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WO2024169577A1 - Double-stranded ribonucleic acid used to inhibit expression of fxii gene, and modifier, conjugate and use thereof - Google Patents

Double-stranded ribonucleic acid used to inhibit expression of fxii gene, and modifier, conjugate and use thereof Download PDF

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Publication number
WO2024169577A1
WO2024169577A1 PCT/CN2024/074466 CN2024074466W WO2024169577A1 WO 2024169577 A1 WO2024169577 A1 WO 2024169577A1 CN 2024074466 W CN2024074466 W CN 2024074466W WO 2024169577 A1 WO2024169577 A1 WO 2024169577A1
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seq
nucleotide sequence
sequence shown
strand comprises
antisense strand
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PCT/CN2024/074466
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French (fr)
Chinese (zh)
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王书成
黄河
王岩
汪小君
荣梅
王忠宇
林国良
产运霞
耿玉先
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北京福元医药股份有限公司
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Publication of WO2024169577A1 publication Critical patent/WO2024169577A1/en

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    • 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
    • C12N15/1137—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 against enzymes
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    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present disclosure belongs to the field of biomedicine. Specifically, the present disclosure relates to a double-stranded ribonucleic acid, a double-stranded ribonucleic acid modification, a double-stranded ribonucleic acid conjugate, a pharmaceutical composition and use for inhibiting the expression of coagulation factor XII gene, and a method for inhibiting the expression of coagulation factor XII gene in cells.
  • Coagulation factor XII (also known as FXII, F12 or Hagemann factor), a serine protease expressed primarily in the liver and found in the blood, has dual functions in the intrinsic coagulation pathway and the kinin-kallikrein system, which plays a role in inflammation, blood pressure control, coagulation and pain.
  • the active form of coagulation factor XII binds to and cleaves coagulation factor XI in the coagulation cascade and prekallikrein in the kinin-kallikrein system to produce the active forms FXI and kallikrein, respectively.
  • Coagulation factor FXII is a protease involved in the coagulation pathway in vivo. It does not participate in physiological hemostasis, but participates in fibrinolysis and pathological thrombus formation; therefore, FXII plays an important role in the propagation stage of pathological clot formation, which is not required for normal hemostasis.
  • This paradigm shift of separating thrombosis and hemostasis has led to the concept of anticoagulation using drugs with minimal bleeding side effects (i.e., the development of FXIIa and FXIa inhibitors) as the next generation of anticoagulants. It has been shown that the prophylactic use of FXIIa inhibitors has anticoagulant and anti-inflammatory effects with minimal bleeding side effects.
  • coagulation factor XII is also one of the key targets for the treatment of hereditary angioedema (HAE).
  • HAE hereditary angioedema
  • HAE hereditary angioedema
  • the most common areas of swelling in the body are the limbs, face, intestines, and airways. Attacks may be spontaneous or caused by physical trauma or stress. Laryngeal (airway) edema can be life-threatening as it may lead to death from suffocation.
  • Most of the drugs currently on the market are given during an HAE attack. If gene expression can be silenced at the genetic level and the production of coagulation factor XII can be blocked, the occurrence of HAE can be effectively inhibited.
  • the present invention aims to provide a siRNA composition, which affects the cleavage of RNA transcripts of the FXII gene mediated by the RNA-induced silencing complex (RISC), thereby inhibiting the expression of the FXII gene and achieving the purpose of disease treatment.
  • RISC RNA-induced silencing complex
  • FXII inhibitors need to be developed for the treatment of FXII-related diseases including multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory reactions of COVID-19), microscopic polyangiitis (MPA), neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
  • MS multiple sclerosis
  • AD Alzheimer's disease
  • HAE hereditary angioedema
  • DVT deep vein thrombosis
  • CAP community-acquired pneumonia
  • MPA microscopic polyangiitis
  • RA rheumatoid arthritis
  • the present disclosure aims to provide a series of double-stranded RNAs, double-stranded RNA modifications, double-stranded RNA conjugates and pharmaceutical compositions for inhibiting FXII gene expression, which can inhibit FXII gene expression and have important application prospects in the treatment of clinical diseases.
  • a double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand and/or substantially reverse complementary to form a double-stranded region of the double-stranded RNA;
  • the sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence, and the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence;
  • the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 1 to 15, 641 to 648.
  • nucleotide sequence of the positive strand is a sequence A which differs by no more than 1 nucleotide from a sequence consisting of 15 to 28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666, preferably 18 to 25 consecutive nucleotides, more preferably 18 to 23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.
  • [5] The double-stranded ribonucleic acid according to any one of [1] to [4], wherein the antisense strand consists of 15-28 nucleotides, preferably 18-25 nucleotides, more preferably 18-23 nucleotides, and more preferably 19, 21 or 23 nucleotides.
  • nucleotide sequence of the antisense strand is a sequence B having a difference of no more than 1 nucleotide compared to the reverse complementary sequence of a sequence consisting of 15 to 28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666, preferably 18 to 25 consecutive nucleotides, more preferably 18 to 23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.
  • the double-stranded ribonucleic acid according to any one of [1] to [6], wherein the length of the double-stranded region is 15-25 nucleotides, preferably 18-23 nucleotides, more preferably 18-21 nucleotides, and more preferably 19, 21 or 23 nucleotides.
  • the sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end; or,
  • the sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end; or,
  • the sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region; or,
  • the sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.
  • the double-stranded RNA according to any one of [1] to [8], wherein the combination of the sense strand and the antisense strand is selected from siRNA 1 to 7, siRNA 9 to 17, siRNA 19 to 21, siRNA 23 to 26, siRNA 29 to 30, siRNA 32 to 41, siRNA 43 to 49, siRNA 52, siRNA 56, siRNA 58 to 61, siRNA 63, siRNA 65, siRNA 68, siRNA 70, siRNA 72 to 77, siRNA 79 to 83, siRNA 89 to 93, siRNA 96, siRNA 100 to 102, siRNA 105 to 106, siRNA 107 to 108, siRNA 109 to 110 08.
  • the difference between the sense strand and the target sequence does not exceed 3 nucleotides, preferably, the number of nucleotides is 1, 2 or 3;
  • the sense strand includes a region complementary to the antisense strand, and the nucleotide sequence of the sense strand is completely identical to the sequence of the antisense strand in the target sequence binding region;
  • the 3' end of the sense strand is an adenine nucleotide, which is an adenine nucleotide obtained by replacing a guanine nucleotide, a cytosine nucleotide, or a uracil nucleotide of the target sequence;
  • the third to last nucleotide at the 3' end of the sense strand is an adenine nucleotide, which is an adenine nucleotide obtained by replacing a guanine nucleotide or a cytosine nucleotide of the target sequence;
  • the 5' end of the sense strand is a cytosine nucleotide, which is obtained by replacing the adenine nucleotide of the target sequence.
  • the third to last nucleotide at the 3' end of the sense strand is a uracil nucleotide, which is obtained by replacing the adenine nucleotide of the target sequence;
  • the 5' end of the antisense strand is an adenine nucleotide, which is obtained by replacing the uracil nucleotide of the target sequence, and the 3' end of the antisense strand is a guanine nucleotide, which is obtained by replacing the cytosine nucleotide of the target sequence;
  • the combination of the sense strand and the antisense strand is selected from siRNA8, siRNA18, siRNA22, siRNA27-28, siRNA31, siRNA42, siRNA50-51, siRNA53-55, siRNA57, siRNA62, siRNA64, siRNA66-67, siRNA69, siRNA71, siRNA78, siRNA84-88, siRNA94-95, siRNA97-99, siRNA103-104, siRNA109, siRNA112, siRNA298, siRNA304, siRNA310, siRNA320, siRNA331, siRNA332, siRNA340, siRNA350, siRNA360, siRNA370, siRNA380, siRNA390, siRNA401, siRNA410 A combination of the sense strand and the antisense strand of any one of iRNA301-302, siRNA304, siRNA306, siRNA308, siRNA310, siRNA312-313, siRNA317, siRNA319, siRNA321-322, siRNA325, siRNA327-331, siRNA333-334, siRNA
  • each nucleotide in the sense strand is independently a modified nucleotide or an unmodified nucleotide
  • each nucleotide in the antisense strand is independently a modified nucleotide or an unmodified nucleotide.
  • Double-stranded RNA according to any one of [1] to [14], wherein the double-stranded RNA is siRNA for inhibiting the expression of the coagulation factor XII gene.
  • a modified double-stranded RNA which is a modified double-stranded RNA as described in any one of [1] to [15], wherein the modified double-stranded RNA comprises at least one of the following chemical modifications:
  • the 3' end of sequence A in the sense strand of the double-stranded RNA is connected to a sequence D consisting of 1-2 nucleotides, preferably a sequence D consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence B in the antisense strand of the double-stranded RNA is connected to a sequence E consisting of 1-2 nucleotides, preferably a sequence E consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence A in the sense strand of the double-stranded RNA excludes 1-2 nucleotides to form sequence A';
  • the sense strand and antisense strand of the double-stranded RNA modification are selected from the following sequence combinations:
  • nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
  • nucleotide sequence of the sense strand is the sequence shown in sequence A
  • nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E;
  • nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
  • nucleotide sequence of the sense strand is the sequence shown by sequence A connected to sequence D
  • nucleotide sequence of the antisense strand is the sequence shown by sequence B connected to sequence E;
  • nucleotide sequence of the sense strand is the sequence shown in sequence A'
  • nucleotide sequence of the antisense strand is the sequence shown in sequence B;
  • nucleotide sequence of the sense strand is the sequence shown in sequence A'
  • nucleotide sequence of the antisense strand is the sequence B'.
  • sequence shown in column E is the sequence shown in column E.
  • nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.
  • a double-stranded ribonucleic acid modified substance according to any one of [16] to [19], wherein, from the 5' end to the 3' end, the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the sense strand are 2'-O-CH 3 modified ribonucleotides.
  • the sense strand contains phosphorothioate diester bonds located at the positions shown below:
  • the positive strand is located between the second and third nucleotides starting from the 5' end.
  • the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
  • the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
  • the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides
  • the ribonucleotide at position 7 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA
  • the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides
  • the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides
  • the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA
  • the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides
  • the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides
  • the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides
  • the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide
  • the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides
  • the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides
  • the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
  • [23] A double-stranded RNA modification according to any one of [16] to [22], wherein the nucleotide at the 5’ end of the antisense strand is linked to a 5’ phosphate group or a 5’ phosphate derivative group in the direction from the 5’ end to the 3’ end.
  • N 1 -N 23 are independently selected from ribonucleotides whose base is A, U, C or G,
  • the capital letter T represents a deoxyribonucleotide whose base is thymine.
  • the lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.
  • the lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.
  • N 1 -N 23 are independently selected from ribonucleotides whose bases are A, U, C or G,
  • the capital letter T represents a deoxyribonucleotide whose base is thymine.
  • the lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.
  • the lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.
  • P1 means that the nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide.
  • EVP means that the nucleotide adjacent to the right of this letter is a 5'-trans vinylphosphonate nucleotide.
  • GNA indicates that the adjacent ribonucleotide on the right is a ribonucleotide with GNA modification.
  • double-stranded RNA modified substance according to any one of [16] to [26], wherein the double-stranded RNA modified substance is a siRNA modified substance.
  • Double-stranded RNA modified substance according to any one of [16] to [27], wherein the double-stranded RNA modified substance is a siRNA modified substance for inhibiting the expression of the coagulation factor XII gene.
  • a double-stranded RNA conjugate wherein the double-stranded RNA conjugate comprises the double-stranded RNA as described in any one of [1]-[15], or the double-stranded RNA modification as described in any one of [16]-[29]; and a conjugated group conjugated to the double-stranded RNA or the double-stranded RNA modification.
  • the sense strand and the antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region;
  • the sense strand and antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.
  • the double helix structure is double-stranded RNA or a modified double-stranded RNA.
  • Double-stranded RNA conjugate according to any one of [30] to [35], wherein the double-stranded RNA conjugate is a siRNA conjugate for inhibiting the expression of the coagulation factor XII gene.
  • the combination of the sense strand and the antisense strand is selected from the sense strand and the antisense strand of any one of siRNA225-296, siRNA415-441, and siRNA454-464 shown in Table 3.
  • a pharmaceutical composition wherein the pharmaceutical composition comprises at least one of the following: a double-stranded Ribonucleic acid, such as the modified double-stranded ribonucleic acid described in any one of [16]-[29], such as the double-stranded ribonucleic acid conjugate described in any one of [30]-[37].
  • a double-stranded Ribonucleic acid such as the modified double-stranded ribonucleic acid described in any one of [16]-[29]
  • the double-stranded ribonucleic acid conjugate described in any one of [30]-[37 such as the double-stranded ribonucleic acid conjugate described in any one of [30]-[37].
  • composition according to [38] wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.
  • sclerosis Multiple sclerosis, atherosclerosis, Alzheimer's disease, hereditary angioedema, sepsis, deep vein thrombosis, community-acquired pneumonia, thrombotic inflammation of COVID-19, microscopic polyangiitis, neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis and colitis, and other related conditions, pathologies or syndromes not yet identified.
  • a method for inhibiting the expression of the coagulation factor XII gene in cells comprising contacting the cells with the double-stranded RNA according to any one of [1]-[15], the double-stranded RNA modification according to any one of [16]-[29], the double-stranded RNA conjugate according to any one of [30]-[37], or the pharmaceutical composition according to any one of [38]-[39].
  • the double-stranded RNA provided by the present disclosure can bind to form an RNA-induced silencing complex (RISC) in cells, cut the mRNA transcribed by the FXII gene, and efficiently and specifically inhibit the expression of the FXII gene, and is used to treat FXII-related diseases including multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory responses of COVID-19), microscopic polyangiitis (MPA), neurological inflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
  • RISC RNA-induced silencing complex
  • the double-stranded RNA in the present disclosure is siRNA, which targets and degrades the transcription product mRNA of the FXII gene, exerts the effect of RNA interference, and inhibits the protein expression of the FXII gene. It is a FXII inhibitor with high inhibition rate and good specificity.
  • the present disclosure modifies double-stranded RNA to obtain modified double-stranded RNA, which has high stability and is suitable for use in in vivo disease treatment.
  • the double-stranded RNA modification is a siRNA modification, which has high stability and good inhibitory activity.
  • the present invention discloses a conjugate of double-stranded RNA or double-stranded RNA modified substance obtained by connecting a conjugation group to double-stranded RNA or double-stranded RNA modified substance, which can be used for efficient targeted delivery to tissues and cells, reducing the impact of double-stranded RNA or double-stranded RNA modified substance on non-targeted normal tissues and cells, and improving its safety in clinical disease treatment.
  • the double-stranded RNA conjugate is a siRNA conjugate, which has organ or tissue targeting while maintaining the inhibitory activity and stability of siRNA, can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs.
  • the conjugated group in the present disclosure is a group (GalNAc) of the structure shown in Formula I.
  • GalNAc can be used to transfer to liver cells, Targeted delivery within tissues is used to efficiently inhibit the expression of FXII gene in the liver.
  • the word “a” or “an” or “the” may mean “one”, but may also mean “one or more”, “at least one” and “one or more than one”.
  • FXII refers to the well-known gene and polypeptide.
  • the FXII gene and FXII mRNA sequence are easily obtained using, for example, the following: Gene Bank (GenBank), database (UniProt), Online Mendelian Inheritance in Man (OMIM), etc.
  • FXII gene may be a wild-type FXII gene, or a FXII gene mutant with sequence variation. Many sequence variations in the FXII gene have been identified and can be found in, for example, NCBIdbSNP and UniProt (see, for example, ncbi.nlm.nih.gov/snp).
  • polypeptide refers to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides.
  • a polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
  • target sequence refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcript.
  • the target sequence may include another shorter target sequence. In some embodiments, the target sequence may include one or more shorter target sequences. It should be considered that two or more shorter target sequences included in the same target sequence have the same characteristics.
  • the target gene is the FXII gene.
  • the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the FXII gene.
  • G", “C”, “A”, “T” and “U” generally represent bases containing guanine, cytosine, adenine, thymine and uracil, respectively, but it is also generally known in the art that “G”, “C”, “A”, “T” and “U” each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively, which is a common way to represent deoxyribonucleic acid sequences and/or ribonucleic acid sequences, so in the context of the present disclosure, the meanings represented by “G”, “C”, “A”, “T”, “U” include the above-mentioned various possible situations.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide (as further described below) or an alternative replacement part.
  • guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement part).
  • a nucleotide comprising inosine as its base can be base paired with a nucleotide comprising adenine, cytosine or uracil.
  • a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing, for example, inosine in the nucleotide sequence of the dsRNA characterized by the present invention.
  • adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U wobble base pairing with the target mRNA. Sequences containing such replacement parts are suitable for the compositions and methods characterized by the present invention.
  • RNAi agent RNA agent
  • RNA interfering agent RNA interfering agent
  • RISC RNA induced silencing complex
  • iRNAs direct sequence-specific degradation of mRNAs through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • iRNAs modulate, e.g., inhibit, expression of a target gene in a cell, e.g., a cell of a subject, e.g., a mammalian subject.
  • double-stranded ribonucleic acid double-stranded RNA (dsRNA) molecule
  • dsRNA double-stranded RNA
  • dsRNA double-stranded RNA
  • double-stranded ribonucleic acid triggers degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).
  • dsRNA double-stranded ribonucleic acid
  • RNAi post-transcriptional gene silencing mechanism
  • each or both of the two strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and/or modified nucleotides.
  • double-stranded RNA may include ribonucleotides, phosphate backbones, etc., with chemical modifications. These modifications may include all types of modifications disclosed herein or known in the art.
  • nucleotide refers to a compound formed by a change in the position of the base on the ribose ring in a nucleotide, for example, a compound formed by a base not being attached to the 1'-position of the ribose ring but being attached to the 2'-position or 3'-position of the ribose ring.
  • the double-stranded ribonucleic acid disclosed herein is siRNA, which interacts with the mRNA sequence transcribed by the target gene (e.g., the mRNA sequence transcribed by the C gene) to guide the cutting of the target RNA.
  • the long double-stranded RNA introduced into the cell is broken down into siRNA by a type III nuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485).
  • Dicer ribonuclease III-like enzyme processes dsRNA into 19-23 base pairs of short interfering RNA with characteristic double base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). These siRNAs are then incorporated into RNA-induced silencing complexes (RISC), in which one or more helicases unwind the siRNA duplex, which makes it possible for complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107: 309). Once bound to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al. (2001) Genes Dev. 15:188).
  • RISC RNA-induced silencing complexes
  • overhanging nucleotides refers to one or more unpaired nucleotides that protrude from the duplex structure of a double-stranded ribonucleic acid when a 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa.
  • “Blunt end” or “blunt end” means that there are no unpaired nucleotides at that end of the double-stranded ribonucleic acid, i.e., no nucleotide overhang.
  • a "blunt-ended" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its length, i.e., has no nucleotide overhangs at either end of the molecule.
  • antisense strand refers to a strand of a double-stranded RNA that is substantially complementary to a target sequence (e.g., derived from human FXII mRNA). Where the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if mismatches occur, they are typically within one or more regions of the termini, e.g., 5, 4, 3, 2, or 1 nucleotides of the 5' and/or 3' termini.
  • sense strand refers to the nucleic acid strand of a double-stranded RNA that contains a region that is substantially complementary to a region of the antisense strand.
  • the terms "complementary" or “reverse complement” are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base cytosine (G).
  • Each base pair includes a purine and a pyrimidine.
  • mismatch means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
  • substantially reverse complementary means that there are no more than three base mismatches between the two nucleotide sequences involved, that is, there are 1, 2 or 3 base mismatches between the two nucleotide sequences involved; “completely complementary” means that there are no base mismatches between the two nucleotide sequences.
  • inhibitor may be used interchangeably with “reduce,””silence,””downregulate,””suppress,” and other similar terms, and includes any level of inhibition.
  • the term "inhibiting the expression of a FXII gene” includes inhibiting the expression of any FXII gene (e.g., a mouse FXII gene, a rat FXII gene, a monkey FXII gene, or a human FXII gene) and variants (e.g., naturally occurring variants) or mutants of a FXII gene.
  • the FXII gene may be a wild-type FXII gene, a mutant FXII gene, or a transgenic FXII gene in the context of a genetically manipulated cell, cell group, or organism.
  • “Inhibiting FXII gene expression” includes any level of inhibition of the FXII gene, such as at least partial inhibition of the expression of the FXII gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
  • substituent X and substituent Y are independently hydrogen, hydroxyl, alkyl or aryl.
  • substituent X is hydrogen
  • substituent Y can be either hydrogen, hydroxyl, alkyl or aryl
  • substituent X can be either hydrogen, hydroxyl, alkyl or aryl.
  • alkyl includes straight chain, branched or cyclic saturated alkyl groups.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl and the like.
  • C 1-6 in "C 1-6 alkyl” refers to a group having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched or cyclic form.
  • alkoxy herein refers to an alkyl group attached to the remainder of the molecule via an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein.
  • alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, and the like.
  • treatment means that after suffering from a disease, a subject is exposed to (e.g., administered) double-stranded RNA, a double-stranded RNA modified substance, a double-stranded RNA conjugate, or a pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when the subject is not exposed to the disease, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.
  • prevention means that before a subject develops a disease, by contacting (e.g., administering) the double-stranded RNA, double-stranded RNA modified material, double-stranded RNA conjugate, or pharmaceutical composition of the present disclosure, the symptoms after the subject develops the disease are alleviated compared to when the subject does not develop the disease. It does not mean that the disease must be completely suppressed.
  • the term "effective amount” refers to such an amount or dosage of the double-stranded RNA, double-stranded RNA modification, double-stranded RNA conjugate or pharmaceutical composition of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses.
  • the effective amount can be easily determined by the attending physician who is a person skilled in the art by considering a variety of factors such as the species of the mammal; its size, age and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
  • disease associated with abnormal expression of the FXII gene is a disease or disorder associated with the synthesis of cholesterol, steroids and other lipids.
  • disease associated with abnormal expression of the FXII gene includes diseases, disorders or conditions that will benefit from reducing the expression of FXII (i.e., "FXII-related diseases”). Such diseases are typically associated with coagulation and inflammation.
  • Non-limiting examples of diseases associated with abnormal expression of the FXII gene include: multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory responses of COVID-19), microscopic polyangiitis (MPA), neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis (Miroslava Didiasova et al., Factor XII in coagulation, inflammation and beyond.
  • MS multiple sclerosis
  • AD Alzheimer's disease
  • HAE hereditary angioedema
  • DVT deep vein thrombosis
  • CAP community-acquired pneumonia
  • pharmaceutically acceptable excipient or “pharmaceutically acceptable carrier” refers to auxiliary materials widely used in the field of drug production.
  • the main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be dissolved at a desired rate, or to promote the effective absorption of the active ingredient in the subject receiving the drug.
  • Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a certain function to the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition).
  • Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.
  • compositions of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, embedding and/or lyophilizing processes.
  • the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.
  • mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domestic animals e.g., cows, sheep, cats, dogs and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • the first aspect of the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the FXII gene.
  • dsRNA double-stranded ribonucleic acid
  • One strand of the double-stranded ribonucleic acid is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the FXII gene) and is used to guide the cutting of the target mRNA (i.e., the transcription product of the FXII gene).
  • the other sense strand in the double-stranded ribonucleic acid includes a double-stranded region that is partially complementary and completely complementary to the antisense strand to form the double-stranded ribonucleic acid.
  • the double-stranded RNA is used as a substrate for the endonuclease (Dicer) and is cut into small fragments of dsRNA, i.e., siRNA.
  • the double-stranded RNA is siRNA.
  • the siRNA assembles to form an RNA-induced silencing complex (RISC) RISC complex, cuts the target mRNA, and inhibits the expression of the FXII gene.
  • RISC RNA-induced silencing complex
  • siRNA that binds to the target mRNA is designed.
  • the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 1 to 15, 641 to 648.
  • the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666.
  • the nucleotide sequence shown in SEQ ID NO:1 contains the nucleotide sequence shown in SEQ ID NO:16-17.
  • the nucleotide sequence shown in SEQ ID NO:3 contains the nucleotide sequence shown in SEQ ID NO:18-21.
  • the nucleotide sequence shown in SEQ ID NO:7 contains the nucleotide sequence shown in SEQ ID NO:22-23.
  • the nucleotide sequence shown in SEQ ID NO:11 contains the nucleotide sequence shown in SEQ ID NO:24-25.
  • the nucleotide sequence shown in SEQ ID NO:12 contains the nucleotide sequence shown in SEQ ID NO:26-27.
  • the nucleotide sequence shown in SEQ ID NO:15 contains the nucleotide sequence shown in SEQ ID NO:28-29.
  • the nucleotide sequence shown in SEQ ID NO:641 contains the nucleotide sequence shown in SEQ ID NO:649-650.
  • the nucleotide sequence shown in SEQ ID NO:642 contains the nucleotide sequence shown in SEQ ID NO:651 to 656.
  • the nucleotide sequence shown in SEQ ID NO:643 contains the nucleotide sequence shown in SEQ ID NO:657 to 659.
  • the nucleotide sequence shown in SEQ ID NO:646 contains the nucleotide sequence shown in SEQ ID NO:660-662.
  • the nucleotide sequence shown in SEQ ID NO:648 contains the nucleotide sequence shown in SEQ ID NO:663 to 666.
  • the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence. Specifically, along the direction from the 5' end to the 3' end, the starting nucleotide is selected in the target sequence, and at least 15 nucleotides extending in the 3' direction including the starting nucleotide are used as the binding region of the siRNA.
  • the antisense strand comprises the reverse complementary sequence of the nucleotide sequence corresponding to the binding region.
  • the starting nucleotide can be a nucleotide at any position of the target sequence, as long as at least 15 consecutive nucleotides (including the nucleotide at the starting position) can be obtained by extending in the 3' direction of the target sequence based on the starting nucleotide.
  • the nucleotide sequence of the antisense strand and the target sequence can be completely complementary or substantially complementary.
  • the nucleotide sequence of the antisense strand and the target sequence are substantially complementary, there are no more than 3 mismatched bases in the nucleotide sequence of the antisense strand and the target sequence.
  • the mismatched bases are 1, 2 or 3.
  • the nucleotide sequence of the antisense strand and the target sequence are completely complementary, there are no mismatched bases in the nucleotide sequence of the antisense strand and the target sequence.
  • the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.
  • the antisense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, most preferably 19, 21 or 23 nucleotides.
  • the sequence B included in the antisense strand is identical to the reverse complementary sequence of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.
  • the sequence B included in the antisense strand is identical to the reverse complementary sequence of a sequence consisting of 18-25 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
  • the sequence B comprised in the antisense strand differs by 1 nucleotide from the reverse complementary sequence of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.
  • the sequence B included in the antisense strand differs by 1 nucleotide from the reverse complementary sequence of a sequence consisting of 18-25 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
  • the sequence B included in the antisense strand differs by 2 nucleotides from the reverse complementary sequence of a sequence consisting of 15-28 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
  • the different nucleotides are located at the 3' end of sequence B. In other specific embodiments, the different nucleotides are located at the 5' end of sequence B. In still other specific embodiments, the different nucleotides are located at both the 3' end and the 5' end of sequence B.
  • the sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence.
  • the sense strand includes a region complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the region where the antisense strand binds to the target sequence.
  • the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that bind to the antisense strand; or, the nucleotide sequence of the sense strand is compared with at least 15 consecutive nucleotides in the target sequence that bind to the antisense strand, and there are 1, 2, or 3 different nucleotides in the nucleotide sequence of the sense strand.
  • the sense strand is composed of at least 15 nucleotides. In some embodiments, the sense strand is composed of 15-28 nucleotides. For example, the length of the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.
  • the sense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, most preferably 19, 21 or 23 nucleotides.
  • the sequence A included in the sense strand is identical to a sequence consisting of at least 15 consecutive nucleotides in the target sequence.
  • the sequence A included in the sense strand is identical to a sequence consisting of 18-25 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides on the target sequence.
  • the sequence A included in the sense strand differs by 1 nucleotide from a sequence consisting of at least 15 consecutive nucleotides in the target sequence.
  • the sequence A included in the sense strand differs by 1 nucleotide from a sequence consisting of 18-25 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides on the target sequence.
  • the sequence A included in the sense strand differs by 2 nucleotides from the reverse complementary sequence of a sequence consisting of 18-25 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
  • the different nucleotides are located at the 3' end of sequence A. In other specific embodiments, the different nucleotides are located at the 5' end of sequence A. In still other specific embodiments, the different nucleotides are located at both the 3' end and the 5' end of sequence A.
  • the length of the sense strand and the length of the antisense strand may be the same or different.
  • the sense strand and the antisense strand are the same length, specifically, the length ratio of the sense strand/antisense strand is 15/15, 16/16, 17/17, 18/18, 19/19, 20/20, 21/21, 22/22, 23/23, 24/24, 25/25, 26/26, 27/27 or 28/28.
  • the length ratio of the sense strand/antisense strand is 18/18, 19/19, 20/20, 21/21, 22/22, 23/23, 24/24 or 25/25, more preferably 19/19, 20/20, 21/21, 22/22 or 23/23, and most preferably 18/18, 19/19, 21/21 or 23/23.
  • the sense strand is different from the antisense strand in length.
  • the sense strand/antisense strand length ratio is 18/19, 18/20, 18/21, 18/22, 18/23, 18/24, 18/25, 18/26, 19/18, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/19, 20/21, 20/22, 20/23, 20/2 4.
  • the length ratio of the sense strand/antisense strand is 19/21, 20/22 or 21/23.
  • the sense strand and the antisense strand may be completely complementary or substantially complementary. When the two are substantially complementary, there are no more than 3 mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.
  • the sense strand, the antisense strand, or a combination thereof has protruding nucleotides extending out of the double-stranded region.
  • the number of protruding nucleotides may be 1 or more, for example, 1 or 2.
  • the protruding 1-2 nucleotides may be located at the 5' end, 3' end, or both ends of any antisense strand or sense strand, and each protruding nucleotide may be any type of nucleotide.
  • the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end.
  • the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end.
  • the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region.
  • the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.
  • the nucleotide sequence of the antisense strand and the target sequence can be completely complementary or substantially complementary; the sense strand and the antisense strand can be completely complementary or substantially complementary. Therefore, for the target sequence and the siRNA that can be complementary to these target sequences, for the antisense strand of each siRNA, the situation that the target sequence complementary thereto is substantially complementary is included, that is, the nucleotide sequence of the antisense strand of each siRNA may have a base mismatch with the corresponding target sequence; for the sense strand of each siRNA, the situation that the target sequence is substantially consistent with the nucleotide sequence of the sense strand of each siRNA may have a base difference with the corresponding target sequence. In some embodiments, the base mismatch or base difference may be a mismatch or difference with the target sequence that does not exceed 3 bases, for example, the mismatch base or the difference base is 1, 2 or 3.
  • the double-stranded RNA is selected from any siRNA as shown in Table 1 or Table 1-1.
  • the siRNA provided by the present disclosure has high specificity for binding to the target mRNA (FXII mRNA), has good silencing activity of the target mRNA, can significantly inhibit FXII gene expression, and is used to treat FXII-related diseases including multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory response of COVID-19), microscopic polyangiitis (MPA), neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
  • MS multiple sclerosis
  • AD Alzheimer's disease
  • the present disclosure provides a siRNA composition comprising any one or a combination of two or more of the siRNAs shown in Table 1 or Table 1-1.
  • each nucleotide of the sense strand is independently a modified nucleotide or an unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified nucleotide or an unmodified nucleotide.
  • any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond. In some embodiments, any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.
  • the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate-derived group. In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.
  • the structure of the 5' phosphate group is:
  • the structures of the 5' phosphate derivative group include but are not limited to: (EVP), wait.
  • Base represents a base, such as A, U, G, C or T.
  • R' is a hydroxyl group or is substituted by various groups known to those skilled in the art, such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.
  • 2'-fluoro (2'-F) modified nucleotides such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides
  • the second aspect of the present disclosure provides a double-stranded RNA modification.
  • the double-stranded RNA modification is a siRNA modification.
  • the siRNA modification can improve the stability of siRNA while maintaining a high FXII mRNA inhibitory activity.
  • the double-stranded RNA modification comprises at least one modification of a nucleotide.
  • the modification of the nucleotide is selected from at least one of the modification of the ribose group and the modification of the base.
  • “modification of the nucleotide” refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base.
  • the modification of the nucleotide does not result in a significant weakening or loss of the function of the siRNA to inhibit gene expression.
  • the modified nucleotide disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.
  • the stability of the siRNA can be improved by modification of the nucleotide, and its high inhibitory efficiency on the FXII gene can be maintained.
  • modified nucleotide has the structure shown below:
  • Base represents a base, such as A, U, G, C or T.
  • the hydroxyl group at the 2' position of the ribose group is substituted by R.
  • the hydroxyl group at the 2' position of these ribose groups can be substituted by various groups known to those skilled in the art, for example, 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxyribonucleotides.
