For example, "about 100" encompasses 90 and 110.

如本文所用，除非另外指出，否则单数形式“一个”，“一种”和“该”包括复数形式。As used herein, the singular forms "a," "an," and "the" include plural forms unless otherwise indicated.

除非本文另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的相同含义。Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

应当理解，本申请包含本文所描述的各种方面、实施方案以及所述方面和/或实施方案的组合。以上描述以及随后的实施例旨在说明而不是限制本申请的范围。在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本申请所公开的内容，均属于本申请的保护范围。It should be understood that the present application includes various aspects, embodiments, and combinations of the aspects and/or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Within the scope of the technical concept of the present application, the technical solution of the present application can be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the contents disclosed in the present application and all fall within the scope of protection of the present application.

除另有说明外，本申请的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的常规技术。现有技术文献记载了所述常规技术。Unless otherwise indicated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are well documented in the art.

应当理解，本申请包含本文所描述的各种方面、实施方案以及所述方面和/或实施方案的组合。以上描述以及随后的实施例旨在说明而不是限制本申请的范围。在本申请范围内的其他方面、改进和修改对于本申请所属领域的技术人员将是显而易见的。因此，本领域的普通技术人员应该认识到，本申请的范围还包括对所述方面和实施方案的所述改进和修改。It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and/or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments.

实施例Example

实施例1：dsRNA的序列设计与合成Example 1: Sequence Design and Synthesis of dsRNA

在本申请具体实施例1-7中，各核苷酸不包含本实施例未标明的修饰。即本申请实施例1-7使用的各核苷酸序列为裸序列或仅包含其所用修饰基序中标明的修饰。In specific Examples 1-7 of the present application, each nucleotide sequence does not contain modifications not indicated in the examples. That is, each nucleotide sequence used in Examples 1-7 of the present application is a naked sequence or contains only the modifications indicated in the modification motif used.

1.1 dsRNA设计1.1 dsRNA design

参考人FXI基因(Gene ID,2160)mRNA序列(NM_000128.4)，选择不同位点设计多个FXI dsRNAs，设计的所有单个dsRNA，所述dsRNA序列经序列相似性软件比对与其他所有非靶标基因序列有最低同源性，具体参见文末序列表。此外，对于修饰的dsRNA，若正义链3’端第一个核苷酸如非A或U，则采用尿嘧啶核糖核苷酸替代原有核苷酸，反义序列的5’端第一个核苷酸如非A或U，则采用腺嘌呤核糖核苷酸替代原有核苷酸，将原有核酸序列从n变为n’进行检测，例如E3、E4、E9、E14、E16、E24、E26、E51、E52、E69、E70的核酸序列分别如文末序列表编号为3’、4’、9’、14’、16’、24’、26’、51’、52’、69’、70’的dsRNA所示。With reference to the human FXI gene (Gene ID, 2160) mRNA sequence (NM_000128.4), multiple FXI dsRNAs were designed at different sites. All designed single dsRNAs had minimal homology with all other non-target gene sequences after sequence similarity comparison using sequence similarity software. For details, please see the sequence listing at the end of the article. In addition, for modified dsRNA, if the first nucleotide at the 3' end of the sense strand is not A or U, a uracil ribonucleotide is used to replace the original nucleotide; if the first nucleotide at the 5' end of the antisense sequence is not A or U, an adenine ribonucleotide is used to replace the original nucleotide, and the original nucleic acid sequence is changed from n to n' for detection. For example, the nucleic acid sequences of E3, E4, E9, E14, E16, E24, E26, E51, E52, E69, and E70 are shown in the dsRNA numbered 3', 4', 9', 14', 16', 24', 26', 51', 52', 69', and 70' in the sequence listing at the end of the article, respectively.

本申请中，我们选择ionis公司专利(专利号：CN 109797150 A)公开的ISIS-416858的ASO序列，选择专利号：CN113227376A公开的对FXI基因抑制率前4的序列(裸序列命名：RB2、RB4、RB6、RB7；修饰序列命名为：ERB2、ERB4、ERB6、ERB7，裸序列及修饰序列分别对应专利中的命名为L10-siFXIa1M1SP、L10-siFXIc1M1SP、L10-siFXIe1M1SP、L10-siFXIg1M1SP分子中无配体偶联的序列)，选择专利号：WO 2022028457A1公开的对FXI基因抑制率前三的序列(裸序列命名：TJ632、TJ645、TJ772；修饰序列命名为：ETJ632、ETJ645、ETJ772；裸序列及修饰序列分别对应专利中命名为TRD0632、TRD0645、TRD0772分子中无配体偶联的序列)序列作为FXI基因阳性对照序列。前述dsRNA序列及阳性对照序列的裸序列如文末序列表所示。In this application, we selected the ASO sequence of ISIS-416858 disclosed in the patent of ionis (patent number: CN 109797150 A), and selected the top 4 sequences of FXI gene inhibition rate disclosed in patent number: CN113227376A (naked sequence named: RB2, RB4, RB6, RB7; modified sequence named: ERB2, ERB4, ERB6, ERB7, naked sequence and modified sequence named L10-siFXIa1M1SP, L10-siFXIc1M1SP, L10- The top three sequences with the highest FXI gene inhibition rates (naked sequences: TJ632, TJ645, and TJ772; modified sequences: ETJ632, ETJ645, and ETJ772; the naked and modified sequences correspond to the ligand-free sequences in the patent molecules named TRD0632, TRD0645, and TRD0772, respectively) were selected as FXI gene positive control sequences. The naked sequences of the aforementioned dsRNA sequences and positive control sequences are shown in the sequence listing at the end of the article.

1.2 dsRNA及其缀合物的合成与纯化1.2 Synthesis and purification of dsRNA and its conjugates

使用经修饰(例如2’-甲氧基或2’-氟修饰或硫代修饰)或未经化学修饰的核糖核苷酸合成dsRNA，其按照理论产量1μmol合成规格完成，选用1μmol通用的Frit载体( 逗点生物)或带保护基的GANLNAC衍生物L96的CPG载体(凯莱英)在LK-192X合成仪(江苏领坤生物科技有限公司)上制备所有的寡核苷酸。根据序列需要，将对应核苷的所有亚磷酰胺单体(均按照1:40(g/mL)的无水乙腈溶剂稀释，偶联时间为3min，共两次偶联。使用3％TCA进行脱保护，采用0.3M苄硫基四唑乙腈溶液进行活化，并通过CAPA/CAPB和50mM I2溶液分别进行盖帽和氧化。在去三苯甲基合成(Trityl-off synthesis)后，将固相载体转移至2mL离心管中，加入1.2mL氨水置于65℃烘箱中加热3h，脱去保护基。然后冷却至室温，真空浓缩30min，将溶液通过0.22um滤膜过滤至进样瓶中，采用半制备反相纯化仪进行单链纯化，洗脱梯度为7％～30％(ACN:100mM TEAA)，时间10min；流速：5mL/min，纯化制备后真空浓缩，室温旋干。最后用水溶解样品，在GE Hi-Trap脱盐柱上将每种溶液脱盐以洗脱最终的寡聚核苷酸产物。分别使用ESI-MS和IEX HPLC确认所有的特性和纯度。采用酶标仪紫外定浓，将等摩尔量的正义链和反义链混合并至新的发货管中，95℃加热5min，并缓慢退火至室温，最后采用真空浓缩仪室温旋干得到最终的dsRNA产品。The dsRNA was synthesized using modified (e.g., 2'-methoxy or 2'-fluoro or thio modified) or unmodified ribonucleotides, and the synthesis was completed according to the theoretical yield of 1 μmol, using 1 μmol of a universal Frit vector ( All oligonucleotides were prepared on an LK-192X synthesizer (Jiangsu Lingkun Biotechnology Co., Ltd.) using CPG vectors (Ailiying) or the protected GANLNAC derivative L96. According to sequence requirements, all phosphoramidite monomers corresponding to the nucleoside were diluted in anhydrous acetonitrile at a ratio of 1:40 (g/mL) and coupled twice for 3 minutes. Deprotection was performed using 3% TCA, activation was performed using 0.3 M benzylthiotetrazolyl in acetonitrile, and capping and oxidation were performed using CAPA/CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated in a 65°C oven for 3 hours to remove the protecting groups. The tube was then cooled to room temperature and concentrated under vacuum for 30 minutes. The solution was then filtered through a 0.22 μm filter into a vial. Single-stranded fragments were purified using a semi-preparative reverse-phase instrument with an elution gradient of 7% to 30% (ACN:100 mM TEAA) over 10 minutes at a flow rate of 5 mL/min. After purification, the fragment was concentrated under vacuum and dried at room temperature. Finally, the sample was dissolved in water and purified on a GE Each solution was desalted on a Hi-Trap desalting column to elute the final oligonucleotide product. All identities and purity were confirmed using ESI-MS and IEX HPLC, respectively. Concentration was determined using a UV-visible microplate reader. Equimolar amounts of the sense and antisense strands were mixed and transferred to a new shipping tube. The mixture was heated at 95°C for 5 minutes, slowly annealed to room temperature, and finally dried using a vacuum concentrator at room temperature to yield the final dsRNA product.