  • the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH 3 ) modified nucleotide, and the like.
  • the modification of the nucleotide is the modification of the base.
  • the modification of the base can be various types of modifications known to those skilled in the art.
  • the modification of the base includes but is not limited to m 6 A, ⁇ , m 1 A, m 5 A, ms 2 i 6 A, i 6 A, m 3 C, m 5 C, ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U, mcm 5 U, ncm 5 U, ncm 5 Um, D, mcm 5 s 2 U, Inosine (I), hm 5 C, s 4 U, s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.
  • a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide.
  • a nucleotide derivative can be an isonucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide.
  • BNA refers to a constrained or inaccessible nucleotide.
  • BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure.
  • the bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.
  • LNA is shown in formula (1)
  • ENA is shown in formula (2)
  • cET is shown in formula (3):
  • Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (4) and GNA is shown in formula (5):
  • UNA unlocked nucleic acid
  • GNA glycerol nucleic acid
  • R is selected from H, OH or alkoxy (O-alkyl).
  • the nucleotide derivative modification refers to that the nucleotide in the nucleic acid is replaced by a nucleotide derivative.
  • the nucleotide derivative is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.
  • nucleotides in the nucleic acid are replaced with isonucleotides, which are also referred to as isonucleoside modifications in the context of the present disclosure.
  • isonucleoside modifications include incorporating isonucleosides at one or more sites of the sense strand and/or antisense strand of the siRNA to be modified to replace natural nucleosides for coupling at the corresponding positions.
  • the isonucleoside modification adopts D-isonucleoside modification. In other embodiments, the isonucleoside modification adopts L-isonucleoside modification. In still other embodiments, the isonucleoside modification adopts D-isonucleoside modification and L-isonucleoside modification.
  • the double-stranded RNA modification comprises a modification of the phosphodiester bond at at least one position.
  • the modification of the phosphodiester bond refers to the replacement of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a thiophosphate diester bond.
  • the thiophosphate diester bond can stabilize the double-stranded structure of the siRNA and maintain the specificity of base pairing. Exemplary, the thiophosphate diester bond structure is shown below:
  • the double-stranded RNA modification comprises at least one of the following chemical modifications:
  • the double-stranded RNA modification product is a siRNA modification product comprising at least one chemical modification among (1) to (4).
  • sequence A in the sense strand and sequence B in the antisense strand complement each other to form a double-stranded region
  • sequence B in the antisense strand complement each other to form a double-stranded region
  • sequence A and sequence B are both blunt-ended;
  • sequence A has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of sequence B forms a blunt end;
  • sequence B has 1-2 protruding nucleotides extending beyond the double-stranded region, and the 3' end of sequence A forms a flat end. end;
  • sequence A has 1-2 protruding nucleotides extending beyond the double-stranded region
  • sequence B has 1-2 protruding nucleotides extending beyond the double-stranded region.
  • nucleotide sequence of the sense strand is the sequence shown in sequence A
  • nucleotide sequence of the antisense strand is the sequence shown in sequence B.
  • nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region
  • 1-2 nucleotides are added to the 3' end of at least one of the sense strand and the antisense strand as protruding nucleotides.
  • the 1-2 nucleotides connected to the 3' end of the sense strand constitute sequence D
  • the 1-2 nucleotides connected to the 3' end of the antisense strand constitute sequence E.
  • the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E
  • the nucleotide sequence of the sense strand is the sequence shown in sequence A
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E
  • the nucleotide sequence of the sense strand is the sequence shown in sequence A
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E
  • the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B.
  • two deoxyribonucleotides are added to the 3' end of the sense strand as sequence D, and two deoxyribonucleotides (TT) are added to the 3' end of the antisense strand as sequence E.
  • two deoxyribonucleotides are added only to the 3' end of the antisense strand as sequence E.
  • two deoxyribonucleotides (TT) are added only to the 3' end of the sense strand as sequence D.
  • nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region
  • a sequence D consisting of 1-2 nucleotides is added to the 3' end of the sense strand as a protruding nucleotide. Then, after the nucleotide sequence formed by connecting sequence A to sequence D is chemically modified, the sequence D consisting of 1-2 nucleotides is excluded.
  • the nucleotide sequence of the sense strand is the sequence shown in sequence A
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B.
  • the nucleotide sequence of the sense strand is the sequence shown in sequence A
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.
  • sequence A when sequence A is complementary to sequence B to form a double-stranded region, the 3' end of sequence A has 1-2 nucleotides protruding out of the double-stranded region, the protruding nucleotides at the 3' end of sequence A are excluded as the nucleotide sequence of the sense strand.
  • sequence A' The sequence excluding the protruding nucleotides at the 3' end is called sequence A'.
  • sequence A' the nucleotide sequence of the sense strand of the double-stranded RNA modified substance is the sequence shown in sequence A'
  • the nucleotide sequence of the antisense strand of the double-stranded RNA modified substance is the sequence shown in sequence B.
  • nucleotide sequence of the sense strand of the double-stranded RNA modified substance is the sequence shown in sequence A'
  • nucleotide sequence of the antisense strand of the double-stranded RNA modified substance is the sequence shown in sequence B connected to sequence E.
  • the sense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotides at other positions in the sense strand are 2'-methoxy-modified ribonucleotides.
  • the sense strand of the siRNA modification includes phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, between the second nucleotide and the third nucleotide starting from the 5' end, between the first nucleotide and the second nucleotide starting from the 3' end, and between the second nucleotide and the third nucleotide starting from the 3' end.
  • the sense strand of the siRNA modification includes phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, and between the second nucleotide and the third nucleotide starting from the 5' end.
  • the sense strand of the siRNA modification has a structure as shown in any one of (a 1 )-(a 3 ):
  • N1 - N23 are independently selected from ribonucleotides whose bases are A, U, C or G, and the capital letter T indicates that the base is thymine.
  • the lowercase letter m indicates that the adjacent ribonucleotide on the right side of the letter m is a 2'-O-CH 3 modified ribonucleotide.
  • the lowercase letter f indicates that the adjacent ribonucleotide on the left side of the letter f is a 2'-F modified ribonucleotide.
  • -(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
  • the sense strand of the siRNA modification has a structure as shown in any one of ( a4 )-( a5 ):
  • N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- CH3 -modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; and -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond.
  • the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotide at any odd position in the antisense strand is a 2'-methoxy-modified ribonucleotide, and the ribonucleotide at any even position in the antisense strand is a 2'-fluoro-modified ribonucleotide.
  • the antisense strand of the siRNA modification comprises the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides.
  • the antisense strand of the siRNA modification comprises the following modifications: the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- CH3 modified ribonucleotides.
  • the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- CH3 modified ribonucleotides.
  • the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- CH3 modified ribonucleotides.
  • the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
  • the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
  • the antisense strand of the siRNA modification includes thiophosphate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, between the second nucleotide and the third nucleotide starting from the 5' end, between the first nucleotide and the second nucleotide starting from the 3' end, and between the second nucleotide and the third nucleotide starting from the 3' end.
  • the nucleotide at the 5' end of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group along the 5' end to the 3' end.
  • the structure of the 5' phosphate group is:
  • the structures of the 5' phosphate derivative group include but are not limited to: wait.
  • the antisense strand of the siRNA modification has a structure as shown in any one of (b 1 ) to (b 27 ):
  • N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- CH3 -modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; P1 represents that a nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide; EVP represents that a nucleotide adjacent to the right of the letter is a 5'-trans-vinylphosphonate nucleotide; -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond; and
  • the double-stranded RNA modifications include, but are not limited to, the siRNA modifications shown in Table 2.
  • the third aspect of the present disclosure provides a double-stranded RNA conjugate, which is obtained by conjugating the double-stranded RNA provided by the first aspect of the present disclosure or the double-stranded RNA modification provided by the second aspect with a conjugation group.
  • the sense strand and the antisense strand of the double-stranded RNA conjugate form a double-stranded region of the double-stranded RNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the double-stranded RNA conjugate.
  • the 3' end of the sense strand of the double-stranded RNA conjugate forms a blunt end
  • the 3' end of the antisense strand of the double-stranded RNA conjugate has 1-2 protruding nucleotides extending out of the double-stranded region.
  • the 3' end of the sense strand of the double-stranded RNA conjugate forms a blunt end
  • the 3' end of the antisense strand of the double-stranded RNA conjugate forms a blunt end.
  • the double-stranded RNA conjugate is obtained by conjugating a double-stranded RNA modification with a conjugation group, wherein the sense strand and the antisense strand of the double-stranded RNA modification are complementary to form a double-stranded region of the double-stranded RNA modification, and the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the conjugation group is conjugated with the 3' end of the sense strand with the blunt end to form a double-stranded RNA conjugate.
  • the sense strand of the double-stranded RNA modification is the sequence shown in sequence A
  • the antisense strand is the sequence shown in sequence B connected to sequence E.
  • the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end
  • the 3' end of the sense strand of the double-stranded RNA modification is connected to a conjugated group to form a double-stranded RNA conjugate.
  • the sense strand of the double-stranded RNA modification is the sequence shown in sequence A
  • the antisense strand is the sequence shown in sequence B.
  • the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end
  • the 3' end of the sense strand of the double-stranded RNA modification is connected to a conjugated group to form a double-stranded RNA conjugate.
  • the sense strand of the double-stranded RNA modification is the sequence shown by sequence A connected to sequence D
  • the antisense strand is the sequence shown by sequence B connected to sequence E.
  • the 3' end of the sense strand of the double-stranded RNA modification has a sequence D consisting of protruding 1-2 nucleotides, and after excluding the sequence D at the 3' end of the sense strand in the double-stranded RNA modification, a conjugation group is connected to the 3' end of sequence A to form a double-stranded RNA conjugate.
  • the sense strand of the double-stranded RNA modification is a sequence A connected to a sequence D
  • the antisense strand is a sequence B
  • the 3' end of the sense strand of the double-stranded RNA modification has a sequence D consisting of 1-2 nucleotides protruding, and after excluding the sequence D at the 3' end of the sense strand in the double-stranded RNA modification, a conjugated group is connected to the 3' end of sequence A to form Double-stranded RNA conjugate.
  • the sense strand of the double-stranded RNA modification is the sequence shown in sequence A
  • the antisense strand is the sequence shown in sequence B connected to sequence E.
  • the 3' end of sequence A has a protruding nucleotide extending out of the double-stranded region
  • the sequence after excluding the protruding nucleotide at the 3' end of sequence A (also known as sequence A') is used as the nucleotide sequence for connecting the conjugated group. Therefore, the nucleotide sequence of the sense strand of the double-stranded RNA conjugate is the sequence shown in sequence A'
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.
  • the sense strand of the double-stranded RNA modification is the sequence shown in sequence A
  • the antisense strand is the sequence shown in sequence B.
  • the 3' end of sequence A has a protruding nucleotide extending out of the double-stranded region
  • the sequence after excluding the protruding nucleotide at the 3' end of sequence A (also known as sequence A') is used as the nucleotide sequence for connecting the conjugated group. Therefore, the nucleotide sequence of the sense strand of the double-stranded RNA conjugate is the sequence shown in sequence A'
  • the nucleotide sequence of the antisense strand is the sequence shown in sequence B.
  • the sense strand of the double-stranded RNA conjugate has a structure as shown in any one of (d 1 )-(d 2 ):
  • N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G
  • the capital letter T represents a deoxyribonucleotide whose base is thymine
  • the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a ribonucleotide modified with 2'-O- CH3
  • the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a ribonucleotide modified with 2'-F
  • -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond.
  • L96 is the conjugate group GalNAc shown in formula I.
  • the antisense strand of the double-stranded RNA conjugate has a structure as shown in any one of (b 1 )-(b 27 ):
  • N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- CH3 -modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; P1 represents that a nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide; EVP represents that a nucleotide adjacent to the right of the letter is a 5'-trans-vinylphosphonate nucleotide; -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond; and
  • the double-stranded RNA conjugate is a siRNA conjugate, wherein the siRNA molecule connected to the conjugation group in the siRNA conjugate can be an unmodified siRNA, or a siRNA modification.
  • the siRNA molecule modified with the conjugation group has good tissue and organ targeting and the ability to promote cell endocytosis while maintaining high inhibitory activity and stability, which can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs.
  • any one of the siRNA molecules shown in Table 1 or Table 1-1 or Table 2 is selected to be connected to the conjugation group to obtain a double-stranded RNA conjugate.
  • the conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end of the siRNA sense strand.
  • the conjugated group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugated group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by 2',5'-phosphodiester bonds.
  • the conjugated group is usually connected to the phosphate group of the nucleotide; when the conjugated group is connected to the internal sequence of the siRNA, the conjugated group is usually connected to the ribose sugar ring or the base.
  • connection methods can be referred to in the literature: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10 (5): 1181-7.
  • the conjugated group can be a ligand conventionally used in the field of siRNA administration.
  • the conjugated group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors
  • lipophilic molecules such
  • the conjugated group has the following structure:
  • GalNAc has liver targeting property and can deliver siRNA molecules to liver tissue with high specificity, thereby specifically inhibiting the high expression of FXII gene in the liver.
  • GalNAc is conjugated to the 3' end of the sense strand via a phosphodiester bond to obtain a siRNA conjugate with the following structure:
  • the double helix structure is unmodified siRNA or siRNA modification.
  • the double-stranded ribonucleic acid conjugates include, but are not limited to, siRNA conjugates as shown in Table 3.
  • the fourth aspect of the present disclosure provides a pharmaceutical composition, comprising one or more of the double-stranded RNA described in the first aspect, the double-stranded RNA modification described in the second aspect, and the double-stranded RNA conjugate described in the third aspect.
  • the pharmaceutical composition contains siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the purpose of using the pharmaceutical composition is to promote administration to an organism, which is beneficial to the absorption of the active ingredient and thus exerts biological activity.
  • the pharmaceutical composition of the present disclosure can be administered in any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid preparations, oral liquid preparations), rectal, inhalation, implantation, local (e.g., eye) administration, etc.
  • Non-limiting examples of oral solid preparations include, but are not limited to, powders, capsules, lozenges, granules, tablets, etc.
  • Non-limiting examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, etc.
  • Non-limiting examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or blood
  • Non-limiting examples of parenteral preparations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, etc.
  • the pharmaceutical composition of the present disclosure can also be prepared into a controlled release or delayed release dosage form (e.g., liposomes or microspheres).
  • the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.
  • the fifth aspect of the present disclosure provides at least one of the following uses of double-stranded RNA, double-stranded RNA modification or double-stranded RNA conjugate:
  • the present disclosure further provides the use of siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or pharmaceutical compositions in at least one of the above (1)-(3).
  • abnormal expression of FXII gene triggers one or more of the following diseases related to abnormal expression of FXII gene: multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory responses of COVID-19), microscopic polyangiitis (MPA), neurological inflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
  • MS multiple sclerosis
  • AD Alzheimer's disease
  • HAE hereditary angioedema
  • DVT deep vein thrombosis
  • CAP community-acquired pneumonia
  • MPA microscopic polyangiitis
  • neurological inflammation cerebrovascular thrombosis
  • the siRNA molecule causes the expression of the FXII gene to be inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, thereby achieving the treatment of diseases related to abnormal expression of the FXII gene.
  • the present disclosure provides a method for inhibiting FXII gene expression in a cell, comprising contacting a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, or a pharmaceutical composition with the cell.
  • the method for inhibiting the expression of FXII gene in cells is to introduce siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or pharmaceutical compositions into cells.
  • the cell is an in vivo cell or an in vitro cell. In some specific embodiments, the cell is in a subject.
  • the present disclosure provides a method for preventing or treating a disease, comprising administering a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, or a pharmaceutical composition to a subject.
  • the method for preventing or treating a disease is to administer siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or pharmaceutical compositions to a subject.
  • subject includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal.
  • the subject may include a transgenic organism. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:
  • capital letters "G”, “C”, “A”, “T” and “U” each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively;
  • mA, mU, mC, mG represent 2'-methoxy modified nucleotides;
  • Af, Gf, Cf, Uf indicate 2'-fluoro modified nucleotides;
  • lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by thiophosphate groups;
  • P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide,
  • EVP indicates that the nucleotide adjacent to the right of this letter is a 5'-trans-vinylphosphonate nucleotide,
  • (GNA) indicates that the ribonucleotide adjacent to the right is a GNA modified ribonucleot
  • each of the capital letters "G”, “C”, “A”, “T” and “U” generally represents a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively;
  • mA, mU, mC, mG represent 2'-methoxy modified nucleotides;
  • Af, Gf, Cf, Uf represent 2'-fluoro modified nucleotides;
  • the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a thiophosphate group;
  • P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide,
  • EVP indicates that the nucleotide adjacent to the right of the letter is a 5'-trans vinylphosphonate nucleotide, (GNA) indicates that the ribonucleotide adjacent to the right is a
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • siRNA, siRNA modifications, and siRNA conjugates involved in the following examples were obtained from Tianlin Biotechnology (Shanghai) Co., Ltd.
  • the cells and reagents used in the examples are shown in Table 4:
  • the target sequence used to design siRNA is shown below.
  • the target sequence is derived from the mRNA sequence of the FXII gene (see NM_000505.4).
  • Target sequence III-1 GATGGGGATACTGTTTGGAGCCCAA (SEQ ID NO: 18)
  • Target sequence III-2 GCAGCAAACACAGCCCCTG (SEQ ID NO: 19)
  • Target sequence III-3 CCAGAAAGGAGGGACCTGTGTGAACATGCCAAGC (SEQ ID NO: 20)
  • Target sequence III-4 CCCACTGTCTCTGTCCACAACACCTCACT (SEQ ID NO: 21)
  • nucleoside monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement.
  • Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization.
  • the connection of the next nucleoside monomer includes four steps of deprotection, coupling, capping, and oxidation.
  • the connection of the next nucleoside monomer includes four steps of deprotection, coupling, capping, and sulfurization.
  • the nucleoside monomer is provided in an acetonitrile solution with a concentration of 0.1 M.
  • the conditions of the deprotection reaction in each step are the same, i.e., the temperature is 25° C., the reaction time is 70 seconds, the deprotection reagent is a dichloroacetic acid dichloromethane solution (3% V/V), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.
  • the coupling reaction conditions for each step were the same, including a temperature of 25° C., a molar ratio of the nucleic acid sequence connected to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence connected to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a coupling reagent of 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.
  • the capping conditions in each step were the same, including a temperature of 25°C and a reaction time of 15 seconds.
  • the oxidation reaction conditions in each step were the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing agent of 0.05 M iodine water.
  • the molar ratio of iodine to the nucleic acid sequence connected to the solid phase support in the coupling step was 30:1.
  • each step of the sulfurization reaction includes a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of hydrogenated xanthan.
  • the molar ratio of the sulfurization reagent to the nucleic acid sequence connected to the solid phase support in the coupling step is 120:1.
  • the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and the antisense chain; finally, the two chains are heated and annealed to obtain the product, and freeze-dried to obtain freeze-dried powder.
  • siRNA conjugate has the structure shown in the following formula II:
  • L96-A is reacted with NH 2 -SPS to obtain L96-B:
  • Preparation process L96-A, O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) are mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to obtain a uniform solution, aminomethyl resin (100-200 mesh) is added to the reaction liquid, and the shaking reaction is started at 25°C. After 18 hours of reaction, the filter cake is filtered and washed with dichloromethane and acetonitrile in turn to obtain a filter cake.
  • HBTU O-benzotriazole-tetramethyluronium hexafluorophosphate
  • DIPEA diisopropylethylamine
  • the obtained filter cake is capped with a CapA/CapB mixed solution to obtain L96-B, which is a solid phase carrier containing a conjugated molecule, and then the nucleoside monomer is connected to the conjugated molecule under a coupling reaction, and then the siRNA sense chain connected to the conjugate molecule is synthesized according to the siRNA molecule synthesis method described above, and the siRNA antisense chain is synthesized using the siRNA molecule synthesis method described above, and annealing is performed to generate the siRNA conjugate of this application.
  • Hep3B cells purchased from ATCC, catalog number HB-8064;
  • Transfection reagent purchased from Invitrogen, catalog number 13778-150;
  • Opti-medium Reduced serum medium, purchased from Gibco, catalog number 31985-070;
  • EMEM medium ATCC, catalog number 30-2003;
  • RNA extraction kit RNeasy 96Kit, catalog number QIAGEN-74182;
  • PBS Phosphate Buffer Saline
  • GAPDH TaqMan probe primer was purchased from Thermo Hs02786624_g1;
  • F12 TaqMan probe primer was purchased from Thermo HS01557542_g1;
  • Hep3B cells were plated in fresh EMEM medium in a 96-well plate and cultured for 24 hours.
  • the cultured cells were resuspended in EMEM medium without PS (penicillin-streptomycin mixture) to prepare a cell suspension with a density of 5.55 ⁇ 10 4 /mL, and plated in a 96-well plate, adding 90 ⁇ L of cell suspension to each well, i.e., 5000 cells/well.
  • siRNA in the experimental process of this example Centrifuge the dry powder of the siRNA to be tested and siRNA modifications (for ease of description, collectively referred to as siRNA in the experimental process of this example) at low temperature and high speed, and then dissolve it with ultrapure distilled water (ULtraPure Distilled Water) to prepare a 100 ⁇ M stock solution.
  • ULP Ultrapure distilled water
  • step b) taking 2 ⁇ L of the 10 ⁇ M siRNA dilution prepared in step a), adding 18 ⁇ L of ultrapure distilled water to obtain a siRNA stock solution Q with a final concentration of 1 ⁇ M;
  • step c) taking 2 ⁇ L of the 1 ⁇ M siRNA stock solution Q prepared in step b), adding 18 ⁇ L of ultrapure distilled water to obtain a siRNA stock solution X with a final concentration of 0.1 ⁇ M;
  • transfection reagent diluent Take 3 ⁇ L of transfection reagent, add 97 ⁇ L of Opti-medium, and get Transfection reagent diluent;
  • the transfection reagent diluent and the 20 nM siRNA diluent Z prepared in step 3.2.3 were mixed in a volume ratio of 1:1 to prepare a transfection mixture, which was allowed to stand for 5 minutes.
  • 10 ⁇ L of the transfection mixture was added to a 96-well plate to transfect the Hep3B cells cultured in step 3.2.1 (final volume 100 ⁇ L, the concentration of siRNA in this system was 1 nM);
  • transfection reagent diluent Take 3 ⁇ L of transfection reagent, add 97 ⁇ L of Opti-medium, and get Transfection reagent diluent;
  • the transfection reagent diluent and the 2nM siRNA dilution W prepared in step 3.2.3 were mixed in a volume ratio of 1:1 to prepare a transfection mixture, which was allowed to stand for 5 minutes.
  • 10 ⁇ L of the transfection mixture was added to a 96-well plate to transfect the Hep3B cells cultured in step 3.2.1 (final volume 100 ⁇ L, the concentration of siRNA in this system was 0.1 nM).
  • the cells were cultured for 48 hours after the above transfection; 2 replicates were set for each concentration (1 nM and 0.1 nM).
  • step b) Add the following reagents to the system obtained in step a) and perform reverse transcription
  • step b) The reverse transcription product obtained in step b) was stored at -20°C for real-time PCR analysis.
  • Inhibition rate (%) (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group ⁇ 100%
  • the Mock group refers to a group to which no siRNA is added compared with the test sample group.
  • siRNA and siRNA modifications provided by the present disclosure show excellent inhibitory effects on the FXII gene.
  • the concentration range of the following siRNA to be tested was set (nM) as follows: 10, 2.5, 0.625, 0.156, 0.039, 0.0097, 0.0024, 0.0006, and IC50 was determined in a similar manner to 3.2.
  • the Mock group refers to a group to which no siRNA is added compared with the test sample group.
  • Inhibition rate (%) (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group ⁇ 100%,
  • the log value of siRNA concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis.
  • the "log (inhibitor) vs. response-variable slope" of the analysis software GraphPad Prism 8 was used to fit the dose-effect curve to obtain the IC50 value of each siRNA.
  • Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%;
  • Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%;
  • HillSlope represents the slope of the percentage inhibition rate curve.
  • the siRNA modifications provided by the present disclosure show excellent inhibitory effects on the FXII gene.
  • PHH medium invitroGRO CP Meduim serum free BIOVIT, catalog number: S03316
  • Transfection reagent purchased from Invitrogen, catalog number: 13778-150;
  • RNA Extraction Kit 96Kit purchased from QIAGEN, catalog number: QIAGEN-74182;
  • Reverse transcription kit FastKing RT Kit (With gDNase), purchased from TianGen, catalog number: KR116-02;
  • FXII and GAPDH primers were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.
  • siRNA conjugates (final concentrations of siRNA conjugates were 10 nM, 2.5 nM, 0.63 nM, 0.16 nM, 0.04 nM, 0.01 nM, 0.0024 nM and 0.0006 nM, in duplicate) were transfected into PHH cells as follows: frozen PHH cells were taken, revived, counted, and the cells were adjusted to 6 ⁇ 10 5 cells/mL, and applied simultaneously RNAiMax transfection reagent was used to transfer siRNA conjugates into cells, and cells were seeded into 96-well plates at a density of 54,000 cells per well, with 100 ⁇ L of culture medium per well. Cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
  • siRNA conjugates enter PHH cells by free uptake, the process is as follows: take frozen PHH cells, revive, count, adjust the cells to 6 ⁇ 10 5 cells/mL, add siRNA conjugates at the same time, and inoculate them into 96-well plates at a density of 54,000 cells per well, with 100 ⁇ L of culture medium per well. The cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
  • the extracted total RNA was reverse transcribed into cDNA by reverse transcription reaction, and the FXII cDNA obtained by reverse transcription was quantitatively amplified by qPCR.
  • GAPDH cDNA was amplified in parallel as an internal control.
  • the PCR reaction program was: 95°C, 10 minutes, then enter the cycle mode, 95°C, 15 seconds, then 60°C, 60 seconds, for a total of 40 cycles.
  • Inhibition rate (%) (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group ⁇ 100%;
  • the log value of the siRNA conjugate concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis.
  • the log (inhibitor) vs. response-variable slope of the analysis software GraphPad Prism 8 was used to fit the dose-effect curve to obtain the IC50 value of each siRNA conjugate.
  • Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%;
  • Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%;
  • HillSlope represents the slope of the percentage inhibition rate curve.
  • L96 in the above table is the conjugate group GalNAc shown in Formula I.
  • the siRNA conjugates provided by the present disclosure show excellent inhibitory effects on the FXII gene.
  • siRNA sequence design concept and method in this example are the same as those in Example 1, and the target sequence used to design siRNA is shown below.
  • the target sequence is derived from the gene mRNA sequence of FXII (see NM_000505.4).
  • the siRNA and siRNA modifications provided by the present disclosure show excellent inhibitory effects on the FXII gene.
  • Example 7 Determination of the inhibition rate of siRNA and siRNA conjugates in inhibiting FXII gene expression
  • Phemocytes Primary human hepatocytes (PHH cells) were provided by Shanghai WuXi AppTec Pharmaceuticals Co., Ltd.;
  • PHH medium invitroGRO CP Meduim serum free BIOVIT, catalog number: S03316;
  • Transfection reagent purchased from Invitrogen, catalog number: 13778-150;
  • RNA Extraction Kit 96Kit purchased from QIAGEN, catalog number: QIAGEN-74182;
  • Reverse transcription kit FastKing RT Kit (With gDNase), purchased from TianGen, catalog number: KR116-02;
  • FXII and GAPDH primers were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.
  • siRNA conjugates (final concentration of siRNA conjugates was 5 nM and 0.5 nM, duplicate wells) were transfected into PHH cells by the following process: frozen PHH cells were taken, revived, counted, and the cells were adjusted to 6 ⁇ 10 5 cells/mL, and then applied RNAiMax transfection reagent was used to transfer siRNA conjugates into cells, and cells were seeded into 96-well plates at a density of 54,000 cells per well, with 100 ⁇ L of culture medium per well. Cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
  • siRNA conjugates (final concentrations of siRNA conjugates were 100 nM and 5 nM, in duplicate) were freely taken into PHH cells, and the process was as follows: frozen PHH cells were taken, revived, counted, and adjusted to 6 ⁇ 10 5 cells/mL, and siRNA conjugates were added at the same time, and inoculated into 96-well plates at a density of 54,000 cells per well, with 100 ⁇ L of culture medium per well. The cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
  • the extracted total RNA was reverse transcribed into cDNA by reverse transcription reaction, and the FXII cDNA obtained by reverse transcription was quantitatively amplified by qPCR.
  • GAPDH cDNA was amplified in parallel as an internal control.
  • the PCR reaction program was: 95°C, 10 minutes, then entered the cycle mode, 95°C, 15 seconds, then 60°C, 60 seconds, for a total of 40 cycles.
  • Inhibition rate (%) (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group ⁇ 100%;
  • the Mock group refers to a group without the addition of siRNA conjugates compared with the test sample group.
  • L96 in the above table is the conjugate group GalNAc shown in Formula I.
  • the siRNA conjugates provided by the present disclosure show excellent inhibitory effects on the FXII gene.
  • Example 8 Inhibitory effect of siRNA conjugates on human FXII (hFXII) gene expression in humanized mice
  • mice C57BL/6-hFXII mice (provided by Shanghai Model Organisms Technology Co., Ltd.) aged 6-8 weeks were placed in the breeding facility. After 7 days of adaptive feeding, the mice were subcutaneously administered N-ER-FY009028M3L96, N-ER-FY009028M7L96, N-ER-FY009017M2L96, N-ER-FY009065M2L96, and N-ER-FY009028M3L96 at a single dose of 1 mg/kg or 3 mg/kg.
  • the siRNA conjugates disclosed in the present invention have high inhibitory activity on the protein expressed by the hFXII gene in vivo, and can reduce the hFXII protein level for a long time.
  • the siRNA conjugate When administered with a single dose of 3 mg/kg, can have an inhibition rate of more than 80% in the inhibition rate results from the 7th day to the 35th day; when administered with a single dose of 1 mg/kg, the siRNA conjugate has an inhibition rate of more than 70% in the inhibition rate results from the 7th day to the 28th day, and even has an inhibition rate of more than 68% when the experimental period is as long as 35 days; This shows that the designed compound can effectively inhibit the expression of hFXII protein.
  • the operations were performed one by one in this order (plasma and serum sample collection, 2 biochemical indices of coagulation function, and wet weight of thrombus after AV-shunt modeling). The blood collection location and test details are described below.
  • N-ER-FY009028M7L96 and N-ER-FY009151M7L96 were subcutaneously administered to cynomolgus monkeys at a single dose of 10 mg/kg (3 cynomolgus monkeys in each group).
  • a special connecting tube (made by cutting two 15 cm long tubes from the infusion extension tube, connecting the two tubes end to end, and using a 10 cm long rough weighed fishing line to wrap and reinforce the connection) was inserted into the proximal end of the femoral vein and the proximal end of the femoral artery in turn, and then removed after blood circulation for 15 minutes, the thrombus in the tube was removed, the wet weight of the thrombus was weighed, the blood vessel was ligated, and the access was closed. After the operation, ceftriaxone 50 mg/kg was injected intramuscularly for 3 consecutive days, and meloxicam 0.1 mL/kg was injected intramuscularly for 3 consecutive days.
  • the siRNA conjugates disclosed in the present invention have high inhibitory activity on the protein expressed by the hFXII gene in cynomolgus monkeys, and can reduce the hFXII protein level for a long time.
  • the inhibition rate effect continued to increase from the 7th day to the 35th day.
  • the conjugate showed an inhibition rate of more than 90%, indicating that the designed compound can effectively inhibit the expression of hFXII protein.
  • the APTT extension rate of the siRNA conjugates disclosed in the present invention continued to increase from the 7th day to the 35th day, and on the 35th day, the conjugates showed an extension rate of more than 160%, indicating that the designed compound can better prolong the activated partial thromboplastin time.
  • mice CD-1 mice, SPF grade, male, about 30 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.
  • siRNA conjugates were administered at a dose of 3 mg/kg (10 mL/kg) and given as a single subcutaneous injection after randomization, with 6 mice in each group.
  • Sample collection Whole blood samples were collected at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, and 48 hours after administration, for a total of 10 points. The first 3 samples in each group were collected at 0.0833, 0.5, 2, 8, and 36 hours, and the last 3 samples were collected at 0.25, 1, 4, 24, and 48 hours. After whole blood was collected, plasma was separated by centrifugation for detection and analysis.
  • siRNA conjugates siRNA225-siRNA296, siRNA415-siRNA441, and siRNA454-siRNA464 disclosed in the present invention have a shorter half-life in plasma and are eliminated faster.
  • mice CD-1 mice, SPF grade, male, about 30 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.
  • siRNA conjugates were administered at a dose of 3 mg/kg (10 mL/kg) and given by a single subcutaneous injection after randomization, with 3 animals at each time point, for a total of 24 mice.
  • LC-MS/MS method was used to detect the concentration of the original drug in plasma and tissue samples at each time point.
  • the AUC in plasma and tissues was calculated by the trapezoidal area method.
  • siRNA conjugates siRNA225-siRNA296, siRNA415-siRNA441, and siRNA454-siRNA464 disclosed in the present invention are mainly enriched in the liver, have a long retention time in the tissue, and have good stability.
  • Example 12 MTD test of single subcutaneous injection of siRNA conjugate into C57B/L mice
  • mice C57 mice, SPF grade, male, about 25g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Animals were randomly divided into groups according to their body weight on the last day of the adaptation period. The specific dosage design and grouping are shown in Table 17 below:
  • Clinical observation Continuous observation for 4 hours on the day of administration, and clinical observation at least once a day during the recovery period.
  • Body weight All surviving animals were weighed twice a week.
  • Immunotoxicity Blood was collected from animals in the MTD dose group at 1h ⁇ 2min, 4h ⁇ 5min, 8h ⁇ 10min, and 24h ⁇ 20min after administration on D1. Three animals/sex/group were collected at each time point for detection of cytokines (IFN- ⁇ , TNF- ⁇ , IL-2/6/8).
  • Toxicokinetics Blood samples were collected from animals in the MTD dose group at 30min ⁇ 2min, 1h ⁇ 2min, 4h ⁇ 5min, 8h ⁇ 10min, and 24h ⁇ 20min before and after administration on D1. Three animals/sex/group were collected at each time point to detect blood drug concentration.
  • Blood biochemistry The animals in the main test group were autopsied on D28, and the animals in the satellite group were autopsied in batches on D7, D14, D21, and D28 to test blood biochemistry.
  • Tissue distribution The animals in the main test group were autopsied on D28, and the animals in the satellite group were autopsied in batches on D7, D14, D21, and D28. Blood and liver were collected to detect tissue drug concentrations.
  • Histopathological examination The animals in the main experimental group were autopsied on D28, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and abnormal tissues or organs were collected and fixed for histopathological examination.
  • siRNA conjugates siRNA225-siRNA296, siRNA415-siRNA441, and siRNA454-siRNA464 disclosed in the present invention have low toxicity and an excellent safety window for use.

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Abstract

The present disclosure relates to a double-stranded ribonucleic acid used to inhibit the expression of a coagulation factor XII gene, and a modifier, conjugate and use thereof. Specifically, the present disclosure relates to a double-stranded ribonucleic acid, a double-stranded ribonucleic acid modifier, a double-stranded ribonucleic acid conjugate, a pharmaceutical composition and a use for inhibiting the expression of a coagulation factor XII gene, and a method for inhibiting the expression of a coagulation factor XII gene in a cell. The double-stranded ribonucleic acid provided in the present disclosure can be combined in cells to form an RNA-induced silencing compound (RISC) and cut the mRNA transcribed by the coagulation factor XII gene, thereby efficiently and specifically inhibiting the expression of the coagulation factor XII gene. The double-stranded ribonucleic acid is used to treat diseases mediated by the coagulation factor XII gene, and has important prospects for use in clinical disease treatment.

Description

用于抑制FXII基因表达的双链核糖核酸及其修饰物、缀合物和用途Double-stranded ribonucleic acid for inhibiting FXII gene expression and its modified product, conjugate and use 技术领域Technical Field
本公开属于生物医药领域，具体来说，本公开涉及一种用于抑制凝血因子XII基因表达的双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物、药物组合物和用途，以及用于抑制细胞内凝血因子XII基因表达的方法。The present disclosure belongs to the field of biomedicine. Specifically, the present disclosure relates to a double-stranded ribonucleic acid, a double-stranded ribonucleic acid modification, a double-stranded ribonucleic acid conjugate, a pharmaceutical composition and use for inhibiting the expression of coagulation factor XII gene, and a method for inhibiting the expression of coagulation factor XII gene in cells.
背景技术Background Art
凝血因子XII(也称为FXII，F12或哈格曼因子)，一种主要在肝脏中表达，并在血液中发现的丝氨酸蛋白酶，在内源性凝血途径和激肽-激肽释放酶系统中有双重功能，激肽-激肽释放酶系统在炎症、血压控制、凝血和疼痛中起作用。凝血因子XII的活性形式结合并裂解凝血级联中的凝血因子XI和激肽-激肽释放酶系统中的前激肽释放酶，分别产生活性形式FXI和激肽释放酶。Coagulation factor XII (also known as FXII, F12 or Hagemann factor), a serine protease expressed primarily in the liver and found in the blood, has dual functions in the intrinsic coagulation pathway and the kinin-kallikrein system, which plays a role in inflammation, blood pressure control, coagulation and pain. The active form of coagulation factor XII binds to and cleaves coagulation factor XI in the coagulation cascade and prekallikrein in the kinin-kallikrein system to produce the active forms FXI and kallikrein, respectively.
凝血因子FXII是体内凝血途径中参与促凝的一种蛋白酶，其不参加生理性止血，参与纤维蛋白溶解及病理血栓的形成；因此，FXII在病理性凝块形成的传播阶段起着重要作用，而正常止血则不需要。这种将血栓形成和止血分离的范式转变导致了使用具有最小出血副作用的药物(即开发出FXIIa和FXIa抑制剂)作为下一代抗凝剂的抗凝概念。目前已证明预防性使用FXIIa抑制剂具有抗凝和抗炎功效，且出血副作用最小。Silasi等人发现通过抑制FXII功能，补体和炎性细胞因子的激活减少，从而使受到热灭活金黄色葡萄球菌(HI-SA,heat-inactivated Staphylococcus aureus)挑战的狒狒的器官功能和存活率得以保留(Blood.2021Jul 15；138(2):178-189.)。这些发现表明，使用FXIIa抑制剂对病理性凝血和炎症反应风险增加的患者进行预防性治疗的潜在益处。此类适应症包括预防败血症或缓解严重感染。Coagulation factor FXII is a protease involved in the coagulation pathway in vivo. It does not participate in physiological hemostasis, but participates in fibrinolysis and pathological thrombus formation; therefore, FXII plays an important role in the propagation stage of pathological clot formation, which is not required for normal hemostasis. This paradigm shift of separating thrombosis and hemostasis has led to the concept of anticoagulation using drugs with minimal bleeding side effects (i.e., the development of FXIIa and FXIa inhibitors) as the next generation of anticoagulants. It has been shown that the prophylactic use of FXIIa inhibitors has anticoagulant and anti-inflammatory effects with minimal bleeding side effects. Silasi et al. found that by inhibiting FXII function, the activation of complement and inflammatory cytokines was reduced, thereby preserving the organ function and survival rate of baboons challenged with heat-inactivated Staphylococcus aureus (HI-SA) (Blood. 2021 Jul 15; 138 (2): 178-189.). These findings suggest the potential benefit of prophylactic treatment with FXIIa inhibitors in patients at increased risk for pathological coagulation and inflammation. Such indications include preventing sepsis or mitigating severe infections.
此外，凝血因子XII也是治疗遗传性血管性水肿(HAE)的关键靶点之一。HAE是一种罕见疾病，其特征在于严重肿胀的反复发作。身体最常见的肿胀区域是四肢、面部、肠道和气道。发作可能是自发性的，也可能是由于身体创伤或压力引起的。喉部(气道)水肿可能会危及生命，因为它可能导致窒息死亡。目前市场上大部分的药物是选择在HAE发作时给予，若能从基因水平沉默基因表达，阻断凝血因子XII的生成，就能够有效抑制HAE的发生。In addition, coagulation factor XII is also one of the key targets for the treatment of hereditary angioedema (HAE). HAE is a rare disease characterized by recurrent episodes of severe swelling. The most common areas of swelling in the body are the limbs, face, intestines, and airways. Attacks may be spontaneous or caused by physical trauma or stress. Laryngeal (airway) edema can be life-threatening as it may lead to death from suffocation. Most of the drugs currently on the market are given during an HAE attack. If gene expression can be silenced at the genetic level and the production of coagulation factor XII can be blocked, the occurrence of HAE can be effectively inhibited.
本发明旨在提供siRNA组合物，该组合物影响RNA诱导的沉默复合体(RISC)介导的FXII基因的RNA转录物的切割，从而可以抑制FXII基因的表达，实现疾病治疗的目的。The present invention aims to provide a siRNA composition, which affects the cleavage of RNA transcripts of the FXII gene mediated by the RNA-induced silencing complex (RISC), thereby inhibiting the expression of the FXII gene and achieving the purpose of disease treatment.
发明内容Summary of the invention
发明要解决的问题Problem that the invention aims to solve
鉴于现有技术中存在的问题，例如，需要开发更多FXII抑制剂，用于治疗包括多发性硬化症(MS)、动脉粥样硬化、阿尔兹海默症(AD)、遗传性血管水肿(HAE)、脓毒症、深静脉血栓形成(DVT)、社区获得性肺炎(CAP)、新冠肺炎的血栓性炎症(参与新冠肺炎的促凝血和促炎反应)、显微镜下型多血管炎(MPA)、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎(RA)及结肠炎的FXII相关疾病。本公开旨在提供一系列用于抑制FXII基因表达的双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物及药物组合物，能够抑制FXII基因表达，在临床疾病治疗中具有重要应用前景。In view of the problems existing in the prior art, for example, more FXII inhibitors need to be developed for the treatment of FXII-related diseases including multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory reactions of COVID-19), microscopic polyangiitis (MPA), neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis. The present disclosure aims to provide a series of double-stranded RNAs, double-stranded RNA modifications, double-stranded RNA conjugates and pharmaceutical compositions for inhibiting FXII gene expression, which can inhibit FXII gene expression and have important application prospects in the treatment of clinical diseases.
用于解决问题的方案Solutions for solving problems
[1].一种双链核糖核酸，所述双链核糖核酸包括正义链和反义链，所述正义链与所述反义链互补和/或基本上反向互补形成所述双链核糖核酸的双链区；[1] A double-stranded RNA, comprising a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand and/or substantially reverse complementary to form a double-stranded region of the double-stranded RNA;
其中，所述正义链包含与靶标序列中至少15个连续核苷酸的差异不超过3个核苷酸的序列A，所述反义链包含与靶标序列中至少15个连续核苷酸的反向互补序列的差异不超过3个核苷酸的序列B；The sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence, and the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence;
所述靶标序列选自如SEQ ID NO:1～15，641～648任一项所示的核苷酸序列。The target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 1 to 15, 641 to 648.
[2].根据[1]所述的双链核糖核酸，其中，所述靶标序列选自如SEQ ID NO:2,4～6,8～10,13～14, 16～29,644,645,647,649～666任一项所示的核苷酸序列，所述正义链包含如SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中至少15个连续核苷酸组成的序列A，所述反义链包含如SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中至少15个连续核苷酸组成的序列反向互补和/或基本上反向互补的序列B。[2] The double-stranded RNA according to [1], wherein the target sequence is selected from SEQ ID NO: 2, 4-6, 8-10, 13-14, The nucleotide sequence as shown in any one of SEQ ID NOs: 16 to 29, 644, 645, 647, 649 to 666, the sense strand comprises a sequence A consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NOs: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666, and the antisense strand comprises a sequence B that is reverse complementary and/or substantially reverse complementary to a sequence consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NOs: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666.
[3].根据[1]或[2]所述的双链核糖核酸，其中，所述正义链由15-28个核苷酸组成，优选18-25个核苷酸，更优选18-23个核苷酸，更优选19、21或23个核苷酸。[3] The double-stranded RNA according to [1] or [2], wherein the sense strand consists of 15-28 nucleotides, preferably 18-25 nucleotides, more preferably 18-23 nucleotides, and more preferably 19, 21 or 23 nucleotides.
[4].根据[3]所述的双链核糖核酸，其中，所述正义链的核苷酸序列是与SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中的15-28个连续核苷酸组成的序列相比差异不超过1个核苷酸的序列A，优选18-25个连续核苷酸，更优选18-23个连续核苷酸，更优选19、21或23个核苷酸。[4]. The double-stranded RNA according to [3], wherein the nucleotide sequence of the positive strand is a sequence A which differs by no more than 1 nucleotide from a sequence consisting of 15 to 28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666, preferably 18 to 25 consecutive nucleotides, more preferably 18 to 23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.
[5].根据[1]-[4]任一项所述的双链核糖核酸，其中，所述反义链由15-28个核苷酸组成，优选18-25个核苷酸，更优选18-23个核苷酸，更优选19、21或23个核苷酸。[5] The double-stranded ribonucleic acid according to any one of [1] to [4], wherein the antisense strand consists of 15-28 nucleotides, preferably 18-25 nucleotides, more preferably 18-23 nucleotides, and more preferably 19, 21 or 23 nucleotides.
[6].根据[5]所述的双链核糖核酸，其中，所述反义链的核苷酸序列是与SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中的15-28个连续核苷酸组成的序列的反向互补序列相比差异不超过1个核苷酸的序列B，优选18-25个连续核苷酸，更优选18-23个连续核苷酸，更优选19、21或23个核苷酸。[6]. The double-stranded RNA according to [5], wherein the nucleotide sequence of the antisense strand is a sequence B having a difference of no more than 1 nucleotide compared to the reverse complementary sequence of a sequence consisting of 15 to 28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666, preferably 18 to 25 consecutive nucleotides, more preferably 18 to 23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.
[7].根据[1]-[6]任一项所述的双链核糖核酸，其中，所述双链区的长度为15-25个核苷酸，优选18-23个核苷酸，更优选18-21个核苷酸，更优选19、21或23个核苷酸。[7]. The double-stranded ribonucleic acid according to any one of [1] to [6], wherein the length of the double-stranded region is 15-25 nucleotides, preferably 18-23 nucleotides, more preferably 18-21 nucleotides, and more preferably 19, 21 or 23 nucleotides.
[8].根据[1]-[7]任一项所述的双链核糖核酸，其中，[8] The double-stranded RNA according to any one of [1] to [7], wherein
所述正义链与所述反义链互补形成所述双链区，且所述正义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，所述反义链的3’末端形成平末端；或者，The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end; or,
所述正义链与所述反义链互补形成所述双链区，且所述反义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，所述正义链的3’末端形成平末端；或者，The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end; or,
所述正义链与所述反义链互补形成所述双链区，且所述正义链与所述反义链的3’末端均具有1-2个延伸出所述双链区的突出的核苷酸；或者，The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region; or,
所述正义链与所述反义链互补形成所述双链区，且所述正义链与所述反义链的3’末端均形成平末端。The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.
[9].根据[1]-[8]任一项所述的双链核糖核酸，其中，所述正义链与所述反义链的组合为选自表1和表1-1中所示的siRNA1～7、siRNA9～17、siRNA19～21、siRNA23～26、siRNA29～30、siRNA32～41、siRNA43～49、siRNA52、siRNA56、siRNA58～61、siRNA63、siRNA65、siRNA68、siRNA70、siRNA72～77、siRNA79～83、siRNA89～93、siRNA96、siRNA100～102、siRNA105～108、siRNA110～111、siRNA113～114、siRNA297、siRNA299、siRNA300、siRNA303、siRNA305、siRNA307、siRNA309、siRNA311、siRNA314～316、siRNA318、siRNA320、siRNA323～324、siRNA326、siRNA332、siRNA335、siRNA343～348、siRNA350、siRNA359、siRNA364中的任一者的正义链和反义链的组合；优选地，所述正义链与所述反义链选自siRNA326。[9]. The double-stranded RNA according to any one of [1] to [8], wherein the combination of the sense strand and the antisense strand is selected from siRNA 1 to 7, siRNA 9 to 17, siRNA 19 to 21, siRNA 23 to 26, siRNA 29 to 30, siRNA 32 to 41, siRNA 43 to 49, siRNA 52, siRNA 56, siRNA 58 to 61, siRNA 63, siRNA 65, siRNA 68, siRNA 70, siRNA 72 to 77, siRNA 79 to 83, siRNA 89 to 93, siRNA 96, siRNA 100 to 102, siRNA 105 to 106, siRNA 107 to 108, siRNA 109 to 110 08. A combination of the sense strand and the antisense strand of any one of siRNA110-111, siRNA113-114, siRNA297, siRNA299, siRNA300, siRNA303, siRNA305, siRNA307, siRNA309, siRNA311, siRNA314-316, siRNA318, siRNA320, siRNA323-324, siRNA326, siRNA332, siRNA335, siRNA343-348, siRNA350, siRNA359, and siRNA364; preferably, the sense strand and the antisense strand are selected from siRNA326.
[10].根据[1]-[8]任一项所述的双链核糖核酸，其中，双链区的长度为19个或21个核苷酸，所述反义链的核苷酸序列与靶标序列存在不超过3个的错配碱基，优选的，错配碱基为1个、2个或3个；[10] The double-stranded RNA according to any one of [1] to [8], wherein the length of the double-stranded region is 19 or 21 nucleotides, and the nucleotide sequence of the antisense strand has no more than 3 mismatched bases with the target sequence, preferably, the number of mismatched bases is 1, 2 or 3;
所述正义链与靶标序列存在的差异不超过3个核苷酸，优选的，核苷酸个数为1个、2个或3个；The difference between the sense strand and the target sequence does not exceed 3 nucleotides, preferably, the number of nucleotides is 1, 2 or 3;
所述正义链中包括与反义链互补的区域，正义链的核苷酸序列与反义链在靶标序列结合区域的序列完全相同；The sense strand includes a region complementary to the antisense strand, and the nucleotide sequence of the sense strand is completely identical to the sequence of the antisense strand in the target sequence binding region;
任选地，所述正义链的3’末端为腺嘌呤核苷酸，是由靶标序列的鸟嘌呤核苷酸或胞嘧啶核苷酸或尿嘧啶核苷酸替换得到的腺嘌呤核苷酸；Optionally, the 3' end of the sense strand is an adenine nucleotide, which is an adenine nucleotide obtained by replacing a guanine nucleotide, a cytosine nucleotide, or a uracil nucleotide of the target sequence;
任选地，所述正义链的3’末端倒数第三个核苷酸为腺嘌呤核苷酸，是由靶标序列的鸟嘌呤核苷酸或胞嘧啶核苷酸替换得到的腺嘌呤核苷酸；Optionally, the third to last nucleotide at the 3' end of the sense strand is an adenine nucleotide, which is an adenine nucleotide obtained by replacing a guanine nucleotide or a cytosine nucleotide of the target sequence;
任选地，所述正义链的5’末端为胞嘧啶核苷酸，是由靶标序列的腺嘌呤核苷酸替换得到的胞嘧啶 核苷酸；Optionally, the 5' end of the sense strand is a cytosine nucleotide, which is obtained by replacing the adenine nucleotide of the target sequence. Nucleotides;
任选地，所述正义链的3’末端倒数第三个核苷酸为尿嘧啶核苷酸，是由靶标序列的腺嘌呤核苷酸替换得到的尿嘧啶核苷酸；Optionally, the third to last nucleotide at the 3' end of the sense strand is a uracil nucleotide, which is obtained by replacing the adenine nucleotide of the target sequence;
任选地，所述反义链的5’末端为腺嘌呤核苷酸，是由靶标序列的尿嘧啶核苷酸替换得到的腺嘌呤核苷酸，所述反义链的3’末端为鸟嘌呤核苷酸，是由靶标序列的胞嘧啶核苷酸替换得到的鸟嘌呤核苷酸；Optionally, the 5' end of the antisense strand is an adenine nucleotide, which is obtained by replacing the uracil nucleotide of the target sequence, and the 3' end of the antisense strand is a guanine nucleotide, which is obtained by replacing the cytosine nucleotide of the target sequence;
所述正义链与所述反义链的组合为选自表1中所示的siRNA8、siRNA18、siRNA22、siRNA27～28、siRNA31、siRNA42、siRNA50～51、siRNA53～55、siRNA57、siRNA62、siRNA64、siRNA66～67、siRNA69、siRNA71、siRNA78、siRNA84～88、siRNA94～95、siRNA97～99、siRNA103～104、siRNA109、siRNA112、siRNA298、siRNA301～302、siRNA304、siRNA306、siRNA308、siRNA310、siRNA312～313、siRNA317、siRNA319、siRNA321～322、siRNA325、siRNA327～331、siRNA333～334、siRNA336～342、siRNA349、siRNA351～358、siRNA360～363、siRNA442～443中任一者的正义链和反义链的组合；优选地，所述正义链与所述反义链选自siRNA53。The combination of the sense strand and the antisense strand is selected from siRNA8, siRNA18, siRNA22, siRNA27-28, siRNA31, siRNA42, siRNA50-51, siRNA53-55, siRNA57, siRNA62, siRNA64, siRNA66-67, siRNA69, siRNA71, siRNA78, siRNA84-88, siRNA94-95, siRNA97-99, siRNA103-104, siRNA109, siRNA112, siRNA298, siRNA304, siRNA310, siRNA320, siRNA331, siRNA332, siRNA340, siRNA350, siRNA360, siRNA370, siRNA380, siRNA390, siRNA401, siRNA410 A combination of the sense strand and the antisense strand of any one of iRNA301-302, siRNA304, siRNA306, siRNA308, siRNA310, siRNA312-313, siRNA317, siRNA319, siRNA321-322, siRNA325, siRNA327-331, siRNA333-334, siRNA336-342, siRNA349, siRNA351-358, siRNA360-363, and siRNA442-443; preferably, the sense strand and the antisense strand are selected from siRNA53.
[11].根据[1]-[10]任一项所述的双链核糖核酸，其中，所述正义链中每个核苷酸彼此独立地为修饰的核苷酸或未修饰的核苷酸，和/或，所述反义链中每个核苷酸彼此独立地为修饰的核苷酸或未修饰的核苷酸。[11]. The double-stranded ribonucleic acid according to any one of [1] to [10], wherein each nucleotide in the sense strand is independently a modified nucleotide or an unmodified nucleotide, and/or each nucleotide in the antisense strand is independently a modified nucleotide or an unmodified nucleotide.
[12].根据[1]-[11]任一项所述的双链核糖核酸，其中，所述正义链中任意相连的两个核苷酸由磷酸二酯键或硫代磷酸二酯键连接，和/或，所述反义链中任意相连的两个核苷酸由磷酸二酯键或硫代磷酸二酯键连接。[12]. The double-stranded RNA according to any one of [1] to [11], wherein any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond, and/or any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.
[13].根据[1]-[12]任一项所述的双链核糖核酸，其中，所述正义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团，和/或，所述反义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团。[13] A double-stranded RNA according to any one of [1] to [12], wherein the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group.
[14].根据[1]-[13]任一项所述的双链核糖核酸，其中，所述双链核糖核酸为siRNA。[14]. The double-stranded RNA according to any one of [1] to [13], wherein the double-stranded RNA is siRNA.
[15].根据[1]-[14]任一项所述的双链核糖核酸，其中，所述双链核糖核酸为用于抑制凝血因子XII基因表达的siRNA。[15] The double-stranded RNA according to any one of [1] to [14], wherein the double-stranded RNA is siRNA for inhibiting the expression of the coagulation factor XII gene.
[16].一种双链核糖核酸修饰物，其为如[1]-[15]任一项所述的双链核糖核酸的修饰物，所述双链核糖核酸修饰物包含如下至少一种的化学修饰：[16] A modified double-stranded RNA, which is a modified double-stranded RNA as described in any one of [1] to [15], wherein the modified double-stranded RNA comprises at least one of the following chemical modifications:
(1)正义链中至少一个核苷酸的修饰，(1) modification of at least one nucleotide in the sense strand,
(2)正义链中至少一个位置处的磷酸二酯键的修饰，(2) modification of the phosphodiester bond at at least one position in the sense strand,
(3)反义链中至少一个核苷酸的修饰，(3) modification of at least one nucleotide in the antisense strand,
(4)反义链中至少一个位置处的磷酸二酯键的修饰；(4) modification of the phosphodiester bond at at least one position in the antisense strand;
任选地，所述双链核糖核酸的正义链中序列A的3’末端连接由1-2个核苷酸组成的序列D，优选由1-2个胸腺嘧啶脱氧核糖核苷酸组成的序列D；和/或，所述双链核糖核酸的反义链中序列B的3’末端连接由1-2个核苷酸组成的序列E，优选由1-2个胸腺嘧啶脱氧核糖核苷酸组成的序列E；和/或，所述双链核糖核酸的正义链中序列A的3’末端排除1-2个核苷酸后形成序列A’；Optionally, the 3' end of sequence A in the sense strand of the double-stranded RNA is connected to a sequence D consisting of 1-2 nucleotides, preferably a sequence D consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence B in the antisense strand of the double-stranded RNA is connected to a sequence E consisting of 1-2 nucleotides, preferably a sequence E consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence A in the sense strand of the double-stranded RNA excludes 1-2 nucleotides to form sequence A';
可选地，所述双链核糖核酸修饰物的正义链和反义链选自如下的序列组合：Optionally, the sense strand and antisense strand of the double-stranded RNA modification are selected from the following sequence combinations:
所述正义链的核苷酸序列为序列A所示的序列，所述反义链的核苷酸序列为序列B所示的序列；The nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
或者，所述正义链的核苷酸序列为序列A所示的序列，所述反义链的核苷酸序列为序列B连接序列E所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E;
或者，所述正义链的核苷酸序列为序列A连接序列D所示的序列，所述反义链的核苷酸序列为序列B所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
或者，所述正义链的核苷酸序列为序列A连接序列D所示的序列，所述反义链的核苷酸序列为序列B连接序列E所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown by sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown by sequence B connected to sequence E;
或者，所述正义链的核苷酸序列为序列A’所示的序列，所述反义链的核苷酸序列为序列B所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
或者，所述正义链的核苷酸序列为序列A’所示的序列，所述反义链的核苷酸序列为序列B连接序 列E所示的序列。Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence B'. The sequence shown in column E.
[17].根据[1]-[16]所述的双链核糖核酸修饰物，其中，所述核苷酸的修饰选自2’-氟代修饰、2’-烷氧基修饰、2’-取代的烷氧基修饰、2’-烷基修饰、2’-取代的烷基修饰、2’-脱氧修饰、核苷酸衍生物修饰或其中任意两种以上的组合。[17]. The double-stranded RNA modified substance according to [1]-[16], wherein the modification of the nucleotide is selected from 2’-fluoro modification, 2’-alkoxy modification, 2’-substituted alkoxy modification, 2’-alkyl modification, 2’-substituted alkyl modification, 2’-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.
[18].根据[16]或[17]所述的双链核糖核酸修饰物，其中，所述核苷酸的修饰选自2’-F修饰、2’-O-CH3修饰、2’-O-CH2-CH2-O-CH3修饰、2’-O-CH2-CH＝CH2修饰、2’-CH2-CH2-CH＝CH2修饰、2’-脱氧修饰、核苷酸衍生物修饰或其中任意两种以上的组合。[18]. The modified double-stranded RNA according to [16] or [17], wherein the modification of the nucleotide is selected from 2'-F modification, 2'-O-CH 3 modification, 2'-O-CH 2 -CH 2 -O-CH 3 modification, 2'-O-CH 2 -CH＝CH 2 modification, 2'-CH 2 -CH 2 -CH＝CH 2 modification, 2'-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.
[19].根据[17]或[18]所述的双链核糖核酸修饰物，其中，所述核苷酸衍生物修饰中的核苷酸衍生物选自异核苷酸、LNA、ENA、cET、UNA或GNA。[19]. The double-stranded RNA modification according to [17] or [18], wherein the nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.
[20].根据[16]-[19]任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述正义链中第7位、第9位、第10位和第11位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述正义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸。[20]. A double-stranded ribonucleic acid modified substance according to any one of [16] to [19], wherein, from the 5' end to the 3' end, the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the sense strand are 2'-O-CH 3 modified ribonucleotides.
[21].根据[16]-[20]任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述正义链包含位于如下所示位置处的硫代磷酸二酯键：[21] The double-stranded RNA modified substance according to any one of [16] to [20], wherein the sense strand comprises a phosphorothioate diester bond located at the following position along the 5' end to the 3' end:
所述正义链5’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;
所述正义链5’末端起始的第2个核苷酸与第3个核苷酸之间；Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand;
所述正义链3’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand;
所述正义链3’末端起始的第2个核苷酸与第3个核苷酸之间；Between the second nucleotide and the third nucleotide starting from the 3' end of the sense strand;
或者，or,
所述正义链包含位于如下所示位置处的硫代磷酸二酯键：The sense strand contains phosphorothioate diester bonds located at the positions shown below:
所述正义链5’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;
所述正义链5’末端起始的第2个核苷酸与第3个核苷酸之间。The positive strand is located between the second and third nucleotides starting from the 5' end.
[22].根据[16]-[21]任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述反义链中任意奇数位置处的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，所述反义链中任意偶数位置处的核糖核苷酸为2’-F修饰的核糖核苷酸；[22] The double-stranded ribonucleic acid modified substance according to any one of [16] to [21], wherein, from the 5' end to the 3' end, the ribonucleotide at any odd-numbered position in the antisense strand is a 2'-O-CH 3 -modified ribonucleotide, and the ribonucleotide at any even-numbered position in the antisense strand is a 2'-F-modified ribonucleotide;
或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第8位、第9位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中第7位的核糖核苷酸为核苷酸衍生物GNA修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
或者，沿5’末端向3’末端方向，所述反义链中第2位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中第6位的核糖核苷酸为核苷酸衍生物GNA修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，且所述反义链中第1位的核糖核苷酸为5’-反式乙烯基膦酸酯核苷酸；Alternatively, along the 5' end to the 3' end direction, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide;
或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第8位、第9位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，且所述反义链中第1位的核糖核苷酸为5’-反式乙烯基膦酸酯核苷酸。Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
[23].根据[16]-[22]任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述反义链的5’末端的核苷酸连接5’磷酸基团或5’磷酸衍生基团。[23] A double-stranded RNA modification according to any one of [16] to [22], wherein the nucleotide at the 5’ end of the antisense strand is linked to a 5’ phosphate group or a 5’ phosphate derivative group in the direction from the 5’ end to the 3’ end.
[24].根据[16]-[23]任一项所述的双链核糖核酸修饰物，其中，所述反义链包含位于如下所示位置 处的硫代磷酸二酯键：[24] The double-stranded RNA modification according to any one of [16] to [23], wherein the antisense strand comprises a The phosphorothioate diester bond at:
所述反义链5’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand;
所述反义链5’末端起始的第2个核苷酸与第3个核苷酸之间；Between the second nucleotide and the third nucleotide starting from the 5' end of the antisense strand;
所述反义链3’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand;
所述反义链3’末端起始的第2个核苷酸与第3个核苷酸之间。Between the second and third nucleotides starting from the 3' end of the antisense strand.
[25].根据[16]-[24]任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物的正义链具有如(a1)-(a5)任一项所示的结构：[25] The double-stranded RNA modified substance according to any one of [16] to [24], wherein the sense strand of the double-stranded RNA modified substance has a structure as shown in any one of (a 1 ) to (a 5 ):
(a1)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-(s)-T-(s)-T-3’，(a 1 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(a2)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(a 2 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(a3)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(a 3 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(a4)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-3’，(a 4 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',
(a5)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-3’；(a 5 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，wherein N 1 -N 23 are independently selected from ribonucleotides whose base is A, U, C or G,
大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，The capital letter T represents a deoxyribonucleotide whose base is thymine.
小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.
小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.
-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接。-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
[26].根据[16]-[25]任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物的反义链具有如(b1)-(b27)任一项所示的结构：[26] The double-stranded RNA modified substance according to any one of [16] to [25], wherein the antisense strand of the double-stranded RNA modified substance has a structure as shown in any one of (b 1 ) to (b 27 ):
(b1)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-T-(s)-T-3’，(b 1 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-T-(s)-T-3',
(b2)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 2 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b3)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 3 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b4)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 4 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
(b5)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-N20f-(s)-mN21-3’，(b 5 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-N 20 f-(s)-mN 21 -3',
(b6)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 6 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b7)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 7 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b8)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 8 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b9)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-N20f-mN21-(s)-N22f-(s)-mN23-3’，(b 9 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -N 20 f-mN 21 -(s)-N 22 f-(s)- mN 23 -3',
(b10)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 10 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b11)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f- mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 11 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f- mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b12)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN1 5-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 12 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 1 5 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 - 3',
(b13)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 13 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
(b14)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’(b 14 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3'
(b15)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 15 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3' ,
(b16)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 16 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b17)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 17 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b18)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 18 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b19)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 19 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b20)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 20 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b21)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(b 21 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
(b22)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 22 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b23)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 23 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b24)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 24 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b25)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 25 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b26)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 26 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b27)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(b 27 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，wherein N 1 -N 23 are independently selected from ribonucleotides whose bases are A, U, C or G,
大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，The capital letter T represents a deoxyribonucleotide whose base is thymine.
小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.
小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.
P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸，P1 means that the nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide.
EVP表示该字母其右侧相邻的一个核苷酸为5’-反式乙烯基膦酸酯核苷酸，EVP means that the nucleotide adjacent to the right of this letter is a 5'-trans vinylphosphonate nucleotide.
-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接，-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
(GNA)表示其右侧相邻的一个核糖核苷酸为存在GNA修饰的核糖核苷酸。(GNA) indicates that the adjacent ribonucleotide on the right is a ribonucleotide with GNA modification.
[27].根据[16]-[26]任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物为siRNA修饰物。[27]. The double-stranded RNA modified substance according to any one of [16] to [26], wherein the double-stranded RNA modified substance is a siRNA modified substance.
[28].根据[16]-[27]任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物为用于抑制凝血因子XII基因表达的siRNA修饰物。 [28] The double-stranded RNA modified substance according to any one of [16] to [27], wherein the double-stranded RNA modified substance is a siRNA modified substance for inhibiting the expression of the coagulation factor XII gene.