本申请实施例中使用的L96结构如下式I所示：
The structure of L96 used in the examples of this application is shown in Formula I below:

其中，代表通过磷酸二酯键与RNA分子的有义链或反义链的3’端相连。in, It represents a phosphodiester bond attached to the 3' end of the sense or antisense strand of an RNA molecule.

实施例2：FXI-dsRNA体外活性高通量筛选检测Example 2: High-throughput screening assay for FXI-dsRNA in vitro activity

2.1 FXI dsRNA转染HepG2细胞2.1 FXI dsRNA transfection into HepG2 cells

HepG2细胞在含10％胎牛血清的DMEM培养基中，于5％CO2、37℃恒温培养箱中培养。细胞消化后调整细胞密度为4×105个/ml，按照每孔1ml细胞悬液铺种到12孔板中。配制100ul转染复合物：将45μL Opti-MEM和5μL不同浓度的dsRNA(裸核酸，无修饰)混合，48μL Opti-MEM和2μL RNAiMax转染试剂混合，静置5min，然后将以上两个混合物混合，静置20min形成转染复合物。将上述转染复合物加入12孔板中，于5％CO2、37℃恒温培养箱中继续培养24h。转染后24小时收获细胞，使用TRIZOL法进行RNA提取。HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2 , 37°C constant temperature incubator. After cell digestion, the cell density was adjusted to 4× 105 cells/ml, and 1 ml of cell suspension was plated into 12-well plates per well. Prepare 100ul transfection complex: 45μL Opti-MEM and 5μL dsRNA (naked nucleic acid, unmodified) of different concentrations were mixed, 48μL Opti-MEM and 2μL RNAiMax transfection reagent were mixed, and the two mixtures were mixed and allowed to stand for 20 minutes to form a transfection complex. The above transfection complex was added to a 12-well plate and cultured in a 5% CO2, 37°C constant temperature incubator for 24 hours. Cells were harvested 24 hours after transfection, and RNA was extracted using the TRIZOL method.

2.2实时荧光定量PCR分析2.2 Real-time fluorescence quantitative PCR analysis

转染24h后裂解细胞，采用诺唯赞柱提法FastPure Cell/Tissue Total RNA Isolation Kit V2试剂盒(参考诺唯赞RC112-01说明书)提取细胞总RNA。采用Takara PrimeScript RT Master Mix RR036Q逆转录为cDNA。qPCR引物序列信息如文末序列表引物部分所示，以人GAPDH基因为内参基因，使用美国Bio-Rad公司CFX96荧光定量PCR仪进行PCR反应。实验中以Mock组为对照做归一化处理，使Mock组FXI mRNA表达量为1。24 hours after transfection, cells were lysed and total RNA was extracted using the Novagen FastPure Cell/Tissue Total RNA Isolation Kit V2 (refer to the Novagen RC112-01 instructions). Reverse transcription was performed into cDNA using Takara PrimeScript RT Master Mix RR036Q. The qPCR primer sequence information is shown in the primer section of the sequence table at the end of the article. The human GAPDH gene was used as the internal reference gene, and the PCR reaction was performed using the CFX96 fluorescence quantitative PCR instrument from Bio-Rad, USA. In the experiment, the mock group was used as the control for normalization, so that the expression level of FXI mRNA in the mock group was 1.

2.3数据分析2.3 Data Analysis

PCR反应结束后，以参照基因作为标准并通过CFX96软件进行相对定量分析并通过GarphPad软件进行统计分析。如表1和图1所示，为dsRNA(裸核酸，无修饰)分子在HepG2细胞中的高通量筛选验证结果。After the PCR reaction, relative quantitative analysis was performed using the reference gene as a standard and statistical analysis was performed using CFX96 software and GarphPad software. As shown in Table 1 and Figure 1, the results of high-throughput screening and validation of dsRNA (naked nucleic acid, unmodified) molecules in HepG2 cells are shown.

表1：HepG2细胞中单剂量5nM高通量筛选结果


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


在HepG2细胞中进行的FXI dsRNA筛选发现了28条优选序列，保留了效果较好且序列同时靶向人和食蟹猴FXI的dsRNA分子作为候选序列。进一步，我们将多个候选阳参序列与我们的候选序列进行比较，表2和图2为候选序列和阳性对照序列在HepG2细胞中单剂量下筛选结果。Screening for FXI dsRNA in HepG2 cells revealed 28 optimal sequences. dsRNA molecules with high efficacy and targeting both human and cynomolgus macaque FXI were retained as candidate sequences. We further compared several candidate Yangshen sequences with our candidate sequence. Table 2 and Figure 2 show the results of single-dose screening of candidate sequences and positive control sequences in HepG2 cells.

表2：未修饰dsRNA在HepG2细胞中单剂量筛选

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

结果可见，7条候选阳参中，TJ632表现出最优的效果，而我们的多条候选序列表现出优效或者等效阳参TJ632的效果，例如3、5、8、9、24、26、50、70、82号dsRNA等。The results showed that among the 7 candidate Yang Shen, TJ632 showed the best effect, and many of our candidate sequences showed superior or equivalent effects to Yang Shen TJ632, such as dsRNA No. 3, 5, 8, 9, 24, 26, 50, 70, 82, etc.

实施例3、FXI-dsRNA的优化Example 3: Optimization of FXI-dsRNA

3.1抑制活性检测3.1 Inhibitory activity detection

为进一步确认优选的dsRNA分子，我们分别对上述优选序列采用两种不同的修饰基序，对候选序列不同位置进行氟代和甲氧基修饰组合。To further confirm the preferred dsRNA molecules, we used two different modification motifs on the above preferred sequences, and performed fluorination and methoxy modification combinations on different positions of the candidate sequences.

具体修饰选自以下的任意种：Specific modifications are selected from any of the following:

(1)修饰E(经E修饰的dsRNA以En进行指代，其中n为候选dsRNA分子的编号，例如3号dsRNA分子经E修饰后以E3进行指代)：(1) Modification of E (dsRNA modified with E is referred to as En, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as E3 after modification with E):

正义序列：从5’开始第7、9、10、11位为2’-氟代修饰的核苷酸，其他位点为2’-O-甲基修饰的核苷酸，并且5’端第1及第2个磷酸酯键通过硫代形成硫代磷酸酯键；例如，以N代表任意核糖核苷酸，则正义序列的修饰为：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm；Sense sequence: starting from the 5' end, the 7th, 9th, 10th, and 11th positions are 2'-fluoro modified nucleotides, and the other positions are 2'-O-methyl modified nucleotides, and the first and second phosphate bonds at the 5' end are thiolated to form phosphorothioate bonds; for example, with N representing any ribonucleotide, the modification of the sense sequence is: NmsNmsNmNmNmNmfNmNfNfNmNmNmNmNmNmNmNmsNmsNm;

反义序列：从5’开始第2、6、14、16位为2’-氟代修饰的核苷酸(换言之，从反义序列与正义链互补区的3’末端开始的第2、6、14、16位为2’-氟代修饰的核苷酸)，其他位点为2’-O-甲基修饰的核苷酸，并且5’端及3’端的第1及第2个磷酸之间通过硫代形成硫代磷酸酯键；例如以N代表任意核糖核苷酸，则反义序列的修饰为：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: the 2nd, 6th, 14th and 16th positions starting from the 5’ are 2’-fluoro-modified nucleotides (in other words, the 2nd, 6th, 14th and 16th positions starting from the 3’ end of the complementary region between the antisense sequence and the sense chain are 2’-fluoro-modified nucleotides), other sites are 2’-O-methyl-modified nucleotides, and the first and second phosphates at the 5’ and 3’ ends are connected by thiolation to form a phosphorothioate bond; for example, if N represents any ribonucleotide, the modification of the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm.

(2)修饰E-1(经E-1修饰的dsRNA以En-1进行指代，其中n为候选dsRNA分子的编号，例如3号dsRNA分子经E修饰后以E3-1进行指代)，(2) Modification E-1 (dsRNA modified with E-1 is referred to as En-1, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as E3-1 after modification with E),

修饰E-1与E1修饰的唯一区别在于E-1修饰的dsRNA反义链从5’端开始的第6位核苷酸为2’-O-甲基修饰的核苷酸而不是2′-氟代修饰，即，例如：The only difference between modification E-1 and E1 modification is that the sixth nucleotide from the 5' end of the antisense strand of the E-1 modified dsRNA is a 2'-O-methyl modified nucleotide instead of a 2'-fluoro modified nucleotide, i.e., for example:

正义序列的修饰为：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm；The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm;

反义序列的修饰为：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。The modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm.

在本申请的序列修饰中，s代表硫代磷酸酯键，m代表2′-O-甲基修饰，f代表2′-氟代修饰。In the sequence modifications of the present application, s represents a phosphorothioate bond, m represents a 2′-O-methyl modification, and f represents a 2′-fluoro modification.