[29].根据[16]-[28]任一项所述的双链核糖核酸修饰物，其中，所述正义链与所述反义链的组合为选自如表2所示的siRNA120～224、siRNA365～414、siRNA444～453中任一者的正义链和反义链的组合。[29]. The double-stranded RNA modification product according to any one of [16] to [28], wherein the combination of the sense strand and the antisense strand is a combination of the sense strand and the antisense strand selected from any one of siRNA120 to 224, siRNA365 to 414, and siRNA444 to 453 as shown in Table 2.
[30].一种双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物包括如[1]-[15]任一项所述的双链核糖核酸，或如[16]-[29]任一项所述的双链核糖核酸修饰物；以及，缀合连接于所述双链核糖核酸或所述双链核糖核酸修饰物的缀合基团。[30]. A double-stranded RNA conjugate, wherein the double-stranded RNA conjugate comprises the double-stranded RNA as described in any one of [1]-[15], or the double-stranded RNA modification as described in any one of [16]-[29]; and a conjugated group conjugated to the double-stranded RNA or the double-stranded RNA modification.
[31].根据[30]所述的双链核糖核酸缀合物，其中，所述缀合基团具有如下所示结构：
[31]. The double-stranded RNA conjugate according to [30], wherein the conjugated group has the following structure:
[32].根据[30]或[31]所述的双链核糖核酸缀合物，其中，所述缀合基团连接于正义链的3’末端。[32] The double-stranded RNA conjugate according to [30] or [31], wherein the conjugated group is connected to the 3’ end of the sense strand.
[33].根据[32]所述的双链核糖核酸缀合物，其中，所述缀合基团通过磷酸二酯键与正义链的3’末端缀合连接；[33] The double-stranded RNA conjugate according to [32], wherein the conjugated group is conjugated to the 3' end of the sense strand via a phosphodiester bond;
优选地，所述双链核糖核酸缀合物的正义链与反义链互补形成所述双链核糖核酸缀合物的双链区，且所述正义链的3’末端形成平末端，所述反义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸；Preferably, the sense strand and the antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region;
或者，or,
所述双链核糖核酸缀合物的正义链与反义链互补形成所述双链核糖核酸缀合物的双链区，且所述正义链的3’末端形成平末端，所述反义链的3’末端形成平末端。The sense strand and antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.
[34].根据[30]-[33]任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物具有如下所示结构：
[34]. The double-stranded RNA conjugate according to any one of [30] to [33], wherein the double-stranded RNA conjugate has the following structure:
其中，双螺旋结构为双链核糖核酸或双链核糖核酸修饰物。The double helix structure is double-stranded RNA or a modified double-stranded RNA.
[35].根据[30]-[34]任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物为siRNA缀合物。[35]. The double-stranded RNA conjugate according to any one of [30] to [34], wherein the double-stranded RNA conjugate is a siRNA conjugate.
[36].根据[30]-[35]任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物是用于抑制凝血因子XII基因表达的siRNA缀合物。[36] The double-stranded RNA conjugate according to any one of [30] to [35], wherein the double-stranded RNA conjugate is a siRNA conjugate for inhibiting the expression of the coagulation factor XII gene.
[37].根据[30]-[36]任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物由表1所示的任意一种siRNA与缀合基团连接形成，或者，所述双链核糖核酸缀合物由表1-1所示的任意一种siRNA与缀合基团连接形成，或者，所述双链核糖核酸缀合物由表2所示的任意一种siRNA修饰物与缀合基团连接形成；[37] The double-stranded RNA conjugate according to any one of [30] to [36], wherein the double-stranded RNA conjugate is formed by connecting any one of the siRNAs listed in Table 1 to a conjugation group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNAs listed in Table 1-1 to a conjugation group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNA modifications listed in Table 2 to a conjugation group;
优选地，所述双链核糖核酸缀合物中，其中，所述正义链与所述反义链的组合为选自表3中所示的siRNA225～296、siRNA415～441、siRNA454～464中任一者的正义链和反义链的。Preferably, in the double-stranded ribonucleic acid conjugate, the combination of the sense strand and the antisense strand is selected from the sense strand and the antisense strand of any one of siRNA225-296, siRNA415-441, and siRNA454-464 shown in Table 3.
[38].一种药物组合物，其中，所述药物组合物包括如下至少一项：如[1]-[15]任一项所述的双链 核糖核酸，如[16]-[29]任一项所述的双链核糖核酸修饰物，如[30]-[37]任一项所述的双链核糖核酸缀合物。[38]. A pharmaceutical composition, wherein the pharmaceutical composition comprises at least one of the following: a double-stranded Ribonucleic acid, such as the modified double-stranded ribonucleic acid described in any one of [16]-[29], such as the double-stranded ribonucleic acid conjugate described in any one of [30]-[37].
[39].根据[38]所述的药物组合物，其中，所述药物组合物还包括一种或多种药学上可接受的载体。[39]. The pharmaceutical composition according to [38], wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.
[40].根据[1]-[15]任一项所述的双链核糖核酸，根据[16]-[29]任一项所述的双链核糖核酸修饰物，根据[30]-[37]任一项所述的双链核糖核酸缀合物，或根据[38]-[39]任一项所述的药物组合物在如下至少一项中的用途：[40] Use of the double-stranded RNA according to any one of [1] to [15], the double-stranded RNA modified product according to any one of [16] to [29], the double-stranded RNA conjugate according to any one of [30] to [37], or the pharmaceutical composition according to any one of [38] to [39] in at least one of the following:
(1)抑制凝血因子XII基因表达，或制备用于抑制凝血因子XII基因表达的药物；(1) Inhibiting the expression of coagulation factor XII gene, or preparing a drug for inhibiting the expression of coagulation factor XII gene;
(2)用于预防或治疗与凝血因子XII基因异常表达相关的疾病，或制备用于预防或治疗与凝血因子XII基因异常表达相关的疾病的药物；(2) for preventing or treating diseases related to abnormal expression of coagulation factor XII gene, or for preparing drugs for preventing or treating diseases related to abnormal expression of coagulation factor XII gene;
(3)用于治疗患有将受益于凝血因子XII基因表达降低的疾病的受试者，或制备用于治疗患有将受益于凝血因子XII基因表达降低的疾病的受试者的药物。(3) Use for treating a subject suffering from a disease that would benefit from reduced expression of the coagulation factor XII gene, or for preparing a medicament for treating a subject suffering from a disease that would benefit from reduced expression of the coagulation factor XII gene.
[41].根据[40]所述的用途，其中，所述与凝血因子XII基因异常表达相关的疾病选自如下疾病组成的组：[41]. The use according to [40], wherein the disease associated with abnormal expression of the coagulation factor XII gene is selected from the group consisting of the following diseases:
多发性硬化症、动脉粥样硬化、阿尔兹海默症、遗传性血管水肿、脓毒症、深静脉血栓形成、社区获得性肺炎、新冠肺炎的血栓性炎症、显微镜下型多血管炎、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎及结肠炎以及其他尚未鉴定的相关病症、病理或综合征。Multiple sclerosis, atherosclerosis, Alzheimer's disease, hereditary angioedema, sepsis, deep vein thrombosis, community-acquired pneumonia, thrombotic inflammation of COVID-19, microscopic polyangiitis, neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis and colitis, and other related conditions, pathologies or syndromes not yet identified.
[42].一种用于抑制细胞内凝血因子XII基因表达的方法，其中，所述方法包括将所述细胞与根据[1]-[15]任一项所述的双链核糖核酸，根据[16]-[29]任一项所述的双链核糖核酸修饰物，根据[30]-[37]任一项所述的双链核糖核酸缀合物，或根据[38]-[39]任一项所述的药物组合物接触。[42]. A method for inhibiting the expression of the coagulation factor XII gene in cells, wherein the method comprises contacting the cells with the double-stranded RNA according to any one of [1]-[15], the double-stranded RNA modification according to any one of [16]-[29], the double-stranded RNA conjugate according to any one of [30]-[37], or the pharmaceutical composition according to any one of [38]-[39].
[43].根据[42]所述的方法，其中，所述细胞为体内细胞或体外细胞。[43] The method according to [42], wherein the cells are in vivo cells or in vitro cells.
[44].根据[42]或[43]所述的方法，其中，所述细胞在受试者体内。[44] The method according to [42] or [43], wherein the cell is in a subject.
[45].根据[44]所述的方法，其中，所述受试者为哺乳动物，优选为人。[45] The method according to [44], wherein the subject is a mammal, preferably a human.
[46].根据[44]或[45]所述的方法，其中，所述受试者具有如下至少一种特性：[46] The method according to [44] or [45], wherein the subject has at least one of the following characteristics:
体内凝血因子XII基因异常表达，更具体地为FXII基因异常高表达；Abnormal expression of coagulation factor XII gene in vivo, more specifically abnormally high expression of FXII gene;
患有与凝血因子XII基因异常表达相关的疾病；Suffering from a disease associated with abnormal expression of the coagulation factor XII gene;
患有将受益于凝血因子XII基因表达降低的疾病。Having a disease that would benefit from decreased expression of the factor XII gene.
发明的效果Effects of the Invention
在一些实施方案中，本公开提供的双链核糖核酸，能够在细胞内结合形成RNA诱导沉默复合物(RISC)，切割FXII基因转录的mRNA，高效、特异地抑制FXII基因的表达，用于治疗包括多发性硬化症(MS)、动脉粥样硬化、阿尔兹海默症(AD)、遗传性血管水肿(HAE)、脓毒症、深静脉血栓形成(DVT)、社区获得性肺炎(CAP)、新冠肺炎的血栓性炎症(参与新冠肺炎的促凝血和促炎反应)、显微镜下型多血管炎(MPA)、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎(RA)及结肠炎在内的FXII相关疾病。In some embodiments, the double-stranded RNA provided by the present disclosure can bind to form an RNA-induced silencing complex (RISC) in cells, cut the mRNA transcribed by the FXII gene, and efficiently and specifically inhibit the expression of the FXII gene, and is used to treat FXII-related diseases including multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory responses of COVID-19), microscopic polyangiitis (MPA), neurological inflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
进一步的，本公开中双链核糖核酸为siRNA，siRNA靶向结合并降解FXII基因的转录产物mRNA，发挥RNA干扰的作用，抑制FXII基因的蛋白表达，是一种抑制率高且特异性好的FXII抑制剂。Furthermore, the double-stranded RNA in the present disclosure is siRNA, which targets and degrades the transcription product mRNA of the FXII gene, exerts the effect of RNA interference, and inhibits the protein expression of the FXII gene. It is a FXII inhibitor with high inhibition rate and good specificity.
在一些实施方案中，本公开对双链核糖核酸进行修饰，得到双链核糖核酸修饰物，双链核糖核酸修饰物的稳定性高，适合体内疾病治疗中的应用。In some embodiments, the present disclosure modifies double-stranded RNA to obtain modified double-stranded RNA, which has high stability and is suitable for use in in vivo disease treatment.
进一步的，双链核糖核酸修饰物为siRNA修饰物，具有高的稳定性和较好的抑制活性。Furthermore, the double-stranded RNA modification is a siRNA modification, which has high stability and good inhibitory activity.
在一些实施方案中，本公开在双链核糖核酸、双链核糖核酸修饰物上连接缀合基团得到双链核糖核酸或双链核糖核酸修饰物的缀合物，能够用于向组织、细胞中高效靶向递送，降低双链核糖核酸或双链核糖核酸修饰物对非靶向的正常组织、细胞的影响，提高其在临床疾病治疗中的安全性。In some embodiments, the present invention discloses a conjugate of double-stranded RNA or double-stranded RNA modified substance obtained by connecting a conjugation group to double-stranded RNA or double-stranded RNA modified substance, which can be used for efficient targeted delivery to tissues and cells, reducing the impact of double-stranded RNA or double-stranded RNA modified substance on non-targeted normal tissues and cells, and improving its safety in clinical disease treatment.
进一步的，双链核糖核酸缀合物为siRNA缀合物，在保持siRNA抑制活性、稳定性的同时，兼具器官或组织靶向性，可降低对其他组织或器官的影响以及减少siRNA分子使用量，可达到减轻毒性和降低成本的目的。Furthermore, the double-stranded RNA conjugate is a siRNA conjugate, which has organ or tissue targeting while maintaining the inhibitory activity and stability of siRNA, can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs.
进一步的，本公开中的缀合基团为式I所示结构的基团(GalNAc)，GalNAc可用于向肝脏细胞、 组织内的靶向递送，用于高效抑制肝脏内FXII基因的表达。Furthermore, the conjugated group in the present disclosure is a group (GalNAc) of the structure shown in Formula I. GalNAc can be used to transfer to liver cells, Targeted delivery within tissues is used to efficiently inhibit the expression of FXII gene in the liver.
具体实施方式DETAILED DESCRIPTION
定义definition
除非有相反陈述，否则在本发明中所使用的术语具有下述含义。Unless otherwise stated, the terms used in the present invention have the following meanings.
在本发明的权利要求和/或说明书中，词语“一(a)”或“一(an)”或“一(the)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。In the claims and/or description of the present invention, the word "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。用以界定本发明的数值范围与参数皆是约略的数值，此处已尽可能精确地呈现具体实施例中的相关数值。然而，任何数值本质上不可避免地含有因前述测试方法或装置所致的标准偏差。因此，除非另有明确的说明，应当理解本公开所用的所有范围、数量、数值与百分比均经过“约”的修饰。在此处，“约”通常是指实际数值在一特定数值或范围的正负10％、5％、1％或0.5％之内。Throughout the application document, the term "about" means that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to the aforementioned test methods or devices. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.
本公开上下文中使用的术语“FXII”，是指熟知的基因和多肽。FXII基因、FXII mRNA序列的是例如使用以下容易获得的：基因库(GenBank)、数据库(UniProt)、人类孟德尔遗传在线(OMIM)等。The term "FXII" as used in the context of this disclosure refers to the well-known gene and polypeptide. The FXII gene and FXII mRNA sequence are easily obtained using, for example, the following: Gene Bank (GenBank), database (UniProt), Online Mendelian Inheritance in Man (OMIM), etc.
术语“FXII基因”，可以是野生型FXII基因，或存在序列变异的FXII基因突变体。在FXII基因中的许多序列变异已经被鉴别并且可以发现在例如NCBIdbSNP和UniProt(参见，例如，ncbi.nlm.nih.gov/snp)中。The term "FXII gene" may be a wild-type FXII gene, or a FXII gene mutant with sequence variation. Many sequence variations in the FXII gene have been identified and can be found in, for example, NCBIdbSNP and UniProt (see, for example, ncbi.nlm.nih.gov/snp).
术语“多肽”、“蛋白”可互换地指通过共价键(例如肽键)相互连接的一串至少两个氨基酸残基，可以是重组多肽、天然多肽或合成多肽。多肽可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。The terms "polypeptide", "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. A polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
本公开上下文中使用的术语“靶标序列”是指在靶基因转录期间形成的mRNA分子的核苷酸序列的连续部分，包括作为对初级转录产物进行RNA加工的产物的mRNA。The term "target sequence" as used in the context of the present disclosure refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcript.
在一些可选的实施方案中，靶标序列中可以包含另一段较短的靶标序列。在一些实施方案中，靶标序列中可以包含一个或多个较短的靶标序列。应当认为，被包含于同一段靶标序列中的两个以上的较短靶标序列之间具有相同的特征。In some optional embodiments, the target sequence may include another shorter target sequence. In some embodiments, the target sequence may include one or more shorter target sequences. It should be considered that two or more shorter target sequences included in the same target sequence have the same characteristics.
在一些实施方案中，靶基因为FXII基因。在一些实施方案中，序列的靶部分将会是至少足够地长，以在FXII基因的转录期间形成的mRNA分子的核苷酸序列部分处或其附近充当iRNA指导的切割的底物。In some embodiments, the target gene is the FXII gene.In some embodiments, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the FXII gene.
在本技术领域中，“G”、“C”、“A”、“T”和“U”通常分别代表含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶、尿嘧啶的碱基，但本领域中也通常知晓，“G”、“C”、“A”、“T”和“U”每个通常也代表分别含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶作为碱基的核苷酸，这在表示脱氧核糖核酸序列和/或核糖核酸序列中是常见的方式，因此在本公开的上下文中，“G”、“C”、“A”、“T”、“U”表示的含义包括上述各种可能的情形。然而，应理解术语“核糖核苷酸”或“核苷酸”还可以指一种经修饰的核苷酸(如以下进一步详述)或一种替代性的置换部分。本领域人员可以意识到，鸟嘌呤、胞嘧啶、腺嘌呤以及尿嘧啶可以被其他部分置换而基本上不改变一种寡核苷酸(包括一种具有这种置换部分的核苷酸)的碱基配对特性。例如非限制性地，包括肌苷作为其碱基的核苷酸可以与包括腺嘌呤、胞嘧啶或尿嘧啶的核苷酸进行碱基配对。因此，含有尿嘧啶、鸟嘌呤或腺嘌呤的核苷酸可以在本发明表征的dsRNA的核苷酸序列中由含有例如肌苷的核苷酸替换。在另一个实例中，寡核苷酸中任何地方的腺嘌呤和胞嘧啶可以分别地替换为鸟嘌呤和尿嘧啶，以形成与靶mRNA的G-U摇摆碱基配对。含有这类替换部分的序列适用于本发明表征的组合物和方法。In the art, "G", "C", "A", "T" and "U" generally represent bases containing guanine, cytosine, adenine, thymine and uracil, respectively, but it is also generally known in the art that "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively, which is a common way to represent deoxyribonucleic acid sequences and/or ribonucleic acid sequences, so in the context of the present disclosure, the meanings represented by "G", "C", "A", "T", "U" include the above-mentioned various possible situations. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide (as further described below) or an alternative replacement part. Those skilled in the art can appreciate that guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement part). For example, without limitation, a nucleotide comprising inosine as its base can be base paired with a nucleotide comprising adenine, cytosine or uracil. Therefore, a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing, for example, inosine in the nucleotide sequence of the dsRNA characterized by the present invention. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U wobble base pairing with the target mRNA. Sequences containing such replacement parts are suitable for the compositions and methods characterized by the present invention.
本公开上下文使用的术语“iRNA”、“RNAi试剂”、“iRNA试剂”、“RNA干扰剂”在此可互换使用，是指在此所定义的术语包含siRNA，并且介导通过RNA诱导沉默复合物(RISC)途径的RNA转 录物靶向切割。iRNA通过已知为RNA干扰(RNAi)的过程指导mRNA的序列特异性降解。iRNA调节，例如抑制，靶基因在细胞(如受试者(如哺乳动物受试者)的细胞)中的表达。The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interfering agent" and "RNA interfering agent" used in the context of this disclosure are used interchangeably herein and refer to siRNA as defined herein and mediate RNA transcription through the RNA induced silencing complex (RISC) pathway. iRNAs direct sequence-specific degradation of mRNAs through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of a target gene in a cell, e.g., a cell of a subject, e.g., a mammalian subject.
本公开上下文使用的术语“双链核糖核酸”、“双链RNA(dsRNA)分子”、“dsRNA”可以互换地使用。术语“dsRNA”，是指核糖核酸分子的复合体，其具有双链结构，包含两条反向平行的和基本上互补的核酸链，被称为相对于靶基因，例如FXII基因，具有“正义”和“反义”定向。在一些实施例中，双链核糖核酸(dsRNA)通过转录后基因沉默机制(在此称为RNA干扰或RNAi)触发靶RNA例如mRNA的降解。The terms "double-stranded ribonucleic acid", "double-stranded RNA (dsRNA) molecule", "dsRNA" used in the context of this disclosure can be used interchangeably. The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, referred to as "sense" and "antisense" orientations relative to a target gene, such as a FXII gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).
通常，dsRNA分子的每条链的大部分的核苷酸是核糖核苷酸，但是如在此详述的，两条链的每一者或两者还可以包括一个或多个非核糖核苷酸，例如，脱氧核糖核苷酸和/或修饰的核苷酸。另外，如本公开中所用，“双链核糖核酸”可以包括具有化学修饰的核糖核苷酸、磷酸骨架等等。这些修饰可以包括在此披露的或在本领域中已知的所有类型的修饰。Typically, most of the nucleotides of each strand of the dsRNA molecule are ribonucleotides, but as described in detail herein, each or both of the two strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and/or modified nucleotides. In addition, as used in the present disclosure, "double-stranded RNA" may include ribonucleotides, phosphate backbones, etc., with chemical modifications. These modifications may include all types of modifications disclosed herein or known in the art.
本公开上下文使用的术语“异核苷酸”是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物，例如，碱基不与核糖环的1’-位相连，而是与核糖环的2’-位或3’-位相连而形成的化合物。The term "isonucleotide" as used in the context of the present disclosure refers to a compound formed by a change in the position of the base on the ribose ring in a nucleotide, for example, a compound formed by a base not being attached to the 1'-position of the ribose ring but being attached to the 2'-position or 3'-position of the ribose ring.
在一些实施方案中，本公开的双链核糖核酸是siRNA，其与靶基因转录的mRNA序列(例如C基因转录的mRNA序列)相互作用以指导靶RNA的切割。不希望受理论约束，引入细胞中的长双链RNA被称作Dicer的III型核酸内切酶分解成siRNA(夏普(Sharp)等人，《基因与发育》(Genes Dev.)2001，15:485)。Dicer(核糖核酸酶III样酶)将dsRNA加工成至具有特征性双碱基3’突出端的19-23碱基对短干扰性RNA(Bernstein等人，(2001)自然(Nature)409:363)。这些siRNA随后掺入RNA诱导性沉默复合物(RISC)中，在其中一种或多种解旋酶解开siRNA双链体，这使得互补性反义链指导靶识别成为可能(Nykanen等人，(2001)细胞(Cell)107:309)。一旦与适宜的靶mRNA结合，RISC内部的一种或多种核酸内切酶切割靶以诱导沉默(巴希尔(Elbashir)等人，(2001)《基因与发育》(Genes Dev.)15:188)。In some embodiments, the double-stranded ribonucleic acid disclosed herein is siRNA, which interacts with the mRNA sequence transcribed by the target gene (e.g., the mRNA sequence transcribed by the C gene) to guide the cutting of the target RNA. Without wishing to be bound by theory, the long double-stranded RNA introduced into the cell is broken down into siRNA by a type III nuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485). Dicer (ribonuclease III-like enzyme) processes dsRNA into 19-23 base pairs of short interfering RNA with characteristic double base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). These siRNAs are then incorporated into RNA-induced silencing complexes (RISC), in which one or more helicases unwind the siRNA duplex, which makes it possible for complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107: 309). Once bound to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al. (2001) Genes Dev. 15:188).
本公开上下文使用的术语“突出的核苷酸”是指当双链核糖核酸的一条链的一个3’端延伸超出另一条链的5’端时从该dsRNA的双链体结构突出的一个或多个不成对的核苷酸，或反之亦然。“平端”或“平末端”意指在该双链核糖核酸的那端处不存在不成对的核苷酸，即无核苷酸突出端。一种“平末端的”双链核糖核酸是一种在其整个长度上都是双链、即在该分子的任一端处都无核苷酸突出端的dsRNA。The term "overhanging nucleotides" as used in the context of this disclosure refers to one or more unpaired nucleotides that protrude from the duplex structure of a double-stranded ribonucleic acid when a 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. "Blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded ribonucleic acid, i.e., no nucleotide overhang. A "blunt-ended" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its length, i.e., has no nucleotide overhangs at either end of the molecule.
术语“反义链”是指双链核糖核酸中与靶标序列(例如，来源于人类FXII mRNA)基本上互补的一个区域的链。在该互补性区域不与该靶标序列完全互补的情况下，错配在末端区域是最为可容忍的，并且如果出现错配，它们通常在末端的一个或多个区域，例如5’和/或3’末端的5、4、3、2或1个核苷酸之内。The term "antisense strand" refers to a strand of a double-stranded RNA that is substantially complementary to a target sequence (e.g., derived from human FXII mRNA). Where the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if mismatches occur, they are typically within one or more regions of the termini, e.g., 5, 4, 3, 2, or 1 nucleotides of the 5' and/or 3' termini.
术语“正义链”指的双链核糖核酸中含有与反义链区域基本上互补的区域的核酸链。The term "sense strand" refers to the nucleic acid strand of a double-stranded RNA that contains a region that is substantially complementary to a region of the antisense strand.
术语“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U))相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。The terms "complementary" or "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
术语“基本上反向互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配，即所涉及的两段核苷酸序列之间存在1个、2个或3个的碱基错配；“完全互补”是指两段核苷酸序列之间不存在碱基错配。The term "substantially reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved, that is, there are 1, 2 or 3 base mismatches between the two nucleotide sequences involved; "completely complementary" means that there are no base mismatches between the two nucleotide sequences.
术语“互补”、“完全互补”和“基本上互补”可相对于在dsRNA的正义链与反义链之间，或dsRNA的反义链与靶标序列之间的碱基配对使用，如将从其使用的上下文理解。The terms "complementary," "fully complementary," and "substantially complementary" may be used with respect to base pairing between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as would be understood from the context of their use.
术语“抑制”，可以与“减少”、“沉默”、“下调”、“压制”和其他类似术语交替使用，并且包括任何水平的抑制。 The term "inhibit" may be used interchangeably with "reduce,""silence,""downregulate,""suppress," and other similar terms, and includes any level of inhibition.
术语“抑制FXII基因的表达”包括抑制任何FXII基因(例如小鼠FXII基因、大鼠FXII基因、猴FXII基因、或人类FXII基因)以及FXII基因的变体(例如天然存在的变体)或突变体的表达。因此，该FXII基因可以是野生型FXII基因、突变FXII基因、或在遗传操作的细胞、细胞群组或生物体的情形下的转基因FXII基因。The term "inhibiting the expression of a FXII gene" includes inhibiting the expression of any FXII gene (e.g., a mouse FXII gene, a rat FXII gene, a monkey FXII gene, or a human FXII gene) and variants (e.g., naturally occurring variants) or mutants of a FXII gene. Thus, the FXII gene may be a wild-type FXII gene, a mutant FXII gene, or a transgenic FXII gene in the context of a genetically manipulated cell, cell group, or organism.
“抑制FXII基因表达”包括任何水平的FXII基因的抑制，例如至少部分抑制FXII基因的表达，如抑制至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％。"Inhibiting FXII gene expression" includes any level of inhibition of the FXII gene, such as at least partial inhibition of the expression of the FXII gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
术语“各自独立地”是指结构中存在的取值范围相同或相近的至少两个基团(或环系)可以在特定情形下具有相同或不同的含义。例如，取代基X和取代基Y各自独立地为氢、羟基、烷基或芳基，则当取代基X为氢时，取代基Y既可以为氢，也可以为羟基、烷基或芳基；同理，当取代基Y为氢时，取代基X既可以为氢，也可以为羟基、烷基或芳基。The term "independently" means that at least two groups (or ring systems) with the same or similar value ranges in the structure may have the same or different meanings in specific circumstances. For example, substituent X and substituent Y are independently hydrogen, hydroxyl, alkyl or aryl. When substituent X is hydrogen, substituent Y can be either hydrogen, hydroxyl, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen, hydroxyl, alkyl or aryl.
术语“烷基”包括直链、支链或环状的饱和烷基。例如，烷基包括但不限于甲基、乙基、丙基、环丙基、正丁基、异丁基、仲丁基、叔丁基、环丁基、正戊基、环已基等类似基团。示例性的，“C1-6烷基”中的“C1-6”是指包含有1、2、3、4、5或6个碳原子的直链、支链或环状形式排列的基团。The term "alkyl" includes straight chain, branched or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl and the like. Exemplarily, "C 1-6 " in "C 1-6 alkyl" refers to a group having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched or cyclic form.
术语“烷氧基”在本文中是指烷基基团通过氧原子与分子其余部分相连(-O-烷基)，其中所述烷基如本文中所定义。烷氧基的非限制性实例包括甲氧基、乙氧基、三氟甲氧基、二氟甲氧基、正丙氧基、异丙氧基、正丁氧基、叔丁氧基、正戊氧基等。The term "alkoxy" herein refers to an alkyl group attached to the remainder of the molecule via an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, and the like.
术语“治疗”是指：在罹患疾病之后，使受试者接触(例如给药)双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物、药物组合物，从而与不接触时相比使该疾病的症状减轻，并不意味着必需完全抑制疾病的症状。罹患疾病是指：身体出现了疾病症状。The term "treatment" means that after suffering from a disease, a subject is exposed to (e.g., administered) double-stranded RNA, a double-stranded RNA modified substance, a double-stranded RNA conjugate, or a pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when the subject is not exposed to the disease, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.
术语“预防”是指：在罹患疾病之前，通过使受试者接触(例如给药)本公开的双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物、药物组合物，从而与不接触时相比减轻罹患疾病后的症状，并不意味着必需完全抑制患病。The term "prevention" means that before a subject develops a disease, by contacting (e.g., administering) the double-stranded RNA, double-stranded RNA modified material, double-stranded RNA conjugate, or pharmaceutical composition of the present disclosure, the symptoms after the subject develops the disease are alleviated compared to when the subject does not develop the disease. It does not mean that the disease must be completely suppressed.
术语“有效量”指本发明的双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物或药物组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。The term "effective amount" refers to such an amount or dosage of the double-stranded RNA, double-stranded RNA modification, double-stranded RNA conjugate or pharmaceutical composition of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses. The effective amount can be easily determined by the attending physician who is a person skilled in the art by considering a variety of factors such as the species of the mammal; its size, age and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
术语“与FXII基因异常表达相关的疾病”是与胆固醇、类固醇和其他脂类的合成相关联的疾病或障碍。术语“与FXII基因异常表达相关的疾病”包括将从减少FXII(即“FXII-相关性疾病”)表达受益的疾病、障碍或病症。此类疾病典型地与凝血与炎症相关。FXII基因异常表达相关的疾病的非限制性实例包括：多发性硬化症(MS)、动脉粥样硬化、阿尔兹海默症(AD)、遗传性血管水肿(HAE)、脓毒症、深静脉血栓形成(DVT)、社区获得性肺炎(CAP)、新冠肺炎的血栓性炎症(参与新冠肺炎的促凝血和促炎反应)、显微镜下型多血管炎(MPA)、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎(RA)及结肠炎等(Miroslava Didiasova等，Factor XII in coagulation,inflammation and beyond.Cell Signal.2018Nov；51:257-265.doi:10.1016/j.cellsig.2018.08.006.Epub 2018 Aug 15；Kerstin等,The Coagulation Factors Fibrinogen,Thrombin,and Factor XII in Inflammatory Disorders-A Systematic Review.Front Immunol.2018Jul 26；9:1731.doi:10.3389/fimmu.2018.01731.eCollection 2018Gobel K等,Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells.Nat Commun.2016；7(1):11626.；Sandra Vorlova等,Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice.Thromb Haemost.2017Jan 5；117(1):176-187.；Daria Zamolodchikov等,Activation of the factor XII-driven contact system in Alzheimer's disease patient and mouse model plasma. Proc Natl Acad Sci U S A.2015Mar 31；112(13):4068-73.；Longhurst HJ等,Hereditary angioedema:an update on causes,manifestations and treatment.Br J Hosp Med(Lond).2019；80(7):391-398.；Jansen PM等,Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6and neutrophil elastase.Blood.1996；87(6):2337-2344.；Pixley RA等,The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia.In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons.J Clin Invest.1993；91(1):61-68.；Yan Meng等,FXII regulates the formation of deep vein thrombosis via the PI3K/AKT signaling pathway in mice Int J Mol Med.2021May；47(5):87.；Kristin Ehrlich等,Sex-specific differences in plasma levels of FXII,HK,and FXIIa-C1-esterase inhibitor complexes in community-acquired pneumonia.Am J Physiol Lung Cell Mol Physiol.2021Oct 1；321(4):L764-L774.；Hanna Englert等,Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation.EBioMedicine.2021May；67:103382；罗昊军，FXII/uPAR通路在显微镜下多血管炎NETs形成中的作用和机制研究.电子科技大学,2019；Stavrou E X等,Factor XII and uPAR upregulate neutrophil functions to influence wound healing.J Clin Invest,2018,128(3):944-959.；Gobel K等,Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells.Nature Communications,2016,7:11626.；Katrin F等,Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases.Arteriosclerosis,Thrombosis,and Vascular BiologyVolume 37,Issue 1,January 2017,Pages 13-20；C Maas and T Renné,Coagulation factor XII in thrombosis and inflammation.Blood,Volume 131,Issue 17,26April 2018,Pages 1903-1909)。The term "disease associated with abnormal expression of the FXII gene" is a disease or disorder associated with the synthesis of cholesterol, steroids and other lipids. The term "disease associated with abnormal expression of the FXII gene" includes diseases, disorders or conditions that will benefit from reducing the expression of FXII (i.e., "FXII-related diseases"). Such diseases are typically associated with coagulation and inflammation. Non-limiting examples of diseases associated with abnormal expression of the FXII gene include: multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory responses of COVID-19), microscopic polyangiitis (MPA), neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis (Miroslava Didiasova et al., Factor XII in coagulation, inflammation and beyond. Cell Signal. 2018 Nov; 51: 257-265. doi: 10.1016/j.cellsig.2018.08.006. Epub 2018 Aug 15; Kerstin ... et al., The Coagulation Factors Fibrinogen, Thrombin, and Factor XII in Inflammatory Disorders-A Systematic Review. Front Immunol.2018Jul 26;9:1731.doi:10.3389/fimmu.2018.01731.eCollection 2018Gobel K et al., Blood coagulation factor cells. Nat Commun. 2016; 7(1): 11626.; Sandra Vorlova et al., Coagulation factor Alzheimer's disease patient and mouse model plasma. Proc Natl Acad Sci U S A. 2015 Mar 31; 112(13): 4068-73.; Longhurst HJ et al., Hereditary angioedema: an update on causes, manifestations and treatment. Br J Hosp Med (Lond). 2019; 80(7): 391-398.; Jansen PM et al., Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6and neutrophil elastase. Blood. 1996; 87(6): 2337-2344.; Pixley RA et al., The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons.J Clin Invest.1993;91(1):61-68.; Yan Meng et al., FXII regulates the formation of deep vein thrombosis via the PI3K/AKT signaling pathway in mice Int J Mol Med.2021May;47(5):87.; Kristin Ehrlich et al., Sex-specific differences in plasma levels of FXII,HK,and FXIIa-C1-esterase inhibitor complexes in community-acquired pneumonia.Am J Phy siol Lung Cell Mol Physiol.2021Oct 1;321(4):L764-L774.;Hanna Englert et al., Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation.EBioMedicine.2021May；67:103382；Luo Haojun, Role and mechanism of FXII/uPAR pathway in NETs formation in microscopic polyangiitis.University of Electronic Science and Technology of China,2019；Stavrou E X et al.,Factor XII and uPAR upregulate neutrophil functions to influence wound healing.J Clin Invest,2018,128(3):944-959.；Gobel K et al.,Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells.Nature Communications,2016,7:11626.；Katrin F et al.,Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases.Arteriosclerosis,Thrombosis,and Vascular BiologyVolume 37,Issue 1,January 2017,Pages 13-20; C Maas and T Renné, Coagulation factor XII in thrombosis and inflammation. Blood, Volume 131, Issue 17, 26 April 2018, Pages 1903-1909).