此外，反义化合物ISIS-416858采用CN109797150A中公开的结构及修饰方式，即“5-10-5”的结构，侧翼各5个2′-O-甲氧基乙基修饰核苷酸，中间10个脱氧核苷酸，以及全硫代、甲基胞嘧啶替代胞嘧啶。In addition, the antisense compound ISIS-416858 adopts the structure and modification method disclosed in CN109797150A, namely the "5-10-5" structure, with 5 2'-O-methoxyethyl modified nucleotides on each flank, 10 deoxynucleotides in the middle, and all-thio and methylcytosine replacing cytosine.

转染细胞为HepG2细胞，其合成、转染、定量PCR检测步骤同实施例2。表3和图3为相对空白对照组的靶基因表达水平的平均值(空白对照组的mRNA相对表达水平设为1)。The transfected cells were HepG2 cells, and the synthesis, transfection, and quantitative PCR detection steps were the same as in Example 2. Table 3 and Figure 3 show the average target gene expression levels relative to the blank control group (the relative mRNA expression level of the blank control group was set to 1).

表3：修饰dsRNA在HepG2细胞中的单剂量(1nM)筛选
Table 3: Single-dose (1 nM) screening of modified dsRNA in HepG2 cells

可见，与经化学修饰后的阳性对照序列相比，我们的多条候选序列，如，E8、E14、E19、E24、E26、E50等序列依旧表现出等效或优效于最好的阳参ETJ632的结果。该结果再次证明，我们筛选得到的优选序列具有高效降低FXI基因核酸水平的潜能。As can be seen, compared to the chemically modified positive control sequence, many of our candidate sequences, such as E8, E14, E19, E24, E26, and E50, still showed equivalent or superior efficacy to the best Yangshen ETJ632. This result further demonstrates that the selected sequences have the potential to effectively reduce FXI gene nucleic acid levels.

实施例4GalNAc-dsRNA体外有效性检测Example 4 In vitro effectiveness detection of GalNAc-dsRNA

将候选的修饰dsRNA进行GalNAc偶联形成GAL-dsRNA复合物，即对各dsRNA进行G修饰。The candidate modified dsRNA is subjected to GalNAc coupling to form a GAL-dsRNA complex, that is, each dsRNA is subjected to G modification.

所述G修饰是指将L96缀合至dsRNA正义序列3’末端，包括GE修饰及GE-1修饰，其中：The G modification refers to conjugating L96 to the 3' end of the dsRNA sense sequence, including GE modification and GE-1 modification, wherein:

GE修饰如下所示，其中“-L96”表示使L96缀合至dsRNA正义序列3’末端：The GE modification is shown below, where "-L96" indicates that L96 is conjugated to the 3' end of the dsRNA sense sequence:

正义序列的修饰为：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96;

反义序列的修饰为：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；The modification of the antisense sequence is: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

经过GE修饰的dsRNA以GEn进行指代，其中n为候选dsRNA分子的编号，例如3号dsRNA分子经GE修饰后以GE3进行指代；The dsRNA modified by GE is referred to as GEn, where n is the number of the candidate dsRNA molecule, for example, dsRNA molecule No. 3 is referred to as GE3 after GE modification;

GE-1如下所示：The GE-1 is shown below:

正义序列的修饰为：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96;

反义序列的修饰为：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；The modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

同样，其中“-L96”表示L96通过磷酸酯键缀合至dsRNA正义序列3’末端，经过GE-1修饰的dsRNA以GEn-1进行指代，其中n为候选dsRNA分子的编号，例如3号dsRNA分子经GE-1修饰后以GE3-1进行指代。Similarly, "-L96" indicates that L96 is conjugated to the 3' end of the dsRNA sense sequence through a phosphate bond. The dsRNA modified with GE-1 is referred to as GEn-1, where n is the number of the candidate dsRNA molecule. For example, dsRNA molecule No. 3 is referred to as GE3-1 after modification with GE-1.

我们进一步在HepG2细胞中验证了经G修饰的候选复合物的活性。We further validated the activity of the G-modified candidate complexes in HepG2 cells.

4.1HepG2细胞中mRNA表达水平检测4.1 Detection of mRNA expression levels in HepG2 cells

检测步骤同实施例2，以人GAPDH基因为内参基因，使用的检测引物如文末序列表引物部分所示。表4为使用G修饰的dsRNA处理的HepG2细胞中mRNA水平相对未处理组的靶基因表达水平的平均值(未处理组的mRNA相对表达水平设为1)。其统计结果示于图4。The detection steps were the same as in Example 2, using the human GAPDH gene as the internal reference gene. The detection primers used are listed in the primer section of the sequence listing at the end of the article. Table 4 shows the average mRNA levels of the target gene expression levels in HepG2 cells treated with G-modified dsRNA relative to the untreated group (the relative mRNA expression level of the untreated group was set to 1). The statistical results are shown in Figure 4.

表4部分GalNAc偶联dsRNA在HepG2细胞中的IC50数据
Table 4 IC50 data of some GalNAc-coupled dsRNA in HepG2 cells

可见，GalNAc偶联dsRNA在HepG2细胞中的IC50均低于0.1nM,再次证明候选序列对靶基因的高效抑制活性。It can be seen that the IC50 of GalNAc-coupled dsRNA in HepG2 cells is lower than 0.1 nM, which once again proves the efficient inhibitory activity of the candidate sequence on the target gene.

实施例5体内有效性检测Example 5 In vivo effectiveness test

试验采用6～8周周龄的SPF级人源化FXI纯合子雄性小鼠(南模生物实验动物有限公司)，随机分组，每组6只，皮下单次注射给药，每组注射GE修饰的不同dsRNA，Blank组注射生理盐水。分别于给药后第7天和第13天采血，眼球后部放血收集血清用于FXI蛋白含量检测。The study used 6- to 8-week-old SPF-grade humanized FXI homozygous male mice (Shanghai Model Organism Laboratory Animal Co., Ltd.) and randomly divided them into groups of six. Each group received a single subcutaneous injection of different GE-modified dsRNAs, while the blank group received saline. Blood was collected from the back of the eye on days 7 and 13 after administration for serum analysis of FXI protein levels.

具体给药剂量如表5所示。The specific dosages are shown in Table 5.

表5 3mpk单次给药方案
Table 5 3mpk single dose regimen

5.1 FXI蛋白ELISA检测5.1 FXI protein ELISA detection

血清按1：500稀释后采用Abcam公司ab108834ELISA检测试剂盒检测各组血浆中残留FXI的含量，具体操作步骤参考ab108834ELISA检测说明书，通过酶标仪在450nm波长下检测吸光值，并用GraphPad软件进行分析，抑制率百分比是相对于未给药处理组的一个相对结果，结果如表6所示，图5则显示了各组血浆中以空白对照组做归一化处理后的FXI蛋白相对表达水平。After serum was diluted 1:500, the residual FXI content in the plasma of each group was detected using the Abcam ab108834 ELISA detection kit. For specific operating steps, please refer to the ab108834 ELISA test instructions. The absorbance value was measured at a wavelength of 450 nm using a microplate reader and analyzed using GraphPad software. The percentage inhibition rate is a relative result relative to the non-drug treatment group. The results are shown in Table 6. Figure 5 shows the relative expression level of FXI protein in the plasma of each group after normalization with the blank control group.

表6：3mpk单次给药后血浆中FXI蛋白的抑制率
Table 6: Inhibition rate of plasma FXI protein after a single dose of 3 mpk

可见，在3mpk单次给药中，候选的dsRNA分子给药组小鼠体内血浆中FXI的含量均显著低于空白对照组。在第13天时，GE26、GE50和GE70的抑制率高于80％以上，其中GE50表现出最高的抑制率92.8％。同时，在第13天时，所有给药组的抑制率均＞50％。该结果证明我们针对FXI设计的多条dsRNA分子能够有效抑制肝脏中FXI蛋白的表达分泌，是一种潜在的靶向FXI基因的治疗药物。As can be seen, after a single dose of 3 mpk, the plasma FXI levels in mice treated with the candidate dsRNA molecules were significantly lower than those in the blank control group. On day 13, the inhibition rates of GE26, GE50, and GE70 exceeded 80%, with GE50 exhibiting the highest inhibition rate of 92.8%. Furthermore, the inhibition rates in all treatment groups were >50% on day 13. These results demonstrate that the multiple dsRNA molecules designed to target FXI effectively inhibit the expression and secretion of FXI protein in the liver, making them potential therapeutic agents targeting the FXI gene.

实施例6不同配体的体内药效评价实验Example 6 In vivo efficacy evaluation experiment of different ligands

我们进一步在候选dsRNA中验证了不同配体偶联物的效果。表7为dsRNA序列信息。其使用LN-E05修饰或GN-E05修饰，其中LN-E05修饰同实施例4的GE-1修饰，GN-E05修饰具体如下所示：We further validated the effects of different ligand conjugates on candidate dsRNAs. Table 7 shows the dsRNA sequence information. These dsRNAs were modified with either LN-E05 or GN-E05. The LN-E05 modification was similar to the GE-1 modification described in Example 4. The specific modifications for GN-E05 are as follows:

正义序列的修饰为：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；The modification of the sense sequence is: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1;

反义序列的修饰为：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；The modification of the antisense sequence is: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm;

即GN-E05修饰与LN-E05修饰的区别仅在于缀合于正义链3’端的L96配体被更换为配体1，配体1通过硫代磷酸酯键与正义链的3’末端连接。That is, the only difference between the GN-E05 modification and the LN-E05 modification is that the L96 ligand conjugated to the 3’ end of the sense chain is replaced with ligand 1, and ligand 1 is connected to the 3’ end of the sense chain through a phosphorothioate bond.

表7合成的寡聚核苷酸
Table 7 Synthetic oligonucleotides

根据表7分组，各组按照3mpk皮下单次注射给药，分别于给药后不同时间点采血，血浆用于FXI蛋白含量检测。残留率百分比是相对于药前点的相对结果，结果如图6所示,在3mpk单次给药中，连接配体1的dsRNA对FXI蛋白的抑制率显著高于(使用配对T检验，p＜0.05)连接L96的dsRNA。Groups were divided according to Table 7 and administered a single subcutaneous injection of 3 mpk. Blood was collected at various time points after administration, and plasma was used for FXI protein analysis. The residual percentage is relative to the pre-drug dose. The results are shown in Figure 6. Following a single 3 mpk administration, the inhibition rate of FXI protein expression by dsRNA linked to ligand 1 was significantly higher (p<0.05 using a paired T-test) than that by dsRNA linked to L96.

配体1结构如式II所示：
The structure of ligand 1 is shown in Formula II:

其中波浪线表示与dsRNA的有义链的3’端通过硫代磷酸酯键连接。式II的分子是由两个化合物6及一个化合物8通过本领域常规原料及合成方法制备形成的三价GalNac缀合物。其中化合物6和8的合成方法如下所示：The wavy line indicates the connection to the 3' end of the sense strand of the dsRNA via a phosphorothioate bond. The molecule of Formula II is a trivalent GalNac conjugate prepared from two compounds 6 and one compound 8 using conventional raw materials and synthetic methods in the art. The synthetic methods of compounds 6 and 8 are as follows:

1、化合物6的合成
1. Synthesis of Compound 6

①化合物2的合成：①Synthesis of compound 2:

于三颈烧瓶中加入1.2g化合物1和12mL吡啶。搅拌溶解后加入1.2g DMTrCl后，氮气保护下室温搅拌1小时。向反应混合物中慢慢加入甲醇(0.6mL)淬灭过量的DMTrCl，室温搅拌15min后，再加入303.5mg NaHCO3。浓缩后得到粗品，加入DCM/H2O，搅拌溶解，并用水洗涤，无水硫酸钠干燥有机相并过滤。收集到的有机相在真空下浓缩得到化合物2的粗品，直接用于下一步反应。1.2 g of compound 1 and 12 mL of pyridine were added to a three-necked flask. After stirring and dissolving, 1.2 g of DMTrCl was added and stirred at room temperature for 1 hour under nitrogen protection. Methanol (0.6 mL) was slowly added to the reaction mixture to quench the excess DMTrCl. After stirring at room temperature for 15 minutes, 303.5 mg of NaHCO3 was added. The crude product was obtained after concentration. DCM/H2O was added, stirred to dissolve, and washed with water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product of compound 2, which was used directly in the next reaction.

②化合物3的合成：②Synthesis of compound 3:

向化合物2粗品中加入乙醇(24mL)，缓慢升温至50℃，搅拌溶解后再加入水合肼(0.75mL)，继续在50℃下搅拌过夜后，再降至室温，并在室温下继续搅拌半小时，此时逐渐析出大量白色固体。过滤，滤渣用乙醇洗涤。所得滤液减压浓缩，再复溶于DCM/H2O，分液，分出有机相，用饱和食盐水洗涤，无水硫酸钠干燥有机相并过滤。收集到的有机相在真空下浓缩得到化合物3粗品，直接用于下一步反应。Add ethanol (24 mL) to the crude compound 2, slowly heat to 50 ° C, stir and dissolve, then add hydrazine hydrate (0.75 mL), continue stirring at 50 ° C overnight, then cool to room temperature, and continue stirring at room temperature for half an hour, at which time a large amount of white solid gradually precipitates. Filter, and wash the filter residue with ethanol. The obtained filtrate is concentrated under reduced pressure, then redissolved in DCM/H2O, separated, and the organic phase is separated. It is washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The collected organic phase is concentrated under vacuum to give the crude compound 3, which is directly used in the next reaction.

③化合物5的合成：③Synthesis of compound 5:

向化合物3粗品中依次加入0.9g化合物4、DCM(24mL)、0.84mL Et3N和2.3gHBTU，氮气保护下室温搅拌2小时。加入9mL水淬灭反应，分出有机相，用饱和食盐水洗涤，无水硫酸钠干燥有机相并过滤。收集到的有机相在真空下浓缩并通过反相制备(75％ACN-H2O)纯化得到1.4g化合物5，三步收率44.2％。ESI-MS:m/z 1025.6[M+OAc-]-。To the crude compound 3, 0.9 g of compound 4, DCM (24 mL), 0.84 mL of Et3N, and 2.3 g of HBTU were added sequentially, and the mixture was stirred at room temperature under nitrogen for 2 hours. The reaction was quenched by adding 9 mL of water, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by reverse phase preparative (75% ACN-H2O) to afford 1.4 g of compound 5, with a three-step yield of 44.2%. ESI-MS: m/z 1025.6 [M + OAc - ] - .

④化合物6的合成：④Synthesis of compound 6:

将1.4g化合物5溶于DCM(14mL)中，置于0±2℃中冷却。加入524.7mg 2-氰乙基-N,N,N',N'-四异丙基亚磷酰二胺，随后加入81.3mg 1H-tetrazole。反应液先在0±2℃中搅拌15min后，再升至室温，继续搅拌2小时。将反应液在0±2℃中冷却，加入14mL 5％NaHCO3淬灭，分出有机相，在0±2℃中用饱和食盐水(1x14mL)洗涤，无水硫酸钠干燥有机相并过滤。收集到的有机相在真空下浓缩，所得粗品复溶于DCM/MTBE,小心滴加至剧烈搅拌的正庚烷(hept.)溶液中，此时瓶壁和瓶底逐渐析出大量粘稠油状物，静置10min后，倾出上清液。粗品用EA:hept.＝10:1～2:1洗脱剂层析，浓缩及干燥后得560mg白色固体粉末，收率32％，磷谱纯度98.19％。ESI-MS:m/z 1225.4[M+OAc-]-。Dissolve 1.4 g of compound 5 in DCM (14 mL) and cool at 0±2°C. Add 524.7 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, followed by 81.3 mg of 1H-tetrazole. Stir the reaction mixture at 0±2°C for 15 minutes, then warm to room temperature and continue stirring for 2 hours. Cool the reaction mixture at 0±2°C and quench with 14 mL of 5% NaHCO3. Separate the organic phase, wash with saturated brine (1 x 14 mL) at 0±2°C, dry over anhydrous sodium sulfate, and filter. Concentrate the collected organic phase under vacuum, and the resulting crude product is redissolved in DCM/MTBE and carefully added dropwise to a vigorously stirred solution of heptane. A large amount of viscous oil will gradually precipitate from the walls and bottom of the flask. After standing for 10 minutes, decanter the supernatant. The crude product was chromatographed using EA:hept. = 10:1 to 2:1 as eluent, concentrated, and dried to give 560 mg of a white solid powder with a yield of 32% and a phosphorus purity of 98.19%. ESI-MS: m/z 1225.4 [M + OAc − ] − .

2、化合物8的合成
2. Synthesis of Compound 8

①化合物7的合成：①Synthesis of compound 7:

将2g化合物5溶于DCM(20mL)，依次加入248.4mg丁二酸酐和0.69mL Et3N。最后加入25.3mg DMAP，室温搅拌16小时后，HPLC监测原料剩余较多，补加310.4mg丁二酸酐和0.69mL Et3N。最后加入25.3mg DMAP，继续室温搅拌36小时。将反应液在0±2℃中冷却，向反应中加入冰水，再加入DCM，最后加入40mL 1％HOAc水溶液，搅拌10min后分出有机相，依次用1％HOAc水溶液及水洗涤，无水硫酸钠干燥有机相并过滤。收集到的有机相在真空下浓缩，得到粗品。粗品用DCM:MeOH＝70:1～20:1洗脱剂层析，浓缩及干燥后得0.9g白色固体粉末，收率40.7％，丁二酸检测无残留，丁二酸酐残留为0.26％。ESI-MS:m/z 1065.7[M-H]-。2 g of compound 5 was dissolved in DCM (20 mL), followed by the addition of 248.4 mg of succinic anhydride and 0.69 mL of Et3N. Finally, 25.3 mg of DMAP was added. After stirring at room temperature for 16 hours, HPLC analysis indicated a significant residual starting material. 310.4 mg of succinic anhydride and 0.69 mL of Et3N were added. Finally, 25.3 mg of DMAP was added, and stirring continued at room temperature for 36 hours. The reaction mixture was cooled to 0±2°C, ice water was added, followed by DCM, and finally 40 mL of 1% aqueous HOAc solution. After stirring for 10 minutes, the organic phase was separated and washed sequentially with 1% aqueous HOAc solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to yield the crude product. The crude product was chromatographed using a DCM:MeOH ratio of 70:1 to 20:1 eluent. After concentration and drying, 0.9 g of a white solid powder was obtained, yielding 40.7%. No succinic acid residue was detected, and the residual succinic anhydride was 0.26%. ESI-MS: m/z 1065.7[M - H] - .