术语“药学上可接受的辅料”或“药学上可接受的载体”是指在药物生产领域中广泛采用的辅助物料。使用辅料的主要目的在于提供一种使用安全、性质稳定和/或具有特定功能性的药物组合物，还在于提供一种方法，以便在为受试者施用药物之后，活性成分能够以所期望的速率溶出，或者促进活性成分在接受给药的受试者体内得到有效吸收。药学上可接受的辅料可以是具有惰性的填充剂，也可以是为药用组合物提供某种功能(例如稳定组合物的整体pH值或防止组合物中活性成分的降解)的功效成分。药学上可接受的辅料的非限制性实例包括但不限于粘合剂、助悬剂、乳化剂、稀释剂(或填充剂)、成粒剂、胶粘剂、崩解剂、润滑剂、抗粘着剂、助流剂、润湿剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂、甜味剂等。The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to auxiliary materials widely used in the field of drug production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be dissolved at a desired rate, or to promote the effective absorption of the active ingredient in the subject receiving the drug. Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a certain function to the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.
本公开中的药物组合物可以使用本领域技术人员已知的任何方法来制备。例如，常规混合、溶解、造粒、乳化、磨细、包封、包埋和/或冻干工艺。The pharmaceutical compositions of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, embedding and/or lyophilizing processes.
在本公开中，施用途经能够以任何适用的方式进行变化或调整，以满足药物的性质、患者和医务人员的便利以及其它相关因素的需求。In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.
本公开上下文中使用的术语“个体”、“患者”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。The terms "individual", "patient" or "subject" used in the context of this disclosure include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.
双链核糖核酸dsRNA
本公开的第一方面提供一种双链核糖核酸(dsRNA)，用于抑制FXII基因的表达。双链核糖核酸的一条链为反义链，反义链与靶基因(也即，FXII基因)在表达过程中形成的mRNA序列互补配对，用于指导靶mRNA(也即，FXII基因的转录产物)的切割。双链核糖核酸中另一条正义链包括与反义链部分互补和完全互补形成双链核糖核酸的双链区。The first aspect of the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the FXII gene. One strand of the double-stranded ribonucleic acid is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the FXII gene) and is used to guide the cutting of the target mRNA (i.e., the transcription product of the FXII gene). The other sense strand in the double-stranded ribonucleic acid includes a double-stranded region that is partially complementary and completely complementary to the antisense strand to form the double-stranded ribonucleic acid.
在一些实施方案中，双链核糖核酸作为核酸内切酶(Dicer)的底物，被切割为小片段的dsRNA，也即siRNA。在一些实施方案中，双链核糖核酸为siRNA。siRNA通过装配形成RNA诱导的沉默复合物(RNA-induced silencing complex，RISC)RISC复合体，切割靶mRNA，抑制FXII基因的表达。 In some embodiments, the double-stranded RNA is used as a substrate for the endonuclease (Dicer) and is cut into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded RNA is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC) RISC complex, cuts the target mRNA, and inhibits the expression of the FXII gene.
根据来源于人FXII mRNA(NM_000505.4)中的靶标序列，设计与靶mRNA结合的siRNA。在一些实施方案中，靶标序列选自如SEQ ID NO:1～15，641～648任一项所示的核苷酸序列。在一些更为具体的实施方案中，靶标序列选自如SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列。According to the target sequence derived from human FXII mRNA (NM_000505.4), siRNA that binds to the target mRNA is designed. In some embodiments, the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 1 to 15, 641 to 648. In some more specific embodiments, the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 2, 4 to 6, 8 to 10, 13 to 14, 16 to 29, 644, 645, 647, 649 to 666.
在一些具体的实施方案中，SEQ ID NO:1所示的核苷酸序列包含SEQ ID NO:16～17所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:1 contains the nucleotide sequence shown in SEQ ID NO:16-17.
在一些具体的实施方案中，SEQ ID NO:3所示的核苷酸序列包含SEQ ID NO:18～21所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:3 contains the nucleotide sequence shown in SEQ ID NO:18-21.
在一些具体的实施方案中，SEQ ID NO:7所示的核苷酸序列包含SEQ ID NO:22～23所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:7 contains the nucleotide sequence shown in SEQ ID NO:22-23.
在一些具体的实施方案中，SEQ ID NO:11所示的核苷酸序列包含SEQ ID NO:24～25所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:11 contains the nucleotide sequence shown in SEQ ID NO:24-25.
在一些具体的实施方案中，SEQ ID NO:12所示的核苷酸序列包含SEQ ID NO:26～27所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:12 contains the nucleotide sequence shown in SEQ ID NO:26-27.
在一些具体的实施方案中，SEQ ID NO:15所示的核苷酸序列包含SEQ ID NO:28～29所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:15 contains the nucleotide sequence shown in SEQ ID NO:28-29.
在一些具体的实施方案中，SEQ ID NO:641所示的核苷酸序列包含SEQ ID NO:649～650所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:641 contains the nucleotide sequence shown in SEQ ID NO:649-650.
在一些具体的实施方案中，SEQ ID NO:642所示的核苷酸序列包含SEQ ID NO:651～656所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:642 contains the nucleotide sequence shown in SEQ ID NO:651 to 656.
在一些具体的实施方案中，SEQ ID NO:643所示的核苷酸序列包含SEQ ID NO:657～659所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:643 contains the nucleotide sequence shown in SEQ ID NO:657 to 659.
在一些具体的实施方案中，SEQ ID NO:646所示的核苷酸序列包含SEQ ID NO:660～662所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:646 contains the nucleotide sequence shown in SEQ ID NO:660-662.
在一些具体的实施方案中，SEQ ID NO:648所示的核苷酸序列包含SEQ ID NO:663～666所示的核苷酸序列。In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:648 contains the nucleotide sequence shown in SEQ ID NO:663 to 666.
在一些实施方案中，反义链包含与靶标序列中至少15个连续核苷酸的反向互补序列的差异不超过3个核苷酸的序列B。具体地，沿5’末端向3’末端的方向，在靶标序列中选择起始核苷酸，以包含起始核苷酸在内的向3’方向延伸的至少15个核苷酸作为siRNA的结合区域。反义链包含结合区域对应的核苷酸序列的反向互补序列。需要说明的是，起始核苷酸可以是靶标序列任意位置处的核苷酸，只要基于该起始核苷酸向靶标序列3’方向延伸，可以得到至少15个连续核苷酸(包含起始位置处的核苷酸)即可。In some embodiments, the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence. Specifically, along the direction from the 5' end to the 3' end, the starting nucleotide is selected in the target sequence, and at least 15 nucleotides extending in the 3' direction including the starting nucleotide are used as the binding region of the siRNA. The antisense strand comprises the reverse complementary sequence of the nucleotide sequence corresponding to the binding region. It should be noted that the starting nucleotide can be a nucleotide at any position of the target sequence, as long as at least 15 consecutive nucleotides (including the nucleotide at the starting position) can be obtained by extending in the 3' direction of the target sequence based on the starting nucleotide.
在本公开中，反义链的核苷酸序列与靶标序列可以是完全互补或基本上互补。当反义链的核苷酸序列与靶标序列基本上互补时，反义链的核苷酸序列中存在与靶标序列存在不超过3个的错配碱基。例如，错配碱基为1个、2个或3个。当反义链的核苷酸序列与靶标序列完全互补时，反义链的核苷酸序列与靶标序列不存在错配碱基。In the present disclosure, the nucleotide sequence of the antisense strand and the target sequence can be completely complementary or substantially complementary. When the nucleotide sequence of the antisense strand and the target sequence are substantially complementary, there are no more than 3 mismatched bases in the nucleotide sequence of the antisense strand and the target sequence. For example, the mismatched bases are 1, 2 or 3. When the nucleotide sequence of the antisense strand and the target sequence are completely complementary, there are no mismatched bases in the nucleotide sequence of the antisense strand and the target sequence.
进一步地，反义链由至少15个核苷酸组成。在一些实施方案中，反义链由15-28个核苷酸组成。例如，反义链的长度为15、16、17、18、19、20、21、22、23、24、25、26、27或28个核苷酸。Further, the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.
作为优选，反义链由18-25个核苷酸组成，更优选18-23个核苷酸，最优选19、21或23个核苷酸。Preferably, the antisense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, most preferably 19, 21 or 23 nucleotides.
在一些可选的实施方案中，反义链包含的序列B与靶标序列上至少15个连续核苷酸组成的序列的反向互补序列相同。In some optional embodiments, the sequence B included in the antisense strand is identical to the reverse complementary sequence of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.
在一些具体的实施方案中，反义链包含的序列B与靶标序列上18-25个连续核苷酸组成的序列的反向互补序列相同。优选靶标序列上19-25个连续核苷酸，更优选靶标序列上19-23个连续核苷酸，最优选19、21或23个连续核苷酸。 In some specific embodiments, the sequence B included in the antisense strand is identical to the reverse complementary sequence of a sequence consisting of 18-25 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
在一些可选的实施方案中，反义链包含的序列B与靶标序列上至少15个连续核苷酸组成的序列的反向互补序列存在1个核苷酸的差异。In some optional embodiments, the sequence B comprised in the antisense strand differs by 1 nucleotide from the reverse complementary sequence of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.
在一些具体的实施方案中，反义链包含的序列B与靶标序列上18-25个核苷酸组成的序列的反向互补序列存在1个核苷酸的差异。优选靶标序列上19-25个连续核苷酸，更优选靶标序列上19-23个连续核苷酸，最优选19、21或23个连续核苷酸。In some specific embodiments, the sequence B included in the antisense strand differs by 1 nucleotide from the reverse complementary sequence of a sequence consisting of 18-25 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
在一些具体的实施方案中，反义链包含的序列B与靶标序列上15-28个核苷酸组成的序列的反向互补序列存在2个核苷酸的差异。优选靶标序列上19-25个连续核苷酸，更优选靶标序列上19-23个连续核苷酸，最优选19、21或23个连续核苷酸。In some specific embodiments, the sequence B included in the antisense strand differs by 2 nucleotides from the reverse complementary sequence of a sequence consisting of 15-28 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
在一些具体的实施方案中，存在差异的核苷酸位于序列B的3’末端。在另一些具体的实施方案中，存在差异的核苷酸位于序列B的5’末端。在又一些具体的实施方案中，存在差异的核苷酸位于序列B的3’末端和5’末端。In some specific embodiments, the different nucleotides are located at the 3' end of sequence B. In other specific embodiments, the different nucleotides are located at the 5' end of sequence B. In still other specific embodiments, the different nucleotides are located at both the 3' end and the 5' end of sequence B.
在一些实施方案中，所述正义链包含与靶标序列中至少15个连续核苷酸的差异不超过3个核苷酸的序列A。正义链中包括与反义链互补的区域，正义链的核苷酸序列与反义链在靶标序列上结合区域的序列完全相同或基本完全相同。因此，正义链的核苷酸序列为靶标序列中结合反义链的至少15个连续核苷酸；或者，正义链的核苷酸序列与靶标序列中结合反义链的至少15个连续核苷酸相比，存在1个、2个或3个碱基不同的差异核苷酸。In some embodiments, the sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence. The sense strand includes a region complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the region where the antisense strand binds to the target sequence. Therefore, the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that bind to the antisense strand; or, the nucleotide sequence of the sense strand is compared with at least 15 consecutive nucleotides in the target sequence that bind to the antisense strand, and there are 1, 2, or 3 different nucleotides in the nucleotide sequence of the sense strand.
进一步地，正义链由至少15个核苷酸组成。在一些实施方案中，正义链由15-28个核苷酸组成。例如，正义链的长度为15、16、17、18、19、20、21、22、23、24、25、26、27或28个核苷酸。Further, the sense strand is composed of at least 15 nucleotides. In some embodiments, the sense strand is composed of 15-28 nucleotides. For example, the length of the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.
作为优选，正义链由18-25个核苷酸组成，更优选18-23个核苷酸，最优选19、21或23个核苷酸。Preferably, the sense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, most preferably 19, 21 or 23 nucleotides.
在一些可选的实施方案中，正义链包含的序列A与靶标序列上至少15个连续核苷酸组成的序列相同。In some optional embodiments, the sequence A included in the sense strand is identical to a sequence consisting of at least 15 consecutive nucleotides in the target sequence.
在一些具体的实施方案中，正义链包含的序列A与靶标序列上18-25个连续核苷酸组成的序列相同。优选靶标序列上19-25个连续核苷酸，更优选靶标序列上19-23个连续核苷酸，最优选靶标序列上19、21或23个连续核苷酸。In some specific embodiments, the sequence A included in the sense strand is identical to a sequence consisting of 18-25 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides on the target sequence.
在一些可选的实施方案中，正义链包含的序列A与靶标序列上至少15个连续核苷酸组成的序列存在1个核苷酸的差异。In some optional embodiments, the sequence A included in the sense strand differs by 1 nucleotide from a sequence consisting of at least 15 consecutive nucleotides in the target sequence.
在一些具体的实施方案中，正义链包含的序列A与靶标序列上18-25个连续核苷酸组成的序列存在1个核苷酸的差异。优选靶标序列上19-25个连续核苷酸，更优选靶标序列上19-23个连续核苷酸，最优选靶标序列上19、21或23个连续核苷酸。In some specific embodiments, the sequence A included in the sense strand differs by 1 nucleotide from a sequence consisting of 18-25 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides on the target sequence.
在一些具体的实施方案中，正义链包含的序列A与靶标序列上18-25个核苷酸组成的序列的反向互补序列存在2个核苷酸的差异。优选靶标序列上19-25个连续核苷酸，更优选靶标序列上19-23个连续核苷酸，最优选19、21或23个连续核苷酸。In some specific embodiments, the sequence A included in the sense strand differs by 2 nucleotides from the reverse complementary sequence of a sequence consisting of 18-25 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.
在一些具体的实施方案中，存在差异的核苷酸位于序列A的3’末端。在另一些具体的实施方案中，存在差异的核苷酸位于序列A的5’末端。在又一些具体的实施方案中，存在差异的核苷酸位于序列A的3’末端和5’末端。In some specific embodiments, the different nucleotides are located at the 3' end of sequence A. In other specific embodiments, the different nucleotides are located at the 5' end of sequence A. In still other specific embodiments, the different nucleotides are located at both the 3' end and the 5' end of sequence A.
在本公开中，正义链的长度与反义链长度可以相同或不同。In the present disclosure, the length of the sense strand and the length of the antisense strand may be the same or different.
在一些实施方式中，正义链与反义链的长度相同，具体地，正义链/反义链的长度比为15/15、16/16、17/17、18/18、19/19、20/20、21/21、22/22、23/23、24/24、25/25、26/26、27/27或28/28。作为优选，正义链/反义链的长度比为18/18、19/19、20/20、21/21、22/22、23/23、24/24或25/25，更优选19/19、20/20、21/21、22/22或23/23，最优选18/18、19/19、21/21或23/23。In some embodiments, the sense strand and the antisense strand are the same length, specifically, the length ratio of the sense strand/antisense strand is 15/15, 16/16, 17/17, 18/18, 19/19, 20/20, 21/21, 22/22, 23/23, 24/24, 25/25, 26/26, 27/27 or 28/28. Preferably, the length ratio of the sense strand/antisense strand is 18/18, 19/19, 20/20, 21/21, 22/22, 23/23, 24/24 or 25/25, more preferably 19/19, 20/20, 21/21, 22/22 or 23/23, and most preferably 18/18, 19/19, 21/21 or 23/23.
在一些实施方式中，正义链与反义链的长度不同。例如，正义链/反义链的长度比为18/19、18/20、18/21、18/22、18/23、18/24、18/25、18/26、19/18、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/19、20/21、20/22、20/23、20/24、20/25、20/26、21/18、21/19、21/20、21/22、21/23、21/24、21/25、21/26、22/18、22/19、22/20、22/21、22/23、22/24、22/25、22/26、23/19、23/20、23/21、23/22、23/24、 23/25或23/26等等；在一些优选的实施方式中，正义链/反义链的长度比为19/21、20/22或21/23。In some embodiments, the sense strand is different from the antisense strand in length. For example, the sense strand/antisense strand length ratio is 18/19, 18/20, 18/21, 18/22, 18/23, 18/24, 18/25, 18/26, 19/18, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/19, 20/21, 20/22, 20/23, 20/2 4. 20/25, 20/26, 21/18, 21/19, 21/20, 21/22, 21/23, 21/24, 21/25, 21/26, 22/18, 22/19, 22/20, 22/21, 22/23, 22/24, 22/25, 22/26, 23/19, 23/20, 23/21, 23/22, 23/24, 23/25 or 23/26, etc.; in some preferred embodiments, the length ratio of the sense strand/antisense strand is 19/21, 20/22 or 21/23.
在本公开中，正义链与反义链可以是完全互补或基本上互补，当两者基本上互补时，正义链与反义链形成的双链区内存在不超过3个错配碱基。In the present disclosure, the sense strand and the antisense strand may be completely complementary or substantially complementary. When the two are substantially complementary, there are no more than 3 mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.
在一些实施方案中，正义链与反义链互补形成双链区后，正义链、反义链或其组合具有延伸出所述双链区的突出的核苷酸。突出的核苷酸的数量可以是1个或多个，例如，1个或2个。另外，突出1-2个核苷酸可以位于任意反义链或正义链的5’末端、3’末端或两端，并且，每一个突出的核苷酸可以是任意类型的核苷酸。In some embodiments, after the sense strand and the antisense strand complement each other to form a double-stranded region, the sense strand, the antisense strand, or a combination thereof has protruding nucleotides extending out of the double-stranded region. The number of protruding nucleotides may be 1 or more, for example, 1 or 2. In addition, the protruding 1-2 nucleotides may be located at the 5' end, 3' end, or both ends of any antisense strand or sense strand, and each protruding nucleotide may be any type of nucleotide.
在一些实施方案中，所述正义链与所述反义链互补形成所述双链区，且所述正义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，所述反义链的3’末端形成平末端。In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end.
在一些实施方案中，所述正义链与所述反义链互补形成所述双链区，且所述反义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，所述正义链的3’末端形成平末端。In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end.
在一些实施方案中，所述正义链与所述反义链互补形成所述双链区，且所述正义链与所述反义链的3’末端均具有1-2个延伸出所述双链区的突出的核苷酸。In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region.
在一些实施方案中，所述正义链与所述反义链互补形成所述双链区，且所述正义链与所述反义链的3’末端均形成平末端。In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.
在本公开中，如上所述，反义链的核苷酸序列与靶标序列可以是完全互补或基本上互补；正义链与反义链可以是完全互补或基本上互补。因此，对于靶标序列以及能够与这些靶标序列互补的siRNA，对于每个siRNA的反义链，均包括与其互补的靶标序列基本上互补的情况，也即，每个siRNA的反义链的核苷酸序列中可以存在与相应的靶标序列存在碱基错配的情况；对于每个siRNA的正义链，均包括与其靶标序列基本上一致的情况，也即，每个siRNA的正义链的核苷酸序列中可以存在与相应的靶标序列存在碱基差异的情况。在一些实施方案中，所述碱基错配或碱基差异可以是与靶标序列的差异不超过3个碱基的错配或差异，例如，错配碱基或差异碱基为1个、2个或3个。In the present disclosure, as described above, the nucleotide sequence of the antisense strand and the target sequence can be completely complementary or substantially complementary; the sense strand and the antisense strand can be completely complementary or substantially complementary. Therefore, for the target sequence and the siRNA that can be complementary to these target sequences, for the antisense strand of each siRNA, the situation that the target sequence complementary thereto is substantially complementary is included, that is, the nucleotide sequence of the antisense strand of each siRNA may have a base mismatch with the corresponding target sequence; for the sense strand of each siRNA, the situation that the target sequence is substantially consistent with the nucleotide sequence of the sense strand of each siRNA may have a base difference with the corresponding target sequence. In some embodiments, the base mismatch or base difference may be a mismatch or difference with the target sequence that does not exceed 3 bases, for example, the mismatch base or the difference base is 1, 2 or 3.
在一些具体的实施方案中，双链核糖核酸选自如表1或表1-1中所示的任一siRNA。本公开提供的siRNA，其与靶mRNA(FXII mRNA)结合的特异性高，具有较好的靶mRNA的沉默活性，可以显著抑制FXII基因表达，用于治疗包括多发性硬化症(MS)、动脉粥样硬化、阿尔兹海默症(AD)、遗传性血管水肿(HAE)、脓毒症、深静脉血栓形成(DVT)、社区获得性肺炎(CAP)、新冠肺炎的血栓性炎症(参与新冠肺炎的促凝血和促炎反应)、显微镜下型多血管炎(MPA)、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎(RA)及结肠炎在内的FXII相关的疾病。In some specific embodiments, the double-stranded RNA is selected from any siRNA as shown in Table 1 or Table 1-1. The siRNA provided by the present disclosure has high specificity for binding to the target mRNA (FXII mRNA), has good silencing activity of the target mRNA, can significantly inhibit FXII gene expression, and is used to treat FXII-related diseases including multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory response of COVID-19), microscopic polyangiitis (MPA), neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
在一些实施方案中，本公开提供了一种siRNA组合物，其包含表1或表1-1中所示siRNA中的任意一种或两种以上的组合。In some embodiments, the present disclosure provides a siRNA composition comprising any one or a combination of two or more of the siRNAs shown in Table 1 or Table 1-1.
在一些实施方案中，正义链的每个核苷酸彼此独立地为修饰的核苷酸或未修饰的核苷酸。在一些实施方案中，反义链的每个核苷酸彼此独立地为修饰的核苷酸或未修饰的核苷酸。In some embodiments, each nucleotide of the sense strand is independently a modified nucleotide or an unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified nucleotide or an unmodified nucleotide.
在一些实施方案中，正义链中任意相连的两个核苷酸由磷酸二酯键或硫代磷酸二酯键连接。在一些实施方案中，反义链中任意相连的两个核苷酸由磷酸二酯键或硫代磷酸二酯键连接。In some embodiments, any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond. In some embodiments, any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.
在一些实施方案中，正义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团。在一些实施方式中，所述反义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团。In some embodiments, the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate-derived group. In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.
示例性的，5’磷酸基团的结构为：5’磷酸衍生基团的结构包括但不限于：(EVP)，等。Exemplary, the structure of the 5' phosphate group is: The structures of the 5' phosphate derivative group include but are not limited to: (EVP), wait.
位于正义链或反义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团后，形成如下所示结构： After the 5' terminal nucleotide of the sense strand or antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, the following structure is formed:
其中，Base表示碱基，例如A、U、G、C或T。R’为羟基或被本领域技术人员所知晓的各类基团所取代，例如，2’-氟代(2’-F)修饰的核苷酸，2’-烷氧基修饰的核苷酸，2’-取代的烷氧基修饰的核苷酸，2’-烷基修饰的核苷酸，2’-取代的烷基修饰的核苷酸，2’-脱氧核糖核苷酸。 Wherein, Base represents a base, such as A, U, G, C or T. R' is a hydroxyl group or is substituted by various groups known to those skilled in the art, such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.
双链核糖核酸修饰物Double-stranded RNA modification
本公开的第二方面提供一种双链核糖核酸修饰物。进一步地，双链核糖核酸修饰物为siRNA修饰物。siRNA修饰物在保持较高FXII mRNA抑制活性的同时，可提高siRNA的稳定性。The second aspect of the present disclosure provides a double-stranded RNA modification. Further, the double-stranded RNA modification is a siRNA modification. The siRNA modification can improve the stability of siRNA while maintaining a high FXII mRNA inhibitory activity.
在一些实施方案中，双链核糖核酸修饰物包含至少一个核苷酸的修饰。核苷酸的修饰选自核糖基团的修饰和碱基的修饰中的至少一种。在一些实施方案中，“核苷酸的修饰”是指核苷酸的核糖基团2’位羟基被其他基团取代形成的核苷酸或核苷酸衍生物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述核苷酸的修饰不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008,13(19-20):842-55中公开的修饰的核苷酸。通过核苷酸的修饰可以提高siRNA的稳定性，并保持其对FXII基因的高抑制效率。In some embodiments, the double-stranded RNA modification comprises at least one modification of a nucleotide. The modification of the nucleotide is selected from at least one of the modification of the ribose group and the modification of the base. In some embodiments, "modification of the nucleotide" refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modification of the nucleotide does not result in a significant weakening or loss of the function of the siRNA to inhibit gene expression. For example, the modified nucleotide disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected. The stability of the siRNA can be improved by modification of the nucleotide, and its high inhibitory efficiency on the FXII gene can be maintained.
示例性的，修饰的核苷酸具有如下所示结构：Exemplarily, the modified nucleotide has the structure shown below:
其中，Base表示碱基，例如A、U、G、C或T。核糖基团2’位的羟基被R取代。这些核糖基2’位的羟基可以为本领域技术人员所知晓的各类基团所取代，例如，2’-氟代(2’-F)修饰的核苷酸，2’-烷氧基修饰的核苷酸，2’-取代的烷氧基修饰的核苷酸，2’-烷基修饰的核苷酸，2’-取代的烷基修饰的核苷酸，2’-脱氧核糖核苷酸。 Wherein, Base represents a base, such as A, U, G, C or T. The hydroxyl group at the 2' position of the ribose group is substituted by R. The hydroxyl group at the 2' position of these ribose groups can be substituted by various groups known to those skilled in the art, for example, 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxyribonucleotides.
在一些实施方案中，2’-烷氧基修饰的核苷酸为2’-甲氧基(2’-OMe，2’-O-CH3)修饰的核苷酸等等。In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH 3 ) modified nucleotide, and the like.
在一些实施方案中，2’-取代的烷氧基修饰的核苷酸为2’-甲氧基乙氧基(2’-O-CH2-CH2-O-CH3)修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸等。In some embodiments, the 2'-substituted alkoxy modified nucleotide is a 2'-methoxyethoxy (2'-O-CH 2 -CH 2 -O-CH 3 ) modified nucleotide, a 2'-O-CH 2 -CH=CH 2 modified nucleotide, and the like.
在一些实施方案中，2’-取代的烷基修饰的核苷酸为2’-CH2-CH2-CH＝CH2修饰的核苷酸等等。In some embodiments, the 2'-substituted alkyl modified nucleotide is a 2'- CH2 - CH2 -CH= CH2 modified nucleotide and the like.
在一些实施方案中，核苷酸的修饰是碱基的修饰。碱基的修饰可以是本领域技术人员所知晓的各类型修饰。示例性的，碱基的修饰包括但不限于m6A、Ψ、m1A、m5A、ms2i6A、i6A、m3C、m5C、ac4C、m7G、m2,2G、m2G、m1G、Q、m5U、mcm5U、ncm5U、ncm5Um、D、mcm5s2U、Inosine(I)、hm5C、s4U、s2U、偶氮苯、Cm、Um、Gm、t6A、yW、ms2t6A或其衍生物。In some embodiments, the modification of the nucleotide is the modification of the base. The modification of the base can be various types of modifications known to those skilled in the art. Exemplary, the modification of the base includes but is not limited to m 6 A, Ψ, m 1 A, m 5 A, ms 2 i 6 A, i 6 A, m 3 C, m 5 C, ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U, mcm 5 U, ncm 5 U, ncm 5 Um, D, mcm 5 s 2 U, Inosine (I), hm 5 C, s 4 U, s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.
在一些实施方案中，核苷酸衍生物是指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的化合物。在一些实施方案中，核苷酸衍生物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3’-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2’-、4’-位处以提供一个2’,4’-BNA核苷酸，如LNA、ENA、cET等。In some embodiments, a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. In some embodiments, a nucleotide derivative can be an isonucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide. BNA refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.
LNA如式(1)所示，ENA如式(2)所示，cET如式(3)所示：
LNA is shown in formula (1), ENA is shown in formula (2), and cET is shown in formula (3):
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸，如解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(4)所示，GNA如式(5)所示：
Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (4) and GNA is shown in formula (5):
上述式(4)和式(5)中，R选自H、OH或烷氧基(O-烷基)。In the above formula (4) and formula (5), R is selected from H, OH or alkoxy (O-alkyl).
在一些实施方案中，核苷酸衍生物修饰是指核酸中的核苷酸被替代为核苷酸衍生物。示例性的，核苷酸衍生物选自异核苷酸、LNA、ENA、cET、UNA或GNA。In some embodiments, the nucleotide derivative modification refers to that the nucleotide in the nucleic acid is replaced by a nucleotide derivative. Exemplarily, the nucleotide derivative is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.
在一些实施方案中，核酸中的核苷酸被替代为异核苷酸，在本公开的上下文中，也称之为异核苷修饰。在一些实施方案中，异核苷修饰包括在欲修饰的siRNA的正义链和/或反义链的一个或多个位点掺入异核苷，以代替天然核苷在相应位置进行偶联。In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, which are also referred to as isonucleoside modifications in the context of the present disclosure. In some embodiments, isonucleoside modifications include incorporating isonucleosides at one or more sites of the sense strand and/or antisense strand of the siRNA to be modified to replace natural nucleosides for coupling at the corresponding positions.
在一些实施方案中，异核苷修饰采用D-异核苷修饰。在另一些实施方案中，异核苷修饰采用L-异核苷修饰。在又一些实施方案中，异核苷修饰采用D-异核苷修饰和L-异核苷修饰。In some embodiments, the isonucleoside modification adopts D-isonucleoside modification. In other embodiments, the isonucleoside modification adopts L-isonucleoside modification. In still other embodiments, the isonucleoside modification adopts D-isonucleoside modification and L-isonucleoside modification.
在一些实施方案中，双链核糖核酸修饰物包含至少一个位置处的磷酸二酯键的修饰。在一些实施方式中，磷酸二酯键的修饰是指磷酸二酯键中至少一个氧原子被硫原子取代形成硫代磷酸二酯键。硫代磷酸二酯键可以稳定siRNA的双链结构，保持碱基配对的特异性。示例性的，硫代磷酸二酯键结构如下所示：
In some embodiments, the double-stranded RNA modification comprises a modification of the phosphodiester bond at at least one position. In some embodiments, the modification of the phosphodiester bond refers to the replacement of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a thiophosphate diester bond. The thiophosphate diester bond can stabilize the double-stranded structure of the siRNA and maintain the specificity of base pairing. Exemplary, the thiophosphate diester bond structure is shown below:
在一些实施方案中，双链核糖核酸修饰物包含如下至少一种的化学修饰：In some embodiments, the double-stranded RNA modification comprises at least one of the following chemical modifications:
(1)正义链中至少一个核苷酸的修饰，(1) modification of at least one nucleotide in the sense strand,
(2)正义链中至少一个位置处的磷酸二酯键的修饰，(2) modification of the phosphodiester bond at at least one position in the sense strand,
(3)反义链中至少一个核苷酸的修饰，(3) modification of at least one nucleotide in the antisense strand,
(4)反义链中至少一个位置处的磷酸二酯键的修饰。(4) Modification of the phosphodiester bond at at least one position in the antisense strand.
进一步地，双链核糖核酸修饰物是包含(1)-(4)中至少一种化学修饰的siRNA修饰物。Furthermore, the double-stranded RNA modification product is a siRNA modification product comprising at least one chemical modification among (1) to (4).
在本公开中，正义链中的序列A与反义链中的序列B互补形成双链区后，序列A与序列B的3’末端可以为如下任意一种所示：In the present disclosure, after sequence A in the sense strand and sequence B in the antisense strand complement each other to form a double-stranded region, the 3' ends of sequence A and sequence B can be any of the following:
(1)序列A与序列B的3’末端均形成平末端；(1) The 3' ends of sequence A and sequence B are both blunt-ended;
(2)序列A的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，且序列B的3’末端形成平末端；(2) The 3' end of sequence A has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of sequence B forms a blunt end;
(3)序列B的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，且序列A的3’末端形成平末 端；(3) The 3' end of sequence B has 1-2 protruding nucleotides extending beyond the double-stranded region, and the 3' end of sequence A forms a flat end. end;
(4)序列A的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，且序列B的3’末端具有1-2个延伸出所述双链区的突出的核苷酸。(4) The 3’ end of sequence A has 1-2 protruding nucleotides extending beyond the double-stranded region, and the 3’ end of sequence B has 1-2 protruding nucleotides extending beyond the double-stranded region.
在一些实施方案中，正义链的核苷酸序列为序列A所示的序列，反义链的核苷酸序列为序列B所示的序列。In some embodiments, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B.
在一些实施方案中，当正义链与反义链的核苷酸序列互补形成双链区后，正义链与反义链的3’末端不存在突出的核苷酸时，在正义链和反义链中至少一条链的3’末端添加1-2个核苷酸，作为突出的核苷酸。其中，连接于正义链的3’末端的1-2个核苷酸组成序列D，连接于反义链的3’末端的1-2个核苷酸组成序列E。相应地，正义链的核苷酸序列为序列A连接序列D所示的序列，反义链的核苷酸序列为序列B连接序列E所示的序列。或者，正义链的核苷酸序列为序列A所示的序列，反义链的核苷酸序列为序列B连接序列E所示的序列。或者，正义链的核苷酸序列为序列A连接序列D所示的序列，反义链的核苷酸序列为序列B所示的序列。In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region, if there are no protruding nucleotides at the 3' ends of the sense strand and the antisense strand, 1-2 nucleotides are added to the 3' end of at least one of the sense strand and the antisense strand as protruding nucleotides. Among them, the 1-2 nucleotides connected to the 3' end of the sense strand constitute sequence D, and the 1-2 nucleotides connected to the 3' end of the antisense strand constitute sequence E. Accordingly, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E. Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E. Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E. Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B.
示例性的，在正义链的3’末端添加2个脱氧核糖核苷酸(TT)作为序列D，在反义链的3’末端添加2个脱氧核糖核苷酸(TT)作为序列E。或者，仅在反义链的3’末端添加2个脱氧核糖核苷酸(TT)作为序列E。或者，仅在正义链的3’末端添加2个脱氧核糖核苷酸(TT)作为序列D。Exemplarily, two deoxyribonucleotides (TT) are added to the 3' end of the sense strand as sequence D, and two deoxyribonucleotides (TT) are added to the 3' end of the antisense strand as sequence E. Alternatively, two deoxyribonucleotides (TT) are added only to the 3' end of the antisense strand as sequence E. Alternatively, two deoxyribonucleotides (TT) are added only to the 3' end of the sense strand as sequence D.
在一些实施方案中，当正义链与反义链的核苷酸序列互补形成双链区后，正义链的3’末端不存在突出的核苷酸时，在正义链的3’末端添加由1-2个核苷酸组成的序列D，作为突出的核苷酸。然后，当序列A连接序列D形成的核苷酸序列在完成化学修饰后，排除由1-2个核苷酸组成的序列D。相应地，在双链核糖核酸修饰物中，正义链的核苷酸序列为序列A所示的序列，反义链的核苷酸序列为序列B所示的序列。或者，在双链核糖核酸修饰物中，正义链的核苷酸序列为序列A所示的序列，反义链的核苷酸序列为序列B连接序列E所示的序列。In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region, if there is no protruding nucleotide at the 3' end of the sense strand, a sequence D consisting of 1-2 nucleotides is added to the 3' end of the sense strand as a protruding nucleotide. Then, after the nucleotide sequence formed by connecting sequence A to sequence D is chemically modified, the sequence D consisting of 1-2 nucleotides is excluded. Accordingly, in the double-stranded RNA modified substance, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B. Alternatively, in the double-stranded RNA modified substance, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.