②化合物8的合成：②Synthesis of compound 8:

于三颈烧瓶中加入300mg的化合物7，72.7mg的DIPEA，106mg的HBTU和10mL的乙腈，在25℃下搅拌10分钟后，加入650mg的固相载体PS，在25℃下继续搅拌24小时。反应结束后过滤，滤饼用乙腈洗涤，收集滤饼在真空下浓缩除去溶剂得到850mg的固体。从中取700mg加入三颈烧瓶中，再加入1.74g的乙酸酐，4.15mg的DMAP，103mg的三乙胺和10mL的吡啶，在25℃下搅拌4小时。反应结束后过滤，滤饼依次用乙腈，甲醇和乙腈洗涤，收集滤饼在真空下浓缩除去溶剂得到750mg的化合物8。载量的测定值为254.82μmol/g。To a three-necked flask, 300 mg of compound 7, 72.7 mg of DIPEA, 106 mg of HBTU, and 10 mL of acetonitrile were added. After stirring at 25°C for 10 minutes, 650 mg of solid support PS was added and stirring continued at 25°C for 24 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with acetonitrile, and the filter cake was collected and concentrated under vacuum to remove the solvent, yielding 850 mg of solid. 700 mg of the solid was added to a three-necked flask, followed by 1.74 g of acetic anhydride, 4.15 mg of DMAP, 103 mg of triethylamine, and 10 mL of pyridine, and stirred at 25°C for 4 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with acetonitrile, methanol, and acetonitrile, and the filter cake was collected and concentrated under vacuum to remove the solvent, yielding 750 mg of compound 8. The loading was determined to be 254.82 μmol/g.

3、与配体1连接的寡核苷酸序列的制备3. Preparation of oligonucleotide sequence linked to ligand 1

将化合物8顺次与2个化合物6，或将3个化合物6顺次进行反应，通过常规的固相合成循环步骤(脱保护，偶联，氧化和加帽)，即可得到连接至固相载体的配体1的前体(或中间体)。进一步使用对应的2’-修饰单体进行固相合成循环反应。反应完毕后，通过氨解将整个合成的分子从固相载体上解离下来，即得到与配体1连接的寡核苷酸序列。Compound 8 is sequentially reacted with two or three compounds 6, and through conventional solid-phase synthesis cycles (deprotection, coupling, oxidation, and capping), a precursor (or intermediate) of ligand 1 attached to a solid support is obtained. Further solid-phase synthesis cycles are performed using the corresponding 2'-modified monomers. After completion of the reaction, the entire synthesized molecule is dissociated from the solid support by aminolysis, yielding the oligonucleotide sequence attached to ligand 1.

实施例7不同修饰基序的体外、体内药效评价实验Example 7 In vitro and in vivo efficacy evaluation experiments of different modified motifs

我们进一步在碱基序列同双链编号50号和双链编号26号的dsRNA中验证了不同修饰基序的的效果。具体使用的分子具体如表8所示。可见表8中的分子均在正义链3’末端通过硫代磷酸酯键连接配体1，同时反义链5’末端核苷酸使用了5’-vp修饰。We further validated the effects of different modification motifs in dsRNAs with the same base sequence as double-strand number 50 and double-strand number 26. The specific molecules used are listed in Table 8. As can be seen, all molecules in Table 8 were linked to Ligand 1 via a phosphorothioate bond at the 3' end of the sense strand, while the 5' terminal nucleotide of the antisense strand was modified with 5'-vp.

表8：合成的寡聚核苷酸
Table 8: Synthetic oligonucleotides

表8中各修饰基序如下所示：The modified motifs in Table 8 are as follows:

GN-E20VP：GN-E20VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E04VP：GN-E04VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

GN-E05VP：GN-E05VP:

正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1

反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm.

GN-E05修饰同实施例6，其与GN-E05VP修饰的差异仅在于GN-E05在反义链5’末端没有乙烯基磷酸酯修饰。The modification of GN-E05 was the same as in Example 6. The only difference between it and the GN-E05VP modification was that GN-E05 did not have a vinyl phosphate modification at the 5' end of the antisense strand.

7.1不同修饰基序体外药效评价7.1 In vitro efficacy evaluation of different modified motifs

首先，我们在体外HepG2细胞水平验证了表8中不同修饰方式对药效的影响，实验方法参照实施例4。表4为各处理组mRNA水平相对未处理组的靶基因表达水平的平均值(未处理组的mRNA相对表达水平设为1)。体外试验为不同批次的独立重复结果，其统计结果示于表9、表10。First, we verified the effects of the different modifications listed in Table 8 on drug efficacy in vitro using HepG2 cells. The experimental methods were similar to those in Example 4. Table 4 shows the average mRNA levels of each treated group relative to the target gene expression levels of the untreated group (the relative mRNA expression level of the untreated group was set to 1). The in vitro experiments were performed independently in different batches, and the statistical results are shown in Tables 9 and 10.

表9 5VP修饰dsRNA在HepG2细胞中的IC50数据
Table 9 IC50 data of 5VP modified dsRNA in HepG2 cells

表10不同基序修饰dsRNA在HepG2细胞中的IC50数据
Table 10 IC50 data of different motif-modified dsRNA in HepG2 cells

体外结果如表9、图7所示,可见5-VP的修饰有助于药效的进一步提高。同时，在配体和碱基序列均相同的情况下，GN-E05VP、GN-E20VP的修饰基序显著优于GN-E04VP(表10所示)，进一步地，GN-E20VP的修饰基序亦显著优于GN-E05VP。The in vitro results, shown in Table 9 and Figure 7, demonstrate that modification of 5-VP further enhances its efficacy. Furthermore, given the same ligand and base sequences, the modified motifs of GN-E05VP and GN-E20VP significantly outperformed those of GN-E04VP (shown in Table 10). Furthermore, the modified motif of GN-E20VP also significantly outperformed that of GN-E05VP.

7.2不同修饰基序体内药效评价7.2 In vivo efficacy evaluation of different modified motifs

试验方案参照实施例5，参照表8中的各组按照1mpk皮下单次注射给药具有不同修饰的50号dsRNA。分别于给药后第7、14、21、28天采血，血浆用于FXI蛋白含量检测。残留率百分比是相对于药前点的相对结果，结果如表11、图8所示。The experimental protocol followed that of Example 5. For each group, as shown in Table 8, dsRNA No. 50 with various modifications was administered as a single subcutaneous injection at 1 mpk. Blood was collected on days 7, 14, 21, and 28 after administration, and plasma was assayed for FXI protein content. The residual percentages are relative to the pre-drug dose, as shown in Table 11 and Figure 8.

表11：1mpk单次给药后血浆中FXI蛋白的残留率
Table 11: Residual rate of FXI protein in plasma after single administration of 1 mpk

从修饰后的50号的分子结果可见，在1mpk单次给药中，dsRNA碱基序列相同的情况下，E05VP、E20VP修饰基序优于E04VP(P＜0.05)，且E20VP进一步优于E05VP(P＜0.05)。同时，结果显示5’-VP修饰有助于药效的进一步提高。Molecular results from the modified sample 50 revealed that, at a single dose of 1 mpk, with identical dsRNA sequences, the E05VP and E20VP modified motifs outperformed E04VP (P < 0.05), with E20VP further outperforming E05VP (P < 0.05). Furthermore, the results suggest that 5'-VP modification can further enhance drug efficacy.

进一步我们在体内也比较了26号分子不同修饰基序(如表8所示)的药效。分别于给药后第7、14天采血，血浆用于FXI蛋白含量检测。残留率百分比是相对于药前点的相对结果，结果如表12所示。We further compared the in vivo efficacy of molecule 26 with different modified motifs (as shown in Table 8). Blood samples were collected on days 7 and 14 after dosing, and plasma was used to measure FXI protein levels. The residual percentage is relative to the predrug concentration, as shown in Table 12.

表12：1mpk单次给药后血浆中FXI蛋白的残留率
Table 12: Residual rate of FXI protein in plasma after single administration of 1 mpk

在26号分子上再一次证明了E05VP、E20VP药效优于E04VP，且E20VP基序药效是其中最优的。此外，5’-VP的修饰有助于药效的进一步提高。Molecule 26 once again demonstrated that E05VP and E20VP were more effective than E04VP, with the E20VP motif being the most effective. Furthermore, modification of the 5'-VP motif further enhanced efficacy.

本申请以上实施例中使用的序列示于如下序列表中。应当理解，以下序列仅为本申请实施方案的示例性序列，而非对本申请方案的任何限制。以下序列表中的核酸序列可表示DNA序列或RNA序列，当其表示RNA序列时，其中的“T”代表尿苷。并且，在RNA的语境下，如非特别说明“T”和“U”均指代尿嘧啶或尿苷。The sequences used in the above examples of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of this application and do not limit the present invention in any way. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When they represent RNA sequences, "T" represents uridine. Furthermore, in the context of RNA, unless otherwise specified, "T" and "U" both refer to uracil or uridine.

序列表：





Sequence Listing:





Claims (31)

一种通过RNAi抑制细胞中因子11(FXI)基因表达的核酸分子，其包含彼此互补的正义序列和反义序列或由彼此互补的正义序列和反义序列组成，且所述反义序列包含与FXI基因mRNA互补的多核苷酸序列，其中，A nucleic acid molecule for inhibiting the expression of factor 11 (FXI) gene in a cell by RNAi, comprising or consisting of a sense sequence and an antisense sequence that are complementary to each other, and the antisense sequence comprises a polynucleotide sequence that is complementary to FXI gene mRNA, wherein: 当所述反义序列与所述FXI基因mRNA以最大互补率杂交时，由所述反义序列和FXI基因mRNA序列中互补配对的碱基对组成互补区1；When the antisense sequence hybridizes with the FXI gene mRNA at a maximum complementarity rate, complementary region 1 is formed by complementary base pairs in the antisense sequence and the FXI gene mRNA sequence; 当所述反义序列与所述正义序列以最大互补率杂交时，由所述反义序列和所述正义序列中互补配对的碱基对组成互补区2；When the antisense sequence hybridizes with the sense sequence at a maximum complementarity rate, complementary region 2 is formed by complementary base pairs in the antisense sequence and the sense sequence; 其中互补区1与互补区2具有至少18、19或20个相同的碱基对，wherein complementary region 1 and complementary region 2 have at least 18, 19 or 20 identical base pairs, 所述互补区1的碱基对数量为18至30bp，并且所述互补区1包含FXI基因mRNA序列中：The complementary region 1 has a base pair number of 18 to 30 bp, and the complementary region 1 includes the following in the FXI gene mRNA sequence: 418至437位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 418 to 437 or 18 or 19 consecutive nucleotides therein, 1141至1160位的核苷酸或其中的连续18或19个核苷酸；Nucleotides 1141 to 1160 or 18 or 19 consecutive nucleotides therein; 363至382位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 363 to 382 or 18 or 19 consecutive nucleotides therein, 372至391位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 372 to 391 or 18 or 19 consecutive nucleotides therein, 411至430位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 411 to 430 or 18 or 19 consecutive nucleotides therein, 421至440位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 421 to 440 or 18 or 19 consecutive nucleotides therein, 422至441位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 422 to 441 or 18 or 19 consecutive nucleotides therein, 425至444位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 425 to 444 or 18 or 19 consecutive nucleotides therein, 465至484位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 465 to 484 or 18 or 19 consecutive nucleotides therein, 470至489位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 470 to 489 or 18 or 19 consecutive nucleotides therein, 471至490位的核苷酸或其中的连续18或19个核苷酸、Nucleotides 471 to 490 or 18 or 19 consecutive nucleotides therein, 518至537位的核苷酸或其中的连续18或19个核苷酸、或Nucleotides 518 to 537 or 18 or 19 consecutive nucleotides therein, or 其中FXI基因mRNA序列中核苷酸的位点编号为参比序列SEQ ID NO：244中对应核苷酸的编号。The site number of the nucleotide in the FXI gene mRNA sequence is the number of the corresponding nucleotide in the reference sequence SEQ ID NO: 244. 根据权利要求1的核酸分子，其中所述互补区1的碱基对数量为20、21、22或23bp。The nucleic acid molecule according to claim 1, wherein the number of base pairs of said complementary region 1 is 20, 21, 22 or 23 bp. 根据权利要求1或2的核酸分子，其中所述互补区2的碱基对数量为21bp。The nucleic acid molecule according to claim 1 or 2, wherein the number of base pairs of the complementary region 2 is 21 bp. 根据权利要求1至3中任一项所述的核酸分子，其中所述互补区1与互补区2具有20或21个相同的碱基对。The nucleic acid molecule according to any one of claims 1 to 3, wherein the complementary region 1 and the complementary region 2 have 20 or 21 identical base pairs. 根据权利要求1至4任一项所述的核酸分子，其中所述正义序列长度为21nt，所述反义序列长度为23nt。The nucleic acid molecule according to any one of claims 1 to 4, wherein the sense sequence is 21 nt in length and the antisense sequence is 23 nt in length. 根据权利要求1至5中任一项所述的核酸分子，其中所述正义序列和/或反义序列在所述互补区2之外还包含1至2个突出端核苷酸。The 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 overhang nucleotides outside the complementary region 2. 根据权利要求6所述的核酸分子，其中所述突出端核苷酸为2个且位于反义序列中，与所述互补区2的5’末端相邻，所述正义序列不包含突出端核苷酸。The nucleic acid molecule according to claim 6, wherein the overhanging nucleotides are 2 and are located in the antisense sequence, adjacent to the 5' end of the complementary region 2, and the sense sequence does not contain the overhanging nucleotides. 根据权利要求1至7中任一项所述的核酸分子，其中反义序列在互补区1以外的区域仅包含0、1或2个核苷酸，正义序列在互补区2以外仅包含0、1或2个核苷酸。The nucleic acid molecule according to any one of claims 1 to 7, wherein the antisense sequence comprises only 0, 1 or 2 nucleotides outside the complementary region 1, and the sense sequence comprises only 0, 1 or 2 nucleotides outside the complementary region 2. 根据权利要求1至8中任一项所述的核酸分子，其中互补区2的3’末端为AU碱基对。The nucleic acid molecule according to any one of claims 1 to 8, wherein the 3' end of complementary region 2 is an AU base pair. 根据权利要求1至9中任一项所述的核酸分子，其中所述FXI基因为人FXI基因或食蟹猴FXI基因。The nucleic acid molecule according to any one of claims 1 to 9, wherein the FXI gene is a human FXI gene or a cynomolgus monkey FXI gene. 根据权利要求10的核酸分子，其中所述mRNA包含如SEQ ID NO：244所示的多核苷酸序列。The nucleic acid molecule according to claim 10, wherein the mRNA comprises a polynucleotide sequence as shown in SEQ ID NO: 244. 根据权利要求1至11中任一项所述的核酸分子，其中所述反义序列包含或为如SEQ ID NO：153、177、152、122、123、126、129、130、131、136、137、138、143、149、150、200、201或213所示的多核苷酸序列。A nucleic acid molecule according to any one of claims 1 to 11, wherein the antisense sequence comprises or is a polynucleotide sequence as shown in SEQ ID NO: 153, 177, 152, 122, 123, 126, 129, 130, 131, 136, 137, 138, 143, 149, 150, 200, 201 or 213. 根据权利要求1至12中任一项所述的核酸分子，其中：The nucleic acid molecule according to any one of claims 1 to 12, wherein: 所述正义序列包含或为如SEQ ID NO：34所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：153所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 34, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 153; 所述正义序列包含或为如SEQ ID NO：58所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：177所示的多核苷酸序列；所述正义序列包含或为如SEQ ID NO：33所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：152所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 58, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 177; the sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 33, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 152; 所述正义序列包含或为如SEQ ID NO：3所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：122所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 3, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 122; 所述正义序列包含或为如SEQ ID NO：4所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：123所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 4, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 123; 所述正义序列包含或为如SEQ ID NO：7所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：126所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 7, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 126; 所述正义序列包含或为如SEQ ID NO：10所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：129所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 10, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 129; 所述正义序列包含或为如SEQ ID NO：11所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：130所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 11, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 130; 所述正义序列包含或为如SEQ ID NO：12所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：131所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 12, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 131; 所述正义序列包含或为如SEQ ID NO：17所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：136所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 17, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 136; 所述正义序列包含或为如SEQ ID NO：18所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：137所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 18, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 137; 所述正义序列包含或为如SEQ ID NO：19所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：138所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 19, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 138; 所述正义序列包含或为如SEQ ID NO：24所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：143所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 24, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 143; 所述正义序列包含或为如SEQ ID NO：30所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：149所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 30, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 149; 所述正义序列包含或为如SEQ ID NO：31所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：150所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 31, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 150; 所述正义序列包含或为如SEQ ID NO：81所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：200所示的多核苷酸序列；The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 81, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 200; 所述正义序列包含或为如SEQ ID NO：82所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：201所示的多核苷酸序列；或The sense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 82, and the antisense sequence comprises or is the polynucleotide sequence shown in SEQ ID NO: 201; or 所述正义序列包含或为如SEQ ID NO：94所示的多核苷酸序列，且所述反义序列包含或为如SEQ ID NO：213所示的多核苷酸序列。The sense sequence includes or is a polynucleotide sequence as shown in SEQ ID NO: 94, and the antisense sequence includes or is a polynucleotide sequence as shown in SEQ ID NO: 213. 根据权利要求1至13中任一项所述的核酸分子，其为dsRNA或shRNA。The nucleic acid molecule according to any one of claims 1 to 13, which is a dsRNA or shRNA. 根据权利要求14的核酸分子，其为siRNA。The nucleic acid molecule according to claim 14, which is siRNA. 根据权利要求1至15中任一项所述的核酸分子，其中一个或多个核苷酸经过化学修饰。The nucleic acid molecule according to any one of claims 1 to 15, wherein one or more nucleotides are chemically modified. 