在一些实施方案中，当序列A在与序列B互补形成双链区后，序列A的3’末端具有延伸出双链区的突出的1-2个核苷酸时，将序列A中位于3’末端的突出的核苷酸排除后作为正义链的核苷酸序列。排除掉3’末端的突出的核苷酸的序列称为序列A’。相应地，双链核糖核酸修饰物的正义链的核苷酸序列为序列A’所示的序列，双链核糖核酸修饰物的反义链的核苷酸序列为序列B所示的序列。或者，双链核糖核酸修饰物的正义链的核苷酸序列为序列A’所示的序列，双链核糖核酸修饰物的反义链的核苷酸序列为序列B连接序列E所示的序列。In some embodiments, when sequence A is complementary to sequence B to form a double-stranded region, the 3' end of sequence A has 1-2 nucleotides protruding out of the double-stranded region, the protruding nucleotides at the 3' end of sequence A are excluded as the nucleotide sequence of the sense strand. The sequence excluding the protruding nucleotides at the 3' end is called sequence A'. Accordingly, the nucleotide sequence of the sense strand of the double-stranded RNA modified substance is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand of the double-stranded RNA modified substance is the sequence shown in sequence B. Alternatively, the nucleotide sequence of the sense strand of the double-stranded RNA modified substance is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand of the double-stranded RNA modified substance is the sequence shown in sequence B connected to sequence E.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的正义链包括如下修饰：正义链中第7位、第9位、第10位和第11位的核糖核苷酸为2’-氟代修饰的核糖核苷酸；正义链中其他位置的核糖核苷酸为2’-甲氧基修饰的核糖核苷酸。In some embodiments, along the 5' end to the 3' end direction, the sense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotides at other positions in the sense strand are 2'-methoxy-modified ribonucleotides.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的正义链包括如下所示位置处的硫代磷酸二酯键：5’末端起始的第1个核苷酸与第2个核苷酸之间，5’末端起始的第2个核苷酸与第3个核苷酸之间，3’末端起始的第1个核苷酸与第2个核苷酸之间，以及3’末端起始的第2个核苷酸与第3个核苷酸之间。In some embodiments, the sense strand of the siRNA modification includes phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, between the second nucleotide and the third nucleotide starting from the 5' end, between the first nucleotide and the second nucleotide starting from the 3' end, and between the second nucleotide and the third nucleotide starting from the 3' end.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的正义链包括如下位置处的硫代磷酸二酯键：5’末端起始的第1个核苷酸与第2个核苷酸之间、5’末端起始的第2个核苷酸与第3个核苷酸之间。In some embodiments, the sense strand of the siRNA modification includes phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, and between the second nucleotide and the third nucleotide starting from the 5' end.
在一些具体的实施方案中，siRNA修饰物正义链具有如(a1)-(a3)任一项所示的结构：In some specific embodiments, the sense strand of the siRNA modification has a structure as shown in any one of (a 1 )-(a 3 ):
(a1)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-(s)-T-(s)-T-3’，(a 1 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
(a2)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(a 2 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(a3)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(a 3 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，大写字母T表示碱基为胸腺嘧啶 的脱氧核糖核苷酸，小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接。Wherein, N1 - N23 are independently selected from ribonucleotides whose bases are A, U, C or G, and the capital letter T indicates that the base is thymine. The lowercase letter m indicates that the adjacent ribonucleotide on the right side of the letter m is a 2'-O-CH 3 modified ribonucleotide. The lowercase letter f indicates that the adjacent ribonucleotide on the left side of the letter f is a 2'-F modified ribonucleotide. -(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
在另外一些具体的实施方案中，siRNA修饰物正义链具有如(a4)-(a5)任一项所示的结构：In some other specific embodiments, the sense strand of the siRNA modification has a structure as shown in any one of ( a4 )-( a5 ):
(a4)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-3’，(a 4 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',
(a5)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-3’；(a 5 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接。wherein N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- CH3 -modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; and -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中任意奇数位置处的核糖核苷酸为2’-甲氧基修饰的核糖核苷酸，所述反义链中任意偶数位置处的核糖核苷酸为2’-氟代修饰的核糖核苷酸。In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotide at any odd position in the antisense strand is a 2'-methoxy-modified ribonucleotide, and the ribonucleotide at any even position in the antisense strand is a 2'-fluoro-modified ribonucleotide.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸。In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modification comprises the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中第2位、第6位、第8位、第9位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸。In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modification comprises the following modifications: the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- CH3 modified ribonucleotides.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中第7位的核糖核苷酸为核苷酸衍生物GNA修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸。In some embodiments, along the direction from the 5' end to the 3' end, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- CH3 modified ribonucleotides.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中第2位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中第6位的核糖核苷酸为核苷酸衍生物GNA修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸。In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- CH3 modified ribonucleotides.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，且所述反义链中第1位的核糖核苷酸为5’-反式乙烯基膦酸酯核苷酸。In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下修饰：所述反义链中第2位、第6位、第8位、第9位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，且所述反义链中第1位的核糖核苷酸为5’-反式乙烯基膦酸酯核苷酸。In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
在一些实施方案中，沿5’末端向3’末端方向，siRNA修饰物的反义链包括如下所示位置处的硫代磷酸二酯键：5’末端起始的第1个核苷酸与第2个核苷酸之间，5’末端起始的第2个核苷酸与第3个核苷酸之间，3’末端起始的第1个核苷酸与第2个核苷酸之间，以及3’末端起始的第2个核苷酸与第3个核苷酸之间。In some embodiments, the antisense strand of the siRNA modification includes thiophosphate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, between the second nucleotide and the third nucleotide starting from the 5' end, between the first nucleotide and the second nucleotide starting from the 3' end, and between the second nucleotide and the third nucleotide starting from the 3' end.
在一些实施方案中，沿5’末端向3’末端方向，反义链的5’末端的核苷酸连接5’磷酸基团或5’磷酸衍生基团。示例性的，5’磷酸基团的结构为：5’磷酸衍生基团的结构包括但不限于： 等。In some embodiments, the nucleotide at the 5' end of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group along the 5' end to the 3' end. Exemplarily, the structure of the 5' phosphate group is: The structures of the 5' phosphate derivative group include but are not limited to: wait.
在一些具体的实施方案中，siRNA修饰物反义链具有如下(b1)-(b27)任一项所示的结构：In some specific embodiments, the antisense strand of the siRNA modification has a structure as shown in any one of (b 1 ) to (b 27 ):
(b1)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-T-(s)-T-3’，(b 1 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-T-(s)-T-3',
(b2)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 2 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b3)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 3 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b4)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 4 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
(b5)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-N20f-(s)-mN21-3’，(b 5 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-N 20 f-(s)-mN 21 -3',
(b6)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 6 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b7)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 7 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b8)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 8 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b9)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-N20f-mN21-(s)-N22f-(s)-mN23-3’(b 9 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -N 20 f-mN 21 -(s)-N 22 f-(s)- mN 23 -3'
(b10)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 10 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b11)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 11 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b12)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN1 5-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 12 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 1 5 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 - 3',
(b13)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 13 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
(b14)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’(b 14 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3'
(b15)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 15 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3' ,
(b16)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 16 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b17)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 17 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b18)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 18 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b19)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 19 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b20)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 20 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b21)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN 17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 21 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b22)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 22 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b23)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 23 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b24)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 24 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b25)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 25 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b26)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 26 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b27)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(b 27 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸，EVP表示该字母其右侧相邻的一个核苷酸为5’-反式乙烯基膦酸酯核苷酸，-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接，(GNA)表示其右侧相邻的一个核糖核苷酸为存在GNA修饰的核糖核苷酸。wherein N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- CH3 -modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; P1 represents that a nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide; EVP represents that a nucleotide adjacent to the right of the letter is a 5'-trans-vinylphosphonate nucleotide; -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond; and (GNA) represents that a ribonucleotide adjacent to the right of the letter is a GNA-modified ribonucleotide.
在一些实施方案中，双链核糖核酸修饰物包括但不限于如表2中所示的siRNA修饰物。In some embodiments, the double-stranded RNA modifications include, but are not limited to, the siRNA modifications shown in Table 2.
双链核糖核酸缀合物Double-stranded RNA conjugate
本公开的第三方面提供一种双链核糖核酸缀合物，是本公开第一方面提供的双链核糖核酸或第二方面提供的双链核糖核酸修饰物与缀合基团缀合连接得到。The third aspect of the present disclosure provides a double-stranded RNA conjugate, which is obtained by conjugating the double-stranded RNA provided by the first aspect of the present disclosure or the double-stranded RNA modification provided by the second aspect with a conjugation group.
在本公开中，双链核糖核酸缀合物的正义链与反义链形成双链核糖核酸缀合物的双链区，并且，在双链核糖核酸缀合物的正义链的3’末端形成平末端。在一些实施方案中，双链核糖核酸缀合物的正义链的3’末端形成平末端，双链核糖核酸缀合物的反义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸。在另外一些实施方案中，双链核糖核酸缀合物的正义链的3’末端形成平末端，双链核糖核酸缀合物的反义链的3’末端形成平末端。In the present disclosure, the sense strand and the antisense strand of the double-stranded RNA conjugate form a double-stranded region of the double-stranded RNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the double-stranded RNA conjugate. In some embodiments, the 3' end of the sense strand of the double-stranded RNA conjugate forms a blunt end, and the 3' end of the antisense strand of the double-stranded RNA conjugate has 1-2 protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of the sense strand of the double-stranded RNA conjugate forms a blunt end, and the 3' end of the antisense strand of the double-stranded RNA conjugate forms a blunt end.
在一些优选地实施方案中，双链核糖核酸缀合物由双链核糖核酸修饰物与缀合基团缀合连接得到。其中，双链核糖核酸修饰物的正义链与反义链互补形成双链核糖核酸修饰物的双链区，并且，双链核糖核酸修饰物的正义链的3’末端形成平末端，缀合基团与具有平末端的正义链的3’末端缀合连接，形成双链核糖核酸缀合物。In some preferred embodiments, the double-stranded RNA conjugate is obtained by conjugating a double-stranded RNA modification with a conjugation group, wherein the sense strand and the antisense strand of the double-stranded RNA modification are complementary to form a double-stranded region of the double-stranded RNA modification, and the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the conjugation group is conjugated with the 3' end of the sense strand with the blunt end to form a double-stranded RNA conjugate.
示例性地，双链核糖核酸修饰物的正义链为序列A所示的序列，反义链为序列B连接序列E所示的序列。并且，双链核糖核酸修饰物的正义链的3’末端形成平末端，双链核糖核酸修饰物的正义链的3’末端连接缀合基团，形成双链核糖核酸缀合物。Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B connected to sequence E. In addition, the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the 3' end of the sense strand of the double-stranded RNA modification is connected to a conjugated group to form a double-stranded RNA conjugate.
示例性地，双链核糖核酸修饰物的正义链为序列A所示的序列，反义链为序列B所示的序列。并且，双链核糖核酸修饰物的正义链的3’末端形成平末端，双链核糖核酸修饰物的正义链3’末端连接缀合基团，形成双链核糖核酸缀合物。Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B. In addition, the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the 3' end of the sense strand of the double-stranded RNA modification is connected to a conjugated group to form a double-stranded RNA conjugate.
示例性地，双链核糖核酸修饰物的正义链为序列A连接序列D所示的序列，反义链为序列B连接序列E所示的序列。并且，双链核糖核酸修饰物的正义链的3’末端具有突出的1-2个核苷酸组成的序列D，将双链核糖核酸修饰物中正义链的3’末端的序列D排除后，在序列A的3’末端连接缀合基团，形成双链核糖核酸缀合物。Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown by sequence A connected to sequence D, and the antisense strand is the sequence shown by sequence B connected to sequence E. In addition, the 3' end of the sense strand of the double-stranded RNA modification has a sequence D consisting of protruding 1-2 nucleotides, and after excluding the sequence D at the 3' end of the sense strand in the double-stranded RNA modification, a conjugation group is connected to the 3' end of sequence A to form a double-stranded RNA conjugate.
示例性地，双链核糖核酸修饰物的正义链为序列A连接序列D所示的序列，反义链为序列B所示的序列。并且，双链核糖核酸修饰物的正义链的3’末端具有突出的1-2个核苷酸组成的序列D，将双链核糖核酸修饰物中正义链的3’末端的序列D排除后，在序列A的3’末端连接缀合基团，形成 双链核糖核酸缀合物。For example, the sense strand of the double-stranded RNA modification is a sequence A connected to a sequence D, and the antisense strand is a sequence B. In addition, the 3' end of the sense strand of the double-stranded RNA modification has a sequence D consisting of 1-2 nucleotides protruding, and after excluding the sequence D at the 3' end of the sense strand in the double-stranded RNA modification, a conjugated group is connected to the 3' end of sequence A to form Double-stranded RNA conjugate.
示例性地，双链核糖核酸修饰物的正义链为序列A所示的序列，反义链为序列B连接序列E所示的序列。其中，序列A的3’末端具有延伸出双链区的突出的核苷酸，将位于序列A中3’末端的突出的核苷酸排除后的序列(又称，序列A’)作为用于连接缀合基团的核苷酸序列。因此，双链核糖核酸缀合物的正义链的核苷酸序列为序列A’所示的序列，反义链的核苷酸序列为序列B连接序列E所示的序列。Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B connected to sequence E. Wherein, the 3' end of sequence A has a protruding nucleotide extending out of the double-stranded region, and the sequence after excluding the protruding nucleotide at the 3' end of sequence A (also known as sequence A') is used as the nucleotide sequence for connecting the conjugated group. Therefore, the nucleotide sequence of the sense strand of the double-stranded RNA conjugate is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.
示例性地，双链核糖核酸修饰物的正义链为序列A所示的序列，反义链为序列B所示所示的序列。其中，序列A的3’末端具有延伸出双链区的突出的核苷酸，将位于序列A中3’末端的突出的核苷酸排除后的序列(又称，序列A’)作为用于连接缀合基团的核苷酸序列。因此，双链核糖核酸缀合物的正义链的核苷酸序列为序列A’所示的序列，反义链的核苷酸序列为序列B所示的序列。Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B. Wherein, the 3' end of sequence A has a protruding nucleotide extending out of the double-stranded region, and the sequence after excluding the protruding nucleotide at the 3' end of sequence A (also known as sequence A') is used as the nucleotide sequence for connecting the conjugated group. Therefore, the nucleotide sequence of the sense strand of the double-stranded RNA conjugate is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B.
在一些可选地实施方案中，双链核糖核酸缀合物的正义链具有如(d1)-(d2)任一项所示的结构：In some alternative embodiments, the sense strand of the double-stranded RNA conjugate has a structure as shown in any one of (d 1 )-(d 2 ):
(d1)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-L96-3’，(d 1 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -L96-3',
(d2)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-L96-3’；(d 2 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -L96-3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接。L96也即式I所示的缀合物基团GalNAc。Wherein, N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G, the capital letter T represents a deoxyribonucleotide whose base is thymine, the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a ribonucleotide modified with 2'-O- CH3 , the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a ribonucleotide modified with 2'-F, and -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond. L96 is the conjugate group GalNAc shown in formula I.
在一些可选地实施方案中，双链核糖核酸缀合物的反义链具有如(b1)-(b27)任一项所示的结构：In some alternative embodiments, the antisense strand of the double-stranded RNA conjugate has a structure as shown in any one of (b 1 )-(b 27 ):
(b1)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-T-(s)-T-3’，(b 1 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-T-(s)-T-3',
(b2)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 2 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b3)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 3 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b4)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 4 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
(b5)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-N20f-(s)-mN21-3’，(b 5 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-N 20 f-(s)-mN 21 -3',
(b6)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 6 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b7)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 7 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b8)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 8 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b9)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-N20f-mN21-(s)-N22f-(s)-mN23-3’，(b 9 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -N 20 f-mN 21 -(s)-N 22 f-(s)- mN 23 -3',
(b10)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 10 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b11)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 11 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b12)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN1 5-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 12 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 1 5 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 - 3',
(b13)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15 -N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 13 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b14)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’(b 14 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3'
(b15)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 15 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3' ,
(b16)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 16 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b17)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 17 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b18)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 18 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b19)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 19 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b20)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 20 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b21)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(b 21 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
(b22)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 22 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b23)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 23 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b24)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 24 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
(b25)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 25 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
(b26)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 26 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
(b27)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(b 27 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸，EVP表示该字母其右侧相邻的一个核苷酸为5’-反式乙烯基膦酸酯核苷酸，-(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接，(GNA)表示其右侧相邻的一个核糖核苷酸为存在GNA修饰的核糖核苷酸。wherein N1 - N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- CH3 -modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; P1 represents that a nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide; EVP represents that a nucleotide adjacent to the right of the letter is a 5'-trans-vinylphosphonate nucleotide; -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond; and (GNA) represents that a ribonucleotide adjacent to the right of the letter is a GNA-modified ribonucleotide.
进一步地，双链核糖核酸缀合物为siRNA缀合物，其中siRNA缀合物中与缀合基团连接的siRNA分子可以是未修饰的siRNA，或siRNA修饰物。缀合基团修饰的siRNA分子在保持了较高的抑制活性和稳定性的同时，还具有较好的组织、器官靶向性和促进细胞内吞的能力，可降低对其他组织或器官的影响以及减少siRNA分子使用量，可达到减轻毒性和降低成本的目的。可选地，选择表1或表1-1或表2示出的任意一种siRNA分子与缀合基团连接，得到双链核糖核酸缀合物。Further, the double-stranded RNA conjugate is a siRNA conjugate, wherein the siRNA molecule connected to the conjugation group in the siRNA conjugate can be an unmodified siRNA, or a siRNA modification. The siRNA molecule modified with the conjugation group has good tissue and organ targeting and the ability to promote cell endocytosis while maintaining high inhibitory activity and stability, which can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs. Optionally, any one of the siRNA molecules shown in Table 1 or Table 1-1 or Table 2 is selected to be connected to the conjugation group to obtain a double-stranded RNA conjugate.
siRNA与缀合基团的缀合位点可以在siRNA正义链的3’末端或5’末端，也可在反义链的5’端，还可以在siRNA的内部序列中。在一些实施方案中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3’末端。The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end of the siRNA sense strand.
在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2’-位羟基或者碱基上。在一些实施方式中，所述缀合基团还可以连接在3’-位羟基上，此时核苷酸之间采用2’,5’-磷酸二酯键连接。 当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7。In some embodiments, the conjugated group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugated group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by 2',5'-phosphodiester bonds. When the conjugated group is connected to the end of the siRNA chain, the conjugated group is usually connected to the phosphate group of the nucleotide; when the conjugated group is connected to the internal sequence of the siRNA, the conjugated group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10 (5): 1181-7.
在本公开中，缀合基团可以是siRNA给药领域常规使用的配体。在一些实施方式中，所述缀合基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种：亲脂分子，例如胆固醇、胆汁酸、维生素(例如维生素E)、不同链长的脂质分子；聚合物，例如聚乙二醇；多肽，例如透膜肽；适配体；抗体；量子点；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；叶酸(folate)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等。In the present disclosure, the conjugated group can be a ligand conventionally used in the field of siRNA administration. In some embodiments, the conjugated group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.
在一些具体的实施方案中，所述缀合基团具有如下所示结构：
In some specific embodiments, the conjugated group has the following structure:
式I所示的缀合基团为GalNAc，GalNAc具有肝脏靶向性，可以将siRNA分子高特异性地递送于肝脏组织中，特异性抑制肝脏内FXII基因的高表达。The conjugated group shown in Formula I is GalNAc. GalNAc has liver targeting property and can deliver siRNA molecules to liver tissue with high specificity, thereby specifically inhibiting the high expression of FXII gene in the liver.
在一些具体的实施方案中，GalNAc通过磷酸二酯键与正义链的3’末端缀合连接，得到如下所示结构的siRNA缀合物：
In some specific embodiments, GalNAc is conjugated to the 3' end of the sense strand via a phosphodiester bond to obtain a siRNA conjugate with the following structure:
其中，双螺旋结构为未修饰的siRNA或siRNA修饰物。The double helix structure is unmodified siRNA or siRNA modification.
在一些实施方案中，双链核糖核酸缀合物包括但不限于如表3中所示的siRNA缀合物。In some embodiments, the double-stranded ribonucleic acid conjugates include, but are not limited to, siRNA conjugates as shown in Table 3.
药物组合物Pharmaceutical composition
本公开的第四方面提供一种药物组合物，包括第一方面所述的双链核糖核酸、第二方面所述的双链核糖核酸修饰物，以及第三方面所述的双链核糖核酸缀合物中的一种或多种。The fourth aspect of the present disclosure provides a pharmaceutical composition, comprising one or more of the double-stranded RNA described in the first aspect, the double-stranded RNA modification described in the second aspect, and the double-stranded RNA conjugate described in the third aspect.
在一些实施方案中，所述药物组合物含有如上所述的siRNA作为活性成分和药学上可接受的载体。在本公开中，使用药物组合物的目的在于促进针对生物体的给药，有利于活性成分的吸收，进而发挥生物活性。本公开的药物组合物可以通过任何形式给药，包括注射(动脉内、静脉内、肌肉内、腹膜内、皮下)、粘膜、口服(口服固体制剂、口服液体制剂)、直肠、吸入、植入、局部(例如眼部)给药等。口服固体制剂的非限制性实例包括但不限于散剂、胶囊剂、锭剂、颗粒剂、片剂等。口服或粘膜给药的液体制剂的非限制性实例包括但不限于混悬剂、酊剂、酏剂、溶液剂等。局部给药制剂的非限制性实例包括但不限于乳剂、凝胶剂、软膏剂、乳膏剂、贴剂、糊剂、泡沫剂、洗剂、滴剂或血 清制剂。胃肠外给药制剂的非限制性实例包括但不限于注射用溶液剂、注射用干粉剂、注射用悬浮液、注射用乳剂等。本公开的药物组合物还可以制成控制释放或延迟释放剂型(例如脂质体或微球)。In some embodiments, the pharmaceutical composition contains siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier. In the present disclosure, the purpose of using the pharmaceutical composition is to promote administration to an organism, which is beneficial to the absorption of the active ingredient and thus exerts biological activity. The pharmaceutical composition of the present disclosure can be administered in any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid preparations, oral liquid preparations), rectal, inhalation, implantation, local (e.g., eye) administration, etc. Non-limiting examples of oral solid preparations include, but are not limited to, powders, capsules, lozenges, granules, tablets, etc. Non-limiting examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, etc. Non-limiting examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or blood Non-limiting examples of parenteral preparations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, etc. The pharmaceutical composition of the present disclosure can also be prepared into a controlled release or delayed release dosage form (e.g., liposomes or microspheres).
在本公开中，施用途经能够以任何适用的方式进行变化或调整，以满足药物的性质、患者和医务人员的便利以及其它相关因素的需求。In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.
医药用途Medical Uses
本公开的第五方面提供双链核糖核酸，双链核糖核酸修饰物或双链核糖核酸缀合物的如下至少一种用途：The fifth aspect of the present disclosure provides at least one of the following uses of double-stranded RNA, double-stranded RNA modification or double-stranded RNA conjugate:
(1)抑制FXII基因表达，或制备用于抑制FXII基因表达的药物；(1) Inhibiting FXII gene expression, or preparing a drug for inhibiting FXII gene expression;
(2)用于预防或治疗与FXII基因异常表达相关的疾病，或制备用于预防或治疗与FXII基因异常表达相关的疾病的药物；(2) for preventing or treating diseases associated with abnormal expression of FXII gene, or for preparing drugs for preventing or treating diseases associated with abnormal expression of FXII gene;
(3)用于治疗患有将受益于FXII基因表达降低的疾病的受试者，或制备用于治疗患有将受益于FXII基因表达降低的疾病的受试者的药物。(3) Use for treating a subject suffering from a disease that would benefit from decreased expression of the FXII gene, or for preparing a medicament for treating a subject suffering from a disease that would benefit from decreased expression of the FXII gene.
本公开进一步提供了siRNA分子(包括未修饰的siRNA、siRNA修饰物、siRNA缀合物)或药物组合物在上述(1)-(3)至少一种中的用途。The present disclosure further provides the use of siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or pharmaceutical compositions in at least one of the above (1)-(3).
在本公开中，FXII基因异常表达，引发如下一种或多种FXII基因异常表达相关疾病：多发性硬化症(MS)、动脉粥样硬化、阿尔兹海默症(AD)、遗传性血管水肿(HAE)、脓毒症、深静脉血栓形成(DVT)、社区获得性肺炎(CAP)、新冠肺炎的血栓性炎症(参与新冠肺炎的促凝血和促炎反应)、显微镜下型多血管炎(MPA)、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎(RA)及结肠炎。In the present disclosure, abnormal expression of FXII gene triggers one or more of the following diseases related to abnormal expression of FXII gene: multiple sclerosis (MS), atherosclerosis, Alzheimer's disease (AD), hereditary angioedema (HAE), sepsis, deep vein thrombosis (DVT), community-acquired pneumonia (CAP), thrombotic inflammation of COVID-19 (involved in the procoagulant and proinflammatory responses of COVID-19), microscopic polyangiitis (MPA), neurological inflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis (RA) and colitis.
siRNA分子致使FXII基因的表达被抑制至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％，实现对FXII基因异常表达相关疾病的治疗。The siRNA molecule causes the expression of the FXII gene to be inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, thereby achieving the treatment of diseases related to abnormal expression of the FXII gene.
在一些实施方案中，本公开提供一种抑制细胞内FXII基因表达的方法，包括将双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物或药物组合物与细胞接触。In some embodiments, the present disclosure provides a method for inhibiting FXII gene expression in a cell, comprising contacting a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, or a pharmaceutical composition with the cell.
进一步地，抑制细胞内FXII基因表达的方法，是将siRNA分子(包括未修饰的siRNA、siRNA修饰物、siRNA缀合物)或药物组合物引入细胞内。Furthermore, the method for inhibiting the expression of FXII gene in cells is to introduce siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or pharmaceutical compositions into cells.
在一些实施方案中，所述细胞为体内细胞或体外细胞。在一些具体的实施方案中，所述细胞在受试者体内。In some embodiments, the cell is an in vivo cell or an in vitro cell. In some specific embodiments, the cell is in a subject.
在一些实施方案中，本公开提供预防或治疗疾病的方法，包括向受试者施用双链核糖核酸、双链核糖核酸修饰物、双链核糖核酸缀合物或药物组合物。In some embodiments, the present disclosure provides a method for preventing or treating a disease, comprising administering a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, or a pharmaceutical composition to a subject.
进一步地，预防或治疗疾病的方法是向受试者施用siRNA分子(包括未修饰的siRNA、siRNA修饰物、siRNA缀合物)或药物组合物。Furthermore, the method for preventing or treating a disease is to administer siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or pharmaceutical compositions to a subject.
在本公开中，“受试者”包括或者人或者非人类动物，优选脊椎动物，并且更优选哺乳动物。受试者可以包括转基因生物体。最优选地，受试者是人。进一步地，受试者具有如下至少一种特性：In the present disclosure, "subject" includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. The subject may include a transgenic organism. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:
(1)体内FXII基因异常表达，更具体地为FXII基因异常高表达；(1) Abnormal expression of FXII gene in vivo, more specifically, abnormally high expression of FXII gene;
(2)患有与FXII基因异常表达相关的疾病；(2) suffering from diseases related to abnormal expression of FXII gene;
(3)患有将受益于FXII基因表达降低的疾病。如罹患或倾向于患上与FXII基因异常表达相关的疾病的人。(3) Those suffering from diseases that would benefit from decreased FXII gene expression, such as those suffering from or prone to suffering from diseases associated with abnormal FXII gene expression.
表1siRNA序列信息