根据权利要求1至16中任一项所述的核酸分子，其中所述化学修饰选自如下的任一种或多种：The nucleic acid molecule according to any one of claims 1 to 16, wherein the chemical modification is selected from any one or more of the following: 锁核酸修饰、开环或非锁核酸修饰、2′-甲氧基乙基修饰、2′-O-甲基修饰、2′-O-烯丙基修饰、2’-C-烷基修饰、2′-C-烯丙基修饰、2′-氟代修饰、2′-脱氧修饰、硫代磷酸酯键修饰、2’-氨基-修饰、吗啉基修饰、氨基磷酸酯修饰、甲基膦酸酯修饰、四氢吡喃修饰、1,5-脱水己糖醇修饰、5’-乙烯基磷酸酯修饰、以及环己烯基修饰；Locked nucleic acid modification, open ring or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-alkyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, phosphorothioate bond modification, 2′-amino-modification, morpholino modification, phosphoramidate modification, methylphosphonate modification, tetrahydropyranyl modification, 1,5-anhydrohexitol modification, 5′-vinyl phosphate modification, and cyclohexenyl modification; 优选地，所述核酸分子正义链的7、9-12位核苷酸包含2′-氟代修饰和/或反义链2、14、16位核苷酸包含2′-氟代修饰，进一步优选地，其余核苷酸包含2′-O-甲基修饰；Preferably, nucleotides 7, 9-12 of the sense strand of the nucleic acid molecule contain 2′-fluoro modifications and/or nucleotides 2, 14, and 16 of the antisense strand contain 2′-fluoro modifications, and further preferably, the remaining nucleotides contain 2′-O-methyl modifications; 优选地，所述核酸分子正义链的7、9-11位核苷酸包含2′-氟代修饰和/或反义链2、14、16位核苷酸包含2′-氟代修饰；进一步优选地，其余核苷酸包含2′-O-甲基修饰；Preferably, nucleotides 7, 9-11 of the sense strand of the nucleic acid molecule contain 2′-fluoro modifications and/or nucleotides 2, 14, and 16 of the antisense strand contain 2′-fluoro modifications; further preferably, the remaining nucleotides contain 2′-O-methyl modifications; 优选地，所述核酸分子正义链的7、9-11位核苷酸包含2′-氟代修饰，反义链2、6、14、16位核苷酸包含2′-氟代修饰；进一步优选地，其余核苷酸包含2′-O-甲基修饰；Preferably, nucleotides 7, 9-11 of the sense strand of the nucleic acid molecule contain 2'-fluoro modifications, and nucleotides 2, 6, 14, and 16 of the antisense strand contain 2'-fluoro modifications; further preferably, the remaining nucleotides contain 2'-O-methyl modifications; 优选地，所述核酸分子包含选自如下任一种基序：Preferably, the nucleic acid molecule comprises any one of the following motifs: (1)正义序列：NmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm，反义序列：NmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm，(1) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm, (2)正义序列：NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmNfNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm，和(2) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm, and (3)正义序列：NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm，(3) Sense sequence: NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmNfNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm, 其中，Nm表示经2’-O-甲基修饰的核糖核苷酸，Nf表示经2’-氟代修饰的核糖核苷酸；Wherein, Nm represents a 2'-O-methyl modified ribonucleotide, and Nf represents a 2'-fluoro modified ribonucleotide; 进一步优选地，在正义序列5’的第一个和第二核苷酸之间，以及第二个和第三个核苷酸之间，倒数第一个和第二个之间，以及倒数第二个和第三个之间包括至少一个硫代磷酸酯修饰；在反义序列5’的第一个和第二核苷酸之间，第二个和第三个核苷酸之间，倒数第一个和第二个之间，以及倒数第二个和第三个之间包括至少一个硫代磷酸酯修饰。Further preferably, at least one thiophosphate modification is included between the first and second nucleotides, and between the second and third nucleotides, between the first to last and the second to last, and between the second to last and the third nucleotides of the 5' sense sequence; and at least one thiophosphate modification is included between the first and second nucleotides, between the second and third nucleotides, between the first to last and the second to last, and between the second to last and the third nucleotides of the 5' antisense sequence. 根据权利要求17所述的核酸分子，其中所述核酸分子包含选自如下任一种基序：The nucleic acid molecule according to claim 17, wherein the nucleic acid molecule comprises any one of the following motifs: (1)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (2)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (3)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (4)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (5)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；和(5) sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; and (6)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmsNmsNm, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (7)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(7) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; (8)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(8) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (9)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms，反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(9) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms, antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; 其中，Nm表示经2’-O-甲基修饰的核糖核苷酸，Nf表示经2’-氟代修饰的核糖核苷酸，s表示硫代磷酸酯修饰。Wherein, Nm represents a 2'-O-methyl modified ribonucleotide, Nf represents a 2'-fluoro modified ribonucleotide, and s represents a phosphorothioate modified ribonucleotide. 根据权利要求1至18中任一项所述的核酸分子，其连接至少一个去唾液酸糖蛋白受体(ASGPR)配体。The nucleic acid molecule according to any one of claims 1 to 18, which is linked to at least one asialoglycoprotein receptor (ASGPR) ligand. 根据权利要求19的核酸分子，所述ASGPR配体连接于所述正义序列的5’末端或3’末端。According to the nucleic acid molecule of claim 19, the ASGPR ligand is linked to the 5' end or the 3' end of the sense sequence. 根据权利要求19或20的核酸分子，所述ASGPR配体是通过二价或三价支链结构连接的一个或多个GalNAc衍生物。According to the nucleic acid molecule of claim 19 or 20, the ASGPR ligand is one or more GalNAc derivatives connected by a divalent or trivalent branched structure. 根据权利要求21的核酸分子，其中所述GalNac衍生物为L96或配体1，所述L96的结构如下所示：
The nucleic acid molecule according to claim 21, wherein the GalNac derivative is L96 or Ligand 1, and the structure of L96 is as follows:
所述配体1结构如下所示：
The structure of ligand 1 is shown below:
其中式I和式II中的分别代表与所述核酸分子的正义序列或反义序列的3’端连接；优选地，通过磷酸二酯键或硫代磷酸二酯键与3’羟基连接；wherein the Respectively represent connection to the 3' end of the sense sequence or antisense sequence of the nucleic acid molecule; preferably, connected to the 3' hydroxyl group through a phosphodiester bond or a phosphorothioate diester bond; 进一步优选地，所述核酸分子具有如下任一的修饰基序：Further preferably, the nucleic acid molecule has any of the following modification motifs: (1)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(1) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (2)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(2) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmsNmsNm; (3)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(3) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (4)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(4) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (5)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；(5) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-L96; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm; (6)正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；反义序列：NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。(6) Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-ligand 1; antisense sequence: NmsNfsNmNmNmNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmsNmsNm. 根据权利要求1-22的核酸分子，所述核酸分子包含在反义链5’端的磷酸酯或磷酸酯模拟物修饰；优选地，所述5’磷酸酯模拟物为5’-VP修饰；The nucleic acid molecule according to claims 1-22, wherein the nucleic acid molecule comprises a phosphate or phosphate mimic modification at the 5' end of the antisense strand; preferably, the 5' phosphate mimic is a 5'-VP modification; 进一步优选地，所述核酸分子具有如下任一的修饰基序：Further preferably, the nucleic acid molecule has any of the following modification motifs: GN-E20VP：GN-E20VP: 正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-配体1；Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1; 反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; GN-E04VP：GN-E04VP: 正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1; 反义序列：vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm；Antisense sequence: vp-NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm; GN-E05VP：GN-E05VP: 正义序列：NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNms-配体1；Sense sequence: NmsNmsNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-ligand 1; 反义序列：vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm。Antisense sequence: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm. 第二核酸分子，其在细胞中可转录为dsRNA或shRNA前体，所述dsRNA或shRNA为根据权利要求1-23任一项的核酸分子。The second nucleic acid molecule can be transcribed into a dsRNA or shRNA precursor in a cell, wherein the dsRNA or shRNA is a nucleic acid molecule according to any one of claims 1 to 23. 病毒颗粒，其包含根据权利要求24的第二核酸分子的多核苷酸序列。A viral particle comprising the polynucleotide sequence of the second nucleic acid molecule according to claim 24. 细胞，其包含根据权利要求24的第二核酸分子的多核苷酸序列。A cell comprising the polynucleotide sequence of the second nucleic acid molecule according to claim 24. 将根据权利要求1至23中任一项所述的核酸分子或其盐用于抑制因子11的表达的用途。Use of the nucleic acid molecule or a salt thereof according to any one of claims 1 to 23 for inhibiting Factor 11 expression. 将根据权利要求1至23中任一项所述的核酸分子或其盐用于制备抑制受试者体内FXI表达的药物的用途。Use of the nucleic acid molecule or a salt thereof according to any one of claims 1 to 23 for preparing a medicament for inhibiting FXI expression in a subject. 药物组合物，所述组合物包含权利要求1至23中任一项所述的核酸分子或其盐、根据权利要求24的第二核酸分子、根据权利要求25的的病毒颗粒、或根据权利要求26的细胞。A pharmaceutical composition comprising the nucleic acid molecule or a salt thereof according to any one of claims 1 to 23, the second nucleic acid molecule according to claim 24, the viral particle according to claim 25, or the cell according to claim 26. 将根据权利要求1至23中任一项所述的核酸分子或其盐、根据权利要求24的第二核酸分子、根据权利要求25的的病毒颗粒、或根据权利要求26的细胞或根据权利要求29所述的药物组合物用于制备预防或治疗受试者的血栓栓塞性并发症或凝血紊乱的药物的用途，所述血栓栓塞性并发症优选自深静脉血栓形成、肺栓塞、心肌梗塞和中风中的一种或多种。Use of a nucleic acid molecule or a salt thereof according to any one of claims 1 to 23, a second nucleic acid molecule according to claim 24, a viral particle according to claim 25, or a cell according to claim 26, or a pharmaceutical composition according to claim 29 for the preparation of a medicament for preventing or treating thromboembolic complications or coagulation disorders in a subject, wherein the thromboembolic complications are preferably one or more selected from deep vein thrombosis, pulmonary embolism, myocardial infarction and stroke. 治疗或预防受试者的血栓栓塞性并发症或凝血紊乱的方法，其中所述血栓栓塞性并发症优选自深静脉血栓形成、肺栓塞、心肌梗塞和中风中的一种或多种，所述方法包含向有需要的受试者施用有效量的根据权利要求1至23中任一项所述的核酸分子或其盐、根据权利要求24的第二核酸分子、根据权利要求25的病毒颗粒、根据权利要求26的细胞或根据权利要求29所述的药物组合物。A method for treating or preventing thromboembolic complications or coagulation disorders in a subject, wherein the thromboembolic complications are preferably selected from one or more of deep vein thrombosis, pulmonary embolism, myocardial infarction and stroke, the method comprising administering to a subject in need thereof an effective amount of a nucleic acid molecule or a salt thereof according to any one of claims 1 to 23, a second nucleic acid molecule according to claim 24, a viral particle according to claim 25, a cell according to claim 26 or a pharmaceutical composition according to claim 29.
PCT/CN2025/074764 2024-01-26 2025-01-24 Nucleic acid molecule inhibiting f11 gene expression Pending WO2025157271A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202580000106.8A CN120712356A (en) 2024-01-26 2025-01-24 A nucleic acid molecule for inhibiting F11 gene expression

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202410114595 2024-01-26
CN202410114595.7 2024-01-26
CN202410568807.9 2024-05-09
CN202410568807 2024-05-09

Publications (1)

Publication Number Publication Date
WO2025157271A1 true WO2025157271A1 (en) 2025-07-31

Family

ID=96544504

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2025/074764 Pending WO2025157271A1 (en) 2024-01-26 2025-01-24 Nucleic acid molecule inhibiting f11 gene expression

Country Status (2)

Country Link
CN (1) CN120712356A (en)
WO (1) WO2025157271A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025232840A1 (en) * 2024-05-09 2025-11-13 石药集团中奇制药技术（石家庄）有限公司 Galnac derivative and oligonucleotide conjugate thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109797150A (en) * 2008-10-15 2019-05-24 Ionis制药公司 The adjusting of factor 11 expression
CN113227376A (en) * 2019-05-22 2021-08-06 苏州瑞博生物技术股份有限公司 Nucleic acid, pharmaceutical composition and conjugate, preparation method and application
WO2022028457A1 (en) * 2020-08-04 2022-02-10 上海拓界生物医药科技有限公司 Sirna for inhibiting expression of blood coagulation factor xi, and composition and medical use thereof
CN115677810A (en) * 2021-07-23 2023-02-03 苏州瑞博生物技术股份有限公司 Double-stranded oligonucleotide, composition and conjugate containing double-stranded oligonucleotide, preparation method and use
WO2023109945A1 (en) * 2021-12-16 2023-06-22 上海拓界生物医药科技有限公司 Dsrna, and preparation method therefor and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109797150A (en) * 2008-10-15 2019-05-24 Ionis制药公司 The adjusting of factor 11 expression
CN113227376A (en) * 2019-05-22 2021-08-06 苏州瑞博生物技术股份有限公司 Nucleic acid, pharmaceutical composition and conjugate, preparation method and application
WO2022028457A1 (en) * 2020-08-04 2022-02-10 上海拓界生物医药科技有限公司 Sirna for inhibiting expression of blood coagulation factor xi, and composition and medical use thereof
CN115677810A (en) * 2021-07-23 2023-02-03 苏州瑞博生物技术股份有限公司 Double-stranded oligonucleotide, composition and conjugate containing double-stranded oligonucleotide, preparation method and use
WO2023109945A1 (en) * 2021-12-16 2023-06-22 上海拓界生物医药科技有限公司 Dsrna, and preparation method therefor and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAN L., LI C, SUN Y, WIKSTROM J, GUO Z, YU H, TONG C, ZHANG H, LIANG Z, GAO S: "Inhibition of Factor XI using a GalNAc-siRNA RBD5049: a novel antithrombotic drug with high potency and long duration", EUROPEAN HEART JOURNAL, OXFORD UNIVERSITY PRESS, GB, vol. 44, no. supl. 2, 30 October 2023 (2023-10-30), GB , pages 1 - 1, XP093340841, ISSN: 0195-668X, DOI: 10.1093/eurheartj/ehad655.2843 *
MCCLOREY GRAHAM; WOOD MATTHEW J: "An overview of the clinical application of antisense oligonucleotides for RNA-targeting therapies", CURRENT OPINION IN PHARMACOLOGY, ELSEVIER SCIENCE PUBLISHERS,, NL, vol. 24, 14 August 2015 (2015-08-14), NL , pages 52 - 58, XP029334462, ISSN: 1471-4892, DOI: 10.1016/j.coph.2015.07.005 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025232840A1 (en) * 2024-05-09 2025-11-13 石药集团中奇制药技术（石家庄）有限公司 Galnac derivative and oligonucleotide conjugate thereof

Also Published As

Publication number Publication date
CN120712356A (en) 2025-09-26

Similar Documents

Publication Publication Date Title
AU2022202991A1 (en) Organic compositions to treat KRAS-related diseases
CN111511914B (en) Nucleic acid molecules that reduce PAPD5 and PAPD7 mRNA for the treatment of hepatitis B infection
EP3052107B1 (en) Organic compounds to treat hepatitis b virus
TW202006138A (en) Use of FUBP1 inhibitors for treating hepatitis B virus infection
CN119654412A (en) Oligonucleotide modulators that activate complement factor H expression
WO2024140101A1 (en) Modified double-stranded oligonucleotide molecule, modified double-stranded oligonucleotide conjugate, and use thereof
EP3052464B1 (en) 3&#39;end caps for rna-interferring agents for use in rna interference
WO2015051135A2 (en) Organic compositions to treat hepcidin-related diseases
WO2025157271A1 (en) Nucleic acid molecule inhibiting f11 gene expression
CN118984717A (en) ARNATAR compounds and methods for enhancing cellular uptake
JP7499267B2 (en) Oligonucleotides for modulating ATXN2 expression
WO2024153166A1 (en) Modified nucleoside monomer and double-stranded ribonucleic acid
US20250327076A1 (en) Chemically modified antisense oligonucleotides (asos) and compositions for rna editing
CN119816596A (en) A dsRNA molecule for inhibiting C5 gene expression and its application
WO2024240080A1 (en) Double-stranded oligonucleotide, conjugate thereof, and use thereof
WO2025026228A1 (en) Cfb inhibitor composition and use thereof
WO2025232842A1 (en) Nucleic acid molecule inhibiting angptl3 gene expression
JP2022120380A (en) miRNA133-b derivative and its use
CN119119153A (en) Modified nucleotide monomers and their uses
WO2025026124A1 (en) C4b gene inhibitor composition and use thereof
WO2025045194A1 (en) Double-stranded rna targeting coagulation factor xi
CN119698475A (en) Highly efficient RNA targeting of angiotensinogen (ARNATAR)
CN121175420A (en) dsRNA molecules that regulate MASP2 gene activity
HK40033074B (en) Nucleic acid molecule for reduction of papd5 and papd7 mrna for treating hepatitis b infection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25744734

Country of ref document: EP

Kind code of ref document: A1