Table 1 siRNA sequence information









表1-1siRNA序列信息
Table 1-1 siRNA sequence information
表2siRNA修饰物











Table 2 siRNA modifications











上述表格中大写字母“G”、“C”、“A”、“T”和“U”每个通常代表分别含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶作为碱基的核苷酸；mA、mU、mC、mG：表示2’-甲氧基修饰的核苷酸； Af、Gf、Cf、Uf：表示2’-氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；P1：表示该P1右侧相邻的一个核苷酸为5’-磷酸核苷酸，EVP表示该字母其右侧相邻的一个核苷酸为5’-反式乙烯基膦酸酯核苷酸，(GNA)表示其右侧相邻的一个核糖核苷酸为存在GNA修饰的核糖核苷酸。In the above table, capital letters "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively; mA, mU, mC, mG: represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: indicate 2'-fluoro modified nucleotides; lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by thiophosphate groups; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide, EVP indicates that the nucleotide adjacent to the right of this letter is a 5'-trans-vinylphosphonate nucleotide, (GNA) indicates that the ribonucleotide adjacent to the right is a GNA modified ribonucleotide.
表3siRNA缀合物







Table 3 siRNA conjugates







上述表格中大写字母“G”、“C”、“A”、“T”和“U”每个通常代表分别含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶作为碱基的核苷酸；mA、mU、mC、mG：表示2’-甲氧基修饰的核苷酸；Af、Gf、Cf、Uf：表示2’-氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；P1：表示该P1右侧相邻的一个核苷酸为5’-磷酸核苷酸，EVP表示该字母其右侧相邻的一个核苷酸为5’-反式乙烯基膦酸酯核苷酸，(GNA)表示其右侧相邻的一个核糖核苷酸为存在GNA修饰的核糖核苷酸；L96也即式I所示的缀合物基团GalNAc。In the above table, each of the capital letters "G", "C", "A", "T" and "U" generally represents a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively; mA, mU, mC, mG: represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro modified nucleotides; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a thiophosphate group; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide, EVP indicates that the nucleotide adjacent to the right of the letter is a 5'-trans vinylphosphonate nucleotide, (GNA) indicates that the ribonucleotide adjacent to the right is a ribonucleotide modified with GNA; L96 is the conjugate group GalNAc shown in formula I.
实施例Example
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
下述实施例涉及的siRNA、siRNA修饰物、siRNA缀合物由天霖生物科技(上海)有限公司合 成，实施例中使用的细胞、试剂如表4所示：The siRNA, siRNA modifications, and siRNA conjugates involved in the following examples were obtained from Tianlin Biotechnology (Shanghai) Co., Ltd. The cells and reagents used in the examples are shown in Table 4:
表4
Table 4
实施例1：siRNA的合成Example 1: Synthesis of siRNA
1.1siRNA序列设计1.1 siRNA sequence design
根据人FXII基因mRNA序列，选择不同位点设计多对FXII siRNAs，设计的所有单个siRNA均能靶向靶基因的所有转录本(如表5)，这些多对siRNA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。Based on the human FXII gene mRNA sequence, multiple pairs of FXII siRNAs were designed at different sites. All the designed single siRNAs can target all transcripts of the target gene (as shown in Table 5). These multiple pairs of siRNAs have the lowest homology with all other non-target gene sequences after sequence similarity software alignment.
表5
Table 5
用于设计siRNA的靶标序列如下所示，靶标序列来源于FXII的基因mRNA序列(参见NM_000505.4)。The target sequence used to design siRNA is shown below. The target sequence is derived from the mRNA sequence of the FXII gene (see NM_000505.4).
靶标序列I:
Target sequence I:
靶标序列I-1:
Target sequence I-1:
靶标序列I-2:
Target sequence I-2:
靶标序列II：
Target sequence II:
靶标序列III：
Target sequence III:
靶标序列III-1：GATGGGGATACTGTTTGGAGCCCAA(SEQ ID NO:18)Target sequence III-1: GATGGGGATACTGTTTGGAGCCCAA (SEQ ID NO: 18)
靶标序列III-2：GCAGCAAACACAGCCCCTG(SEQ ID NO:19)Target sequence III-2: GCAGCAAACACAGCCCCTG (SEQ ID NO: 19)
靶标序列III-3：CCAGAAAGGAGGGACCTGTGTGAACATGCCAAGC(SEQ ID NO:20)Target sequence III-3: CCAGAAAGGAGGGACCTGTGTGAACATGCCAAGC (SEQ ID NO: 20)
靶标序列III-4：CCCACTGTCTCTGTCCACAACACCTCACT(SEQ ID NO:21) Target sequence III-4: CCCACTGTCTCTGTCCACAACACCTCACT (SEQ ID NO: 21)
靶标序列IV：
Target sequence IV:
靶标序列V：
Target sequence V:
靶标序列VI：
Target sequence VI:
靶标序列VII：
Target sequence VII:
靶标序列VII-1：
Target sequence VII-1:
靶标序列VII-2：
Target sequence VII-2:
靶标序列VIII：
Target sequence VIII:
靶标序列IX：
Target sequence IX:
靶标序列X：
Target sequence X:
靶标序列XI：
Target sequence XI:
靶标序列XI-1
Target sequence XI-1
靶标序列XI-2
Target sequence XI-2
靶标序列XII：
Target sequence XII:
靶标序列XII-1
Target sequence XII-1
靶标序列XII-2
Target sequence XII-2
靶标序列XIII：
Target sequence XIII:
靶标序列XIV：
Target sequence XIV:
靶标序列XV：
Target sequence XV:
靶标序列XV-1：
Target sequence XV-1:
靶标序列XV-2：
Target sequence XV-2:
1.2合成方法描述：1.2 Description of synthesis method:
通过固相亚磷酰胺法，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。其中，两个核苷酸之间采用磷酸酯连接时，连接后一个核苷单体时，包括脱保护、偶联、盖帽、氧化四步反应。两个核苷酸之间采用硫代磷酸酯连接时，连接后一个核苷单体时，包括脱保护、偶联、盖帽、硫化四步反应。Through the solid phase phosphoramidite method, nucleoside monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Among them, when phosphate is used to connect two nucleotides, the connection of the next nucleoside monomer includes four steps of deprotection, coupling, capping, and oxidation. When thiophosphate is used to connect two nucleotides, the connection of the next nucleoside monomer includes four steps of deprotection, coupling, capping, and sulfurization.
1.3合成条件给定如下：1.3 The synthesis conditions are given as follows:
核苷单体以0.1M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为25℃，反应时间为70秒，脱保护试剂为二氯乙酸的二氯甲烷溶液(3％V/V)，二氯乙酸与固相载体上4,4’-二甲氧基三苯甲基保护基的摩尔比为5:1。The nucleoside monomer is provided in an acetonitrile solution with a concentration of 0.1 M. The conditions of the deprotection reaction in each step are the same, i.e., the temperature is 25° C., the reaction time is 70 seconds, the deprotection reagent is a dichloroacetic acid dichloromethane solution (3% V/V), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.
每一步偶联反应条件均相同，包括温度为25℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1:10，固相载体上连接的核酸序列和偶联试剂的摩尔比为1:65，反应时间为600秒，偶联试剂为5-乙硫基-1H-四氮唑的0.5M乙腈溶液。The coupling reaction conditions for each step were the same, including a temperature of 25° C., a molar ratio of the nucleic acid sequence connected to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence connected to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a coupling reagent of 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.
每一步盖帽条件均相同，包括温度为25℃，反应时间为15秒。盖帽试剂溶液为摩尔比为1:1的CapA和CapB的混合溶液，盖帽试剂与固相载体上连接的核酸序列的摩尔比为乙酸酐:N-甲基咪唑:固相载体上连接的核酸序列＝1:1:1。The capping conditions in each step were the same, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a mixed solution of CapA and CapB in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier was acetic anhydride: N-methylimidazole: nucleic acid sequence connected to the solid phase carrier = 1:1:1.
每一步氧化反应条件相同，包括温度为25℃，反应时间为15秒，氧化试剂为浓度为0.05M的碘水。碘与偶联步骤中固相载体上连接的核酸序列的摩尔比为30:1。反应在四氢呋喃:水:吡啶＝3:1:1的混合溶剂中进行。The oxidation reaction conditions in each step were the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing agent of 0.05 M iodine water. The molar ratio of iodine to the nucleic acid sequence connected to the solid phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1.
每一步硫化反应的条件相同，包括温度为25℃，反应时间为300秒，硫化试剂为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为120:1。反应在乙腈:吡啶＝1:1的混合溶剂中进行。The conditions of each step of the sulfurization reaction are the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of hydrogenated xanthan. The molar ratio of the sulfurization reagent to the nucleic acid sequence connected to the solid phase support in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:1.
待最后一个核苷单体连接完成后，依次对固相载体上连接的核酸序列进行切割、脱保护、纯化、脱盐，随后冻干得到正义链和反义链；最后将两条链进行加热退火得到产品，冻干，得到冻干粉。After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and the antisense chain; finally, the two chains are heated and annealed to obtain the product, and freeze-dried to obtain freeze-dried powder.
实施例2：siRNA缀合物(GalNAc-siRNA)的合成Example 2: Synthesis of siRNA conjugate (GalNAc-siRNA)
2.1siRNA缀合物具有如下式II所示的结构：
2.1 The siRNA conjugate has the structure shown in the following formula II:
2.2siRNA缀合物的合成过程 2.2 Synthesis of siRNA conjugates
第一步，通过将DMTr-L96和丁二酸酐反应，得到化合物L96-A：
In the first step, DMTr-L96 and succinic anhydride are reacted to obtain compound L96-A:
制备过程：将DMTr-L96、丁二酸酐、4-二甲基氨基吡啶和二异丙基乙胺加入二氯甲烷中，25℃下搅拌反应24小时，然后用0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷洗涤三次，合并有机相减压蒸干得粗品。然后柱层析纯化得到纯品L96-A。Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine were added to dichloromethane, stirred at 25°C for 24 hours, and then the reaction solution was washed with 0.5M triethylamine phosphate, the aqueous phase was washed three times with dichloromethane, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Then, column chromatography was used for purification to obtain pure L96-A.
第二步，将L96-A与NH2-SPS反应得到L96-B：
In the second step, L96-A is reacted with NH 2 -SPS to obtain L96-B:
制备过程：将L96-A、O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)和二异丙基乙胺(DIPEA)混合溶于乙腈中，室温搅拌5分钟得到均一溶液，加入氨甲基树脂(100-200目)至反应液体中，25℃下开始摇床反应，反应18小时后过滤，滤饼依次用二氯甲烷和乙腈洗涤，得滤饼。所得滤饼用CapA/CapB混合溶液进行盖帽反应得到L96-B，即为含有缀合分子的固相载体，然后在偶联反应下将核苷单体连接至缀合分子，随后按照前文所述的siRNA分子合成方法合成连接至缀合物分子的siRNA正义链，采用前文所述的siRNA分子合成方法合成siRNA反义链，退火生成本申请的siRNA缀合物。Preparation process: L96-A, O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) are mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to obtain a uniform solution, aminomethyl resin (100-200 mesh) is added to the reaction liquid, and the shaking reaction is started at 25°C. After 18 hours of reaction, the filter cake is filtered and washed with dichloromethane and acetonitrile in turn to obtain a filter cake. The obtained filter cake is capped with a CapA/CapB mixed solution to obtain L96-B, which is a solid phase carrier containing a conjugated molecule, and then the nucleoside monomer is connected to the conjugated molecule under a coupling reaction, and then the siRNA sense chain connected to the conjugate molecule is synthesized according to the siRNA molecule synthesis method described above, and the siRNA antisense chain is synthesized using the siRNA molecule synthesis method described above, and annealing is performed to generate the siRNA conjugate of this application.
实施例3：siRNA及siRNA修饰物抑制FXII基因表达Example 3: siRNA and siRNA modifications inhibit FXII gene expression
3.1实验材料：3.1 Experimental Materials:
Hep3B细胞，购自ATCC，货号HB-8064；Hep3B cells, purchased from ATCC, catalog number HB-8064;
转染试剂，购自Invitrogen，货号13778-150； Transfection reagent, purchased from Invitrogen, catalog number 13778-150;
Opti-medium：减血清培养基，购自Gibco，货号31985-070；Opti-medium: Reduced serum medium, purchased from Gibco, catalog number 31985-070;
EMEM培养基：ATCC，货号30-2003；EMEM medium: ATCC, catalog number 30-2003;
RNA提取试剂盒，RNeasy96Kit，货号QIAGEN-74182；RNA extraction kit, RNeasy 96Kit, catalog number QIAGEN-74182;
Fastking RT Kit(with gDNase)，购自TianGen，货号KR116-02；Fastking RT Kit (with gDNase), purchased from TianGen, catalog number KR116-02;
Phosphate Buffer Saline(PBS)，购自Gibco，货号10010-023；Phosphate Buffer Saline (PBS), purchased from Gibco, catalog number 10010-023;
GAPDH TaqMan探针引物购自Thermo Hs02786624_g1；GAPDH TaqMan probe primer was purchased from Thermo Hs02786624_g1;
F12 TaqMan探针引物购自Thermo HS01557542_g1；F12 TaqMan probe primer was purchased from Thermo HS01557542_g1;
FastStart Universal Probe Master，购自Roche，货号04914058001。FastStart Universal Probe Master, purchased from Roche, catalog number 04914058001.
3.2实验方法：3.2 Experimental methods:
3.2.1将Hep3B细胞铺于96孔板的新鲜EMEM培养基中培养24小时。将所培养的细胞用无PS(青霉素链霉素混合液)的EMEM培养基重悬，制成密度为5.55×104/mL的细胞悬液，铺到96孔板中，每孔加90μL细胞悬液，即5000个细胞/孔。3.2.1 Hep3B cells were plated in fresh EMEM medium in a 96-well plate and cultured for 24 hours. The cultured cells were resuspended in EMEM medium without PS (penicillin-streptomycin mixture) to prepare a cell suspension with a density of 5.55×10 4 /mL, and plated in a 96-well plate, adding 90 μL of cell suspension to each well, i.e., 5000 cells/well.
3.2.2将待测siRNA及siRNA修饰物(为便于描述，本实施例实验过程描述中统称为siRNA)的干粉以低温高速离心，然后用超纯蒸馏水(ULtraPure Distilled Water)溶解，配制成100μM母液。3.2.2 Centrifuge the dry powder of the siRNA to be tested and siRNA modifications (for ease of description, collectively referred to as siRNA in the experimental process of this example) at low temperature and high speed, and then dissolve it with ultrapure distilled water (ULtraPure Distilled Water) to prepare a 100 μM stock solution.
3.2.3配制20nM的siRNA稀释液Z和2nM的siRNA稀释液W 3.2.3 Preparation of 20 nM siRNA diluent Z and 2 nM siRNA diluent W
(1)1μM siRNA贮备液Q和0.1μM siRNA贮备液X的制备：(1) Preparation of 1 μM siRNA stock solution Q and 0.1 μM siRNA stock solution X:
a)取上述步骤3.2.2中制得的100μM siRNA母液2μL，加入18μL超纯蒸馏水，得到终浓度为10μM的siRNA稀释液；a) Take 2 μL of the 100 μM siRNA stock solution prepared in step 3.2.2 above, add 18 μL of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 10 μM;
b)取步骤a)中制得的10μM的siRNA稀释液2μL，加入18μL超纯蒸馏水，得到终浓度为1μM的siRNA贮备液Q；b) taking 2 μL of the 10 μM siRNA dilution prepared in step a), adding 18 μL of ultrapure distilled water to obtain a siRNA stock solution Q with a final concentration of 1 μM;
c)取步骤b)中制得的1μM的siRNA贮备液Q 2μL，加入18μL超纯蒸馏水，得到终浓度为0.1μM的siRNA贮备液X；c) taking 2 μL of the 1 μM siRNA stock solution Q prepared in step b), adding 18 μL of ultrapure distilled water to obtain a siRNA stock solution X with a final concentration of 0.1 μM;
(2)取上述配置好的siRNA贮备液Q和siRNA贮备液X各2μL，分别加入98μL Opti-medium，分别得到20nM的siRNA稀释液Z和2nM的siRNA稀释液W。(2) Take 2 μL of the above-prepared siRNA stock solution Q and siRNA stock solution X, respectively, and add 98 μL of Opti-medium to obtain 20 nM siRNA dilution solution Z and 2 nM siRNA dilution solution W, respectively.
3.2.4转染Hep3B细胞3.2.4 Transfection of Hep3B cells
(1)取转染试剂3μL，加入97μL Opti-medium，得到转染试剂稀释液；将转染试剂稀释液与步骤3.2.3中制得的20nM siRNA稀释液Z以1:1体积比混合制备成转染混合物，静置5分钟，取10μL转染混合物加入到96孔板中转染步骤3.2.1中培养的Hep3B细胞(终体积100μL，该体系中siRNA的浓度为1nM)；(1) Take 3 μL of transfection reagent, add 97 μL of Opti-medium, and get Transfection reagent diluent; The transfection reagent diluent and the 20 nM siRNA diluent Z prepared in step 3.2.3 were mixed in a volume ratio of 1:1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10 μL of the transfection mixture was added to a 96-well plate to transfect the Hep3B cells cultured in step 3.2.1 (final volume 100 μL, the concentration of siRNA in this system was 1 nM);
(2)取转染试剂3μL，加入97μL Opti-medium，得到转染试剂稀释液；将转染试剂稀释液与步骤3.2.3中制得的2nM siRNA稀释液W以1:1体积比混合制备成转染混合物，静置5分钟，取10μL转染混合物加入到96孔板中转染步骤3.2.1中培养的Hep3B细胞(终体积100μL，该体系中siRNA的浓度为0.1nM)。(2) Take 3 μL of transfection reagent, add 97 μL of Opti-medium, and get Transfection reagent diluent; The transfection reagent diluent and the 2nM siRNA dilution W prepared in step 3.2.3 were mixed in a volume ratio of 1:1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10 μL of the transfection mixture was added to a 96-well plate to transfect the Hep3B cells cultured in step 3.2.1 (final volume 100 μL, the concentration of siRNA in this system was 0.1 nM).
上述转染后培养48小时；每个浓度(1nM和0.1nM)设置2个重复。The cells were cultured for 48 hours after the above transfection; 2 replicates were set for each concentration (1 nM and 0.1 nM).
3.2.5利用RNeasy96Kit说明书，提取3.2.4中获得的Hep3B细胞中的总RNA。3.2.5 Using RNeasy According to the 96Kit instruction manual, total RNA was extracted from the Hep3B cells obtained in 3.2.4.
3.2.6利用Fastking RT Kit(with gDNase)试剂盒对提取的总RNA进行逆转录至cDNA，按照以下步骤进行：3.2.6 Reverse transcribe the extracted total RNA into cDNA using Fastking RT Kit (with gDNase) according to the following steps:
a)按照下表用gDNA酶除去gDNA；a) Remove gDNA using gDNA enzyme according to the table below;
表6
Table 6
42℃，3min；4℃，静置42℃, 3min; 4℃, stand
b)向步骤a)得到的体系中加入如下所述各试剂并进行逆转录b) Add the following reagents to the system obtained in step a) and perform reverse transcription
表7
Table 7
42℃，15min；95℃，3min。42℃, 15min; 95℃, 3min.
c)将步骤b)所得逆转录产物储存在-20℃以进行实时PCR分析。c) The reverse transcription product obtained in step b) was stored at -20°C for real-time PCR analysis.
3.2.7进行实时PCR分析3.2.7 Real-time PCR analysis
a)如下表所示制备qPCR反应混合物，在整个操作过程中，所有试剂都放置在冰上；a) Prepare qPCR reaction mixture as shown in the table below. Keep all reagents on ice during the entire operation.
表8
Table 8
b)如下所述进行qPCR程序b) Perform qPCR procedure as follows
95℃，10分钟；95°C, 10 minutes;
95℃，15秒；60℃，1分钟(40个循环)。95°C, 15 seconds; 60°C, 1 minute (40 cycles).
3.2.8结果分析3.2.8 Result Analysis
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；a) Use Quant Studio 7 software with default settings to automatically calculate Ct values;
b)使用以下公式计算基因的相对表达量：b) Calculate the relative expression of genes using the following formula:
ΔCt＝Ct(FXII基因)-Ct(GAPDH)ΔCt＝Ct(FXII gene)-Ct(GAPDH)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)ΔΔCt＝ΔCt(test sample group)-ΔCt(Mock group)
相对于Mock组的mRNA表达＝2-ΔΔCt。mRNA expression relative to the Mock group = 2 - ΔΔCt .
抑制率(％)＝(Mock组mRNA相对表达量-检测样品组mRNA相对表达量)/Mock组mRNA相对表达量×100％Inhibition rate (%) = (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group × 100%
Mock组表示：和检测样品组相比，未加入siRNA的组。The Mock group refers to a group to which no siRNA is added compared with the test sample group.
3.3沉默试验结果3.3 Silence test results
选取浓度0.1nM和1nM进行测试，结果如下表9所示。Concentrations of 0.1 nM and 1 nM were selected for testing, and the results are shown in Table 9 below.
表9

Table 9

从表9中可以看出，本公开提供的siRNA、siRNA修饰物显示出对FXII基因的优异的抑制效果。As can be seen from Table 9, the siRNA and siRNA modifications provided by the present disclosure show excellent inhibitory effects on the FXII gene.
3.3.2 IC50测定结果3.3.2 IC50 determination results
下述待测siRNA测定浓度范围设置(nM)为：10、2.5、0.625、0.156、0.039、0.0097、0.0024、0.0006，再按照与3.2相似的方法进行IC50测定。The concentration range of the following siRNA to be tested was set (nM) as follows: 10, 2.5, 0.625, 0.156, 0.039, 0.0097, 0.0024, 0.0006, and IC50 was determined in a similar manner to 3.2.
结果分析Results Analysis
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；a) Use Quant Studio 7 software with default settings to automatically calculate Ct values;
b)使用以下公式计算基因的相对表达量： b) Calculate the relative expression of genes using the following formula:
ΔCt＝Ct(FXII基因)-Ct(GAPDH)ΔCt＝Ct(FXII gene)-Ct(GAPDH)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)ΔΔCt＝ΔCt(test sample group)-ΔCt(Mock group)
相对于Mock组的mRNA表达＝2-ΔΔCt。mRNA expression relative to the Mock group = 2 - ΔΔCt .
Mock组表示：和检测样品组相比，未加入siRNA的组。The Mock group refers to a group to which no siRNA is added compared with the test sample group.
抑制率(％)＝(Mock组mRNA相对表达量-检测样品组mRNA相对表达量)/Mock组mRNA相对表达量×100％，Inhibition rate (%) = (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group × 100%,
以siRNA浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 8的“log(抑制剂)vs.响应-变量斜率”，来拟合量效曲线，从而得出各个siRNA的IC50值。The log value of siRNA concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The "log (inhibitor) vs. response-variable slope" of the analysis software GraphPad Prism 8 was used to fit the dose-effect curve to obtain the IC50 value of each siRNA.
拟合公式为：Y＝Bottom+(Top-Bottom)/(1+10^((logIC50-X)*HillSlope))，The fitting formula is: Y = Bottom + (Top-Bottom) / (1 + 10^((logIC 50 -X)*HillSlope)),
其中：Top表示顶部平台处的百分比抑制率，曲线的Top标准一般在80％至120％；Bottom表示底部平台处的百分比抑制率，曲线的Bottom一般在-20％至20％之间；HillSlope表示百分比抑制率曲线的斜率。Among them: Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.
表10
Table 10
从表10中可以看出，本公开提供的siRNA修饰物显示出对FXII基因的优异的抑制效果。As can be seen from Table 10, the siRNA modifications provided by the present disclosure show excellent inhibitory effects on the FXII gene.
实施例4：递送系统验证Example 4: Delivery System Validation
4.1实验材料：4.1 Experimental Materials:
人原代肝细胞PHH细胞，由上海药明康德新药开发有限公司提供；Human primary hepatocytes PHH cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.;
PHH培养基：invitroGRO CP Meduim serum free BIOVIT，货号：S03316PHH medium: invitroGRO CP Meduim serum free BIOVIT, catalog number: S03316
转染试剂，购自Invitrogen，货号：13778-150； Transfection reagent, purchased from Invitrogen, catalog number: 13778-150;
RNA提取试剂盒96Kit，购自QIAGEN，货号：QIAGEN-74182；RNA Extraction Kit 96Kit, purchased from QIAGEN, catalog number: QIAGEN-74182;
逆转录试剂盒FastKing RT Kit(With gDNase)，购自TianGen，货号：KR116-02；Reverse transcription kit FastKing RT Kit (With gDNase), purchased from TianGen, catalog number: KR116-02;
FastStart Universal Probe master，购自Roche，货号：04914058001；FastStart Universal Probe master, purchased from Roche, catalog number: 04914058001;
FXII及GAPDH引物由上海药明康德新药开发有限公司提供。FXII and GAPDH primers were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.
4.2实验方法：4.2 Experimental methods:
siRNA缀合物(siRNA缀合物终浓度为10nM、2.5nM、0.63nM、0.16nM、0.04nM、0.01nM、0.0024nM和0.0006nM，复孔)通过转染进入PHH细胞，过程如下所述：取冻存的PHH细胞，复苏，计数，调整细胞到6×105细胞/mL，同时应用RNAiMax转染试剂将siRNA缀合物转入细胞，以每孔54,000个细胞的密度接种到96孔板中，每孔培养液为100μL。细胞置于5％CO2、37℃孵箱中培养。48小时后，去除培养基并收集细胞用于总RNA提取。根据试剂盒产品说明书使用96Kit提取总RNA。siRNA conjugates (final concentrations of siRNA conjugates were 10 nM, 2.5 nM, 0.63 nM, 0.16 nM, 0.04 nM, 0.01 nM, 0.0024 nM and 0.0006 nM, in duplicate) were transfected into PHH cells as follows: frozen PHH cells were taken, revived, counted, and the cells were adjusted to 6 × 10 5 cells/mL, and applied simultaneously RNAiMax transfection reagent was used to transfer siRNA conjugates into cells, and cells were seeded into 96-well plates at a density of 54,000 cells per well, with 100 μL of culture medium per well. Cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
siRNA缀合物(siRNA缀合物终浓度为500nM、125nM、31.25nM、7.81nM、1.95nM、0.49nM、 0.12nM和0.03nM，复孔)通过自由摄取进入PHH细胞，过程如下所述：取冻存的PHH细胞，复苏，计数，调整细胞到6×105细胞/mL，同时加入siRNA缀合物，以每孔54,000个细胞的密度接种到96孔板中，每孔培养液为100μL。细胞置于5％CO2、37℃孵箱中培养。48小时后，去除培养基并收集细胞用于总RNA提取。根据试剂盒产品说明书使用96Kit提取总RNA。siRNA conjugates (final concentrations of siRNA conjugates were 500 nM, 125 nM, 31.25 nM, 7.81 nM, 1.95 nM, 0.49 nM, 0.12nM and 0.03nM, duplicate wells) enter PHH cells by free uptake, the process is as follows: take frozen PHH cells, revive, count, adjust the cells to 6×10 5 cells/mL, add siRNA conjugates at the same time, and inoculate them into 96-well plates at a density of 54,000 cells per well, with 100μL of culture medium per well. The cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
采用与实施例3中相似的方法，通过逆转录反应将提取的总RNA逆转录为cDNA，以及通过qPCR对逆转录得到的FXII cDNA进行定量扩增。GAPDH cDNA将作为内部对照进行平行扩增。PCR反应程序为：95℃,10分钟，然后进入循环模式，95℃,15秒，随后60℃，60秒，共40个循环。Using a method similar to that in Example 3, the extracted total RNA was reverse transcribed into cDNA by reverse transcription reaction, and the FXII cDNA obtained by reverse transcription was quantitatively amplified by qPCR. GAPDH cDNA was amplified in parallel as an internal control. The PCR reaction program was: 95°C, 10 minutes, then enter the cycle mode, 95°C, 15 seconds, then 60°C, 60 seconds, for a total of 40 cycles.
4.3结果分析4.3 Results Analysis
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；a) Use Quant Studio 7 software with default settings to automatically calculate Ct values;
b)使用以下公式计算基因的相对表达量：b) Calculate the relative expression of genes using the following formula:
ΔCt＝Ct(FXII基因)-Ct(GAPDH)ΔCt＝Ct(FXII gene)-Ct(GAPDH)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)ΔΔCt＝ΔCt(test sample group)-ΔCt(Mock group)
相对于Mock组的mRNA表达＝2-ΔΔCt，其中Mock组表示和检测样品组相比，未加入siRNA缀合物的组。mRNA expression relative to the Mock group=2 -ΔΔCt , wherein the Mock group represents a group without the addition of siRNA conjugates compared with the test sample group.
抑制率(％)＝(Mock组mRNA相对表达量-检测样品组mRNA相对表达量)/Mock组mRNA相对表达量×100％；Inhibition rate (%) = (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group × 100%;
以siRNA缀合物浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 8的log(抑制剂)vs.响应-变量斜率，来拟合量效曲线，从而得出各个siRNA缀合物的IC50值。The log value of the siRNA conjugate concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The log (inhibitor) vs. response-variable slope of the analysis software GraphPad Prism 8 was used to fit the dose-effect curve to obtain the IC50 value of each siRNA conjugate.
拟合公式为：Y＝Bottom+(Top-Bottom)/(1+10^((logIC50-X)*HillSlope))The fitting formula is: Y = Bottom + (Top-Bottom) / (1 + 10^((logIC 50 -X)*HillSlope))
其中：Top表示顶部平台处的百分比抑制率，曲线的Top标准一般在80％至120％；Bottom表示底部平台处的百分比抑制率，曲线的Bottom一般在-20％至20％之间；HillSlope表示百分比抑制率曲线的斜率。Among them: Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.
表11
Table 11
上表中的L96也即式I所示的缀合物基团GalNAc。L96 in the above table is the conjugate group GalNAc shown in Formula I.
从表11中可以看出，本公开提供的siRNA缀合物显示出对FXII基因的优异的抑制效果。As can be seen from Table 11, the siRNA conjugates provided by the present disclosure show excellent inhibitory effects on the FXII gene.
实施例5：siRNA的合成Example 5: Synthesis of siRNA
5.1siRNA序列设计5.1 siRNA sequence design
本实施例中的siRNA序列设计思想和方法与实施例1中siRNA序列设计思想和方法的相同，且用于设计siRNA的靶标序列如下所示，靶标序列来源于FXII的基因mRNA序列(参见NM_000505.4)。The siRNA sequence design concept and method in this example are the same as those in Example 1, and the target sequence used to design siRNA is shown below. The target sequence is derived from the gene mRNA sequence of FXII (see NM_000505.4).
靶标序列XVI：
Target sequence XVI:
靶标序列XVI-1：
Target sequence XVI-1:
靶标序列XVI-2：
Target sequence XVI-2:
靶标序列XVII
Target sequence XVII
靶标序列XVII-1
Target sequence XVII-1
靶标序列XVII-2
Target sequence XVII-2
靶标序列XVII-3
Target sequence XVII-3
靶标序列XVII-4
Target sequence XVII-4
靶标序列XVII-5
Target sequence XVII-5
靶标序列XVII-6
Target sequence XVII-6
靶标序列XVIII
Target sequence XVIII
靶标序列XVIII-1
Target sequence XVIII-1
靶标序列XVIII-2
Target sequence XVIII-2
靶标序列XVIII-3
Target sequence XVIII-3
靶标序列XIX：
Target sequence XIX:
靶标序列XX：
Target sequence XX:
靶标序列XXI：
Target sequence XXI:
靶标序列XXI-1：
Target sequence XXI-1:
靶标序列XXI-2：
Target sequence XXI-2:
靶标序列XXI-3：
Target sequence XXI-3:
靶标序列XXII
Target sequence XXII
靶标序列XXIII：
Target sequence XXIII:
靶标序列XXIII-1：
Target sequence XXIII-1:
靶标序列XXIII-2：
Target sequence XXIII-2:
靶标序列XXIII-3：
Target sequence XXIII-3:
靶标序列XXIII-4：
Target sequence XXIII-4:
实施例6：siRNA、siRNA修饰物抑制FXII基因表达Example 6: siRNA and siRNA modifications inhibit FXII gene expression
本实施例中使用的实验材料、实验方法与实施例3中的实验材料、实验方法相同。基因表达沉默实验结果如下表12所示。The experimental materials and experimental methods used in this example are the same as those in Example 3. The results of the gene expression silencing experiment are shown in Table 12 below.
表12

Table 12

从表12中可以看出，本公开提供的siRNA、siRNA修饰物显示出对FXII基因的优异的抑制效果。As can be seen from Table 12, the siRNA and siRNA modifications provided by the present disclosure show excellent inhibitory effects on the FXII gene.
实施例7：siRNA、siRNA缀合物抑制FXII基因表达的抑制率测定Example 7: Determination of the inhibition rate of siRNA and siRNA conjugates in inhibiting FXII gene expression
7.1实验材料：7.1 Experimental Materials:
人原代肝细胞(PHH细胞)，由上海药明康德新药开发有限公司提供；Primary human hepatocytes (PHH cells) were provided by Shanghai WuXi AppTec Pharmaceuticals Co., Ltd.;
PHH培养基：invitroGRO CP Meduim serum free BIOVIT，货号：S03316；PHH medium: invitroGRO CP Meduim serum free BIOVIT, catalog number: S03316;
转染试剂，购自Invitrogen，货号：13778-150； Transfection reagent, purchased from Invitrogen, catalog number: 13778-150;
RNA提取试剂盒96Kit，购自QIAGEN，货号：QIAGEN-74182；RNA Extraction Kit 96Kit, purchased from QIAGEN, catalog number: QIAGEN-74182;
逆转录试剂盒FastKing RT Kit(With gDNase)，购自TianGen，货号：KR116-02；Reverse transcription kit FastKing RT Kit (With gDNase), purchased from TianGen, catalog number: KR116-02;
FastStart Universal Probe master，购自Roche，货号：04914058001；FastStart Universal Probe master, purchased from Roche, catalog number: 04914058001;
FXII及GAPDH引物由上海药明康德新药开发有限公司提供。FXII and GAPDH primers were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.
7.2实验方法：7.2 Experimental methods:
siRNA缀合物(siRNA缀合物终浓度为5nM和0.5nM，复孔)通过转染进入PHH细胞，过程如下所述：取冻存的PHH细胞，复苏，计数，调整细胞到6×105细胞/mL，同时应用RNAiMax转染试剂将siRNA缀合物转入细胞，以每孔54,000个细胞的密度接种到96孔板中，每孔培养液为100μL。细胞置于5％CO2、37℃孵箱中培养。48小时后，去除培养基并收集细胞用于总RNA提取。根据试剂盒产品说明书使用96Kit提取总RNA。siRNA conjugates (final concentration of siRNA conjugates was 5 nM and 0.5 nM, duplicate wells) were transfected into PHH cells by the following process: frozen PHH cells were taken, revived, counted, and the cells were adjusted to 6 × 10 5 cells/mL, and then applied RNAiMax transfection reagent was used to transfer siRNA conjugates into cells, and cells were seeded into 96-well plates at a density of 54,000 cells per well, with 100 μL of culture medium per well. Cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
siRNA缀合物(siRNA缀合物终浓度为100nM和5nM，复孔)通过自由摄取进入PHH细胞，过程如下所述：取冻存的PHH细胞，复苏，计数，调整细胞到6×105细胞/mL，同时加入siRNA缀合物，以每孔54,000个细胞的密度接种到96孔板中，每孔培养液为100μL。细胞置于5％CO2、37℃孵箱中培养。48小时后，去除培养基并收集细胞用于总RNA提取。根据试剂盒产品说明书使用96Kit提取总RNA。siRNA conjugates (final concentrations of siRNA conjugates were 100 nM and 5 nM, in duplicate) were freely taken into PHH cells, and the process was as follows: frozen PHH cells were taken, revived, counted, and adjusted to 6 × 10 5 cells/mL, and siRNA conjugates were added at the same time, and inoculated into 96-well plates at a density of 54,000 cells per well, with 100 μL of culture medium per well. The cells were cultured in a 5% CO 2 , 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.
采用与实施例3中相似的方法，通过逆转录反应将提取的总RNA逆转录为cDNA，以及通过qPCR对逆转录得到的FXII cDNA进行定量扩增。GAPDH cDNA将作为内部对照进行平行扩增。PCR反应程序为：95℃，10分钟，然后进入循环模式，95℃，15秒，随后60℃，60秒，共40个循环。Using a method similar to that in Example 3, the extracted total RNA was reverse transcribed into cDNA by reverse transcription reaction, and the FXII cDNA obtained by reverse transcription was quantitatively amplified by qPCR. GAPDH cDNA was amplified in parallel as an internal control. The PCR reaction program was: 95°C, 10 minutes, then entered the cycle mode, 95°C, 15 seconds, then 60°C, 60 seconds, for a total of 40 cycles.
7.3结果分析7.3 Results Analysis
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；a) Use Quant Studio 7 software with default settings to automatically calculate Ct values;
b)使用以下公式计算基因的相对表达量：b) Calculate the relative expression of genes using the following formula:
ΔCt＝Ct(FXII基因)-Ct(GAPDH)ΔCt＝Ct(FXII gene)-Ct(GAPDH)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)ΔΔCt＝ΔCt(test sample group)-ΔCt(Mock group)
相对于Mock组的mRNA表达＝2-ΔΔCt。mRNA expression relative to the Mock group = 2 - ΔΔCt .
抑制率(％)＝(Mock组mRNA相对表达量-检测样品组mRNA相对表达量)/Mock组mRNA相对表达量×100％；Inhibition rate (%) = (relative expression of mRNA in the Mock group - relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group × 100%;
其中Mock组表示和检测样品组相比，未加入siRNA缀合物的组。The Mock group refers to a group without the addition of siRNA conjugates compared with the test sample group.
表13

Table 13

上表中的L96也即式I所示的缀合物基团GalNAc。L96 in the above table is the conjugate group GalNAc shown in Formula I.
从表13中可以看出，本公开提供的siRNA缀合物显示出对FXII基因的优异的抑制效果。As can be seen from Table 13, the siRNA conjugates provided by the present disclosure show excellent inhibitory effects on the FXII gene.
实施例8：siRNA缀合物在人源化小鼠中对人FXII(hFXII)基因表达的抑制作用Example 8: Inhibitory effect of siRNA conjugates on human FXII (hFXII) gene expression in humanized mice
6-8周龄的C57BL/6-hFXII小鼠(由上海南方模式生物科技股份有限公司提供)进入饲养设施，适应性喂养7天后，以1mg/kg或3mg/kg的单一剂量对小鼠分别皮下给药N-ER-FY009028M3L96、N-ER-FY009028M7L96、N-ER-FY009017M2L96、N-ER-FY009065M2L96、N-ER-FY009112M2L96、N-ER-FY009122M3L96、N-ER-FY009151M3L96、N-ER-FY009151M7L96、N-ER-FY009123M3L96、N-ER-FY009154M3L96、N-ER-FY009162M7L96、N-ER-FY009077M7L96和N-ER-FY009183M7L96(每组6只小鼠)。给药后第7日、第14日、第21日、第28日和第35日检测血清hFXII蛋白表达量，由此得出siRNA缀合物对hFXII蛋白表达的抑制率。 C57BL/6-hFXII mice (provided by Shanghai Model Organisms Technology Co., Ltd.) aged 6-8 weeks were placed in the breeding facility. After 7 days of adaptive feeding, the mice were subcutaneously administered N-ER-FY009028M3L96, N-ER-FY009028M7L96, N-ER-FY009017M2L96, N-ER-FY009065M2L96, and N-ER-FY009028M3L96 at a single dose of 1 mg/kg or 3 mg/kg. 09112M2L96, N-ER-FY009122M3L96, N-ER-FY009151M3L96, N-ER-FY009151M7L96, N-ER-FY009123M3L96, N-ER-FY009154M3L96, N-ER-FY009162M7L96, N-ER-FY009077M7L96 and N-ER-FY009183M7L96 (6 mice in each group). The serum hFXII protein expression was detected on the 7th, 14th, 21st, 28th and 35th day after administration, thereby obtaining the inhibition rate of hFXII protein expression by siRNA conjugates.
表14 siRNA缀合物对hFXII蛋白抑制率
Table 14 Inhibition rate of hFXII protein by siRNA conjugates
从表14可以看出，本公开的siRNA缀合物在体内对hFXII基因表达的蛋白具有较高的抑制活性，能够长时间降低hFXII蛋白水平，以3mg/kg的单一剂量给药，在第7天至第35天的抑制率结果中，siRNA缀合物能有高达80％以上的抑制率；以1mg/kg的单一剂量给药，在第7天至第28天的抑制率结果中，siRNA缀合物有70％以上的抑制率，在实验期长达35天时甚至有68％以上的抑制率；说明设计的化合物能较好的抑制hFXII蛋白的表达。As can be seen from Table 14, the siRNA conjugates disclosed in the present invention have high inhibitory activity on the protein expressed by the hFXII gene in vivo, and can reduce the hFXII protein level for a long time. When administered with a single dose of 3 mg/kg, the siRNA conjugate can have an inhibition rate of more than 80% in the inhibition rate results from the 7th day to the 35th day; when administered with a single dose of 1 mg/kg, the siRNA conjugate has an inhibition rate of more than 70% in the inhibition rate results from the 7th day to the 28th day, and even has an inhibition rate of more than 68% when the experimental period is as long as 35 days; This shows that the designed compound can effectively inhibit the expression of hFXII protein.
实施例9：食蟹猴凝血造模实验Example 9: Cynomolgus monkey coagulation modeling experiment
9.1 5-7岁的雄性食蟹猴(体重5.0-7.5kg)(由苏州西山中科实验动物有限公司提供)进入饲养设施，适应性喂养14天。在适应期进行以下操作：9.1 Male cynomolgus monkeys aged 5-7 years (weight 5.0-7.5 kg) (provided by Suzhou Xishan Zhongke Experimental Animal Co., Ltd.) were placed in the breeding facility and fed adaptively for 14 days. The following operations were performed during the adaptation period:
1)体重称量：2次/周；1) Body weight: 2 times/week;
2)笼内临床观察：2次/天。2) Clinical observation in cage: 2 times/day.
3)基线生理指标采集(给药前7天，即D-7)：凝血功能生化指标2项(APTT、PT)，血浆及血清样品采集(用于F12蛋白表达检测)，动静脉分流(Arteriovenous shunt，AV-shunt)造模后称取血栓湿重，以进行凝血功能检测。按此顺序(血浆及血清样品采集、凝血功能生化指标2项、AV-shunt造模后称取血栓湿重)进行逐一操作，血液采集位置及各检测细节详见下文描述。3) Baseline physiological index collection (7 days before administration, i.e. D-7): 2 biochemical indices of coagulation function (APTT, PT), plasma and serum sample collection (for F12 protein expression detection), and wet weight of thrombus after arteriovenous shunt (AV-shunt) modeling for coagulation function detection. The operations were performed one by one in this order (plasma and serum sample collection, 2 biochemical indices of coagulation function, and wet weight of thrombus after AV-shunt modeling). The blood collection location and test details are described below.
9.2适应期结束后(给药当天为D0)以10mg/kg的单一剂量对食蟹猴分别皮下给药N-ER-FY009028M7L96和N-ER-FY009151M7L96(每组3只食蟹猴)。9.2 After the adaptation period (the day of administration was D0), N-ER-FY009028M7L96 and N-ER-FY009151M7L96 were subcutaneously administered to cynomolgus monkeys at a single dose of 10 mg/kg (3 cynomolgus monkeys in each group).
9.3建立凝血模型9.3 Establishing the coagulation model
给药前7天(D-7)和给药后28天(D28)，肌肉注射舒泰50(5mg/kg)麻醉食蟹猴后进行气管插管，接入呼吸麻醉机，通过手术建立AV-shunt凝血模型：在动物左后肢股内侧切开皮肤，进行钝性解剖以暴露股动脉及股静脉，使用特制连通管(制作方法为：将输液延长管剪取两根15cm长的管，两管首尾相连，在连接处使用一根10cm长的粗糙已称重鱼线缠绕加固)依次插入股静脉近心端和股动脉近心端后，待血液流通15分钟后取出，取出管内血栓，称取血栓湿重，结扎血管，关闭通路。 手术完成后，肌肉注射头孢曲松50mg/kg，连续给药3天，肌肉注射美洛昔康0.1mL/kg，连续给药3天。期间每天笼内临床观察2次/天，主要观察动物精神状态、行为活动等指标，必要时进行详细观察。若发现动物采食困难、无法站立、褥疮等异常情况，及时通知兽医进行护理。7 days before administration (D-7) and 28 days after administration (D28), crab-eating macaques were anesthetized by intramuscular injection of Shutai 50 (5 mg/kg), then intubated and connected to a respiratory anesthesia machine. The AV-shunt coagulation model was established by surgery: the skin was incised on the inner side of the left hind limb of the animal, and blunt dissection was performed to expose the femoral artery and femoral vein. A special connecting tube (made by cutting two 15 cm long tubes from the infusion extension tube, connecting the two tubes end to end, and using a 10 cm long rough weighed fishing line to wrap and reinforce the connection) was inserted into the proximal end of the femoral vein and the proximal end of the femoral artery in turn, and then removed after blood circulation for 15 minutes, the thrombus in the tube was removed, the wet weight of the thrombus was weighed, the blood vessel was ligated, and the access was closed. After the operation, ceftriaxone 50 mg/kg was injected intramuscularly for 3 consecutive days, and meloxicam 0.1 mL/kg was injected intramuscularly for 3 consecutive days. During this period, clinical observation was performed twice a day in the cage, mainly observing the animal's mental state, behavioral activities and other indicators, and detailed observation was performed when necessary. If the animal was found to have difficulty eating, unable to stand, bedsores and other abnormal conditions, the veterinarian was notified in time for care.
(4)结果(4) Results
表15 siRNA缀合物对hFXII蛋白抑制率
Table 15 Inhibition rate of hFXII protein by siRNA conjugates
从表15可以看出，本公开的siRNA缀合物在食蟹猴体内对hFXII基因表达的蛋白具有较高的抑制活性，能够长时间降低hFXII蛋白水平，在第7天至第35天的抑制率效果持续增加，在第35天缀合物均出现90％以上的抑制率，说明设计的化合物能较好的抑制hFXII蛋白的表达。As can be seen from Table 15, the siRNA conjugates disclosed in the present invention have high inhibitory activity on the protein expressed by the hFXII gene in cynomolgus monkeys, and can reduce the hFXII protein level for a long time. The inhibition rate effect continued to increase from the 7th day to the 35th day. On the 35th day, the conjugate showed an inhibition rate of more than 90%, indicating that the designed compound can effectively inhibit the expression of hFXII protein.
表16 siRNA缀合物APTT(活化部分凝血活酶时间)延长率
Table 16 siRNA conjugate APTT (activated partial thromboplastin time) extension rate
从表16可以看出，本公开的siRNA缀合物在第7天至第35天的APTT延长率持续增加，在第35天缀合物均出现160％以上的延长率，说明设计的化合物能较好的延长活化部分凝血活酶时间。As can be seen from Table 16, the APTT extension rate of the siRNA conjugates disclosed in the present invention continued to increase from the 7th day to the 35th day, and on the 35th day, the conjugates showed an extension rate of more than 160%, indicating that the designed compound can better prolong the activated partial thromboplastin time.
实施例10：siRNA缀合物在CD-1小鼠血浆动力学研究Example 10: Kinetic study of siRNA conjugates in CD-1 mouse plasma
试验动物：CD-1小鼠，SPF级，雄性，30g左右，购买于斯贝福(北京)生物技术有限公司。Experimental animals: CD-1 mice, SPF grade, male, about 30 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.
给药剂量和方式：siRNA缀合物在3mg/kg(10mL/kg)的剂量下给药，随机分组后单次皮下注射给药，每组6只小鼠。Dosage and administration method: siRNA conjugates were administered at a dose of 3 mg/kg (10 mL/kg) and given as a single subcutaneous injection after randomization, with 6 mice in each group.
样品采集：采集给药后0.0833、0.25、0.5、1、2、4、8、24、36、48h全血样品，共10个点。每组前3只采集0.0833、0.5、2、8、36h，后3只采集0.25、1、4、24、48h，采集全血后离心分离血浆进行检测分析。Sample collection: Whole blood samples were collected at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, and 48 hours after administration, for a total of 10 points. The first 3 samples in each group were collected at 0.0833, 0.5, 2, 8, and 36 hours, and the last 3 samples were collected at 0.25, 1, 4, 24, and 48 hours. After whole blood was collected, plasma was separated by centrifugation for detection and analysis.
样品检测与分析：采用LC-MS/MS方法检测各时间点血浆样品中原形药物的浓度，使用WinNonlin软件计算PK参数：Cmax、Tmax、AUC、MRT、t1/2。Sample detection and analysis: LC-MS/MS method was used to detect the concentration of the original drug in plasma samples at each time point, and WinNonlin software was used to calculate the PK parameters: C max , T max , AUC, MRT, t 1/2 .
从该实验中可以得出，本公开的siRNA缀合物siRNA225-siRNA296、siRNA415-siRNA441、siRNA454-siRNA464在血浆中半衰期较短，清除较快。It can be concluded from this experiment that the siRNA conjugates siRNA225-siRNA296, siRNA415-siRNA441, and siRNA454-siRNA464 disclosed in the present invention have a shorter half-life in plasma and are eliminated faster.
实施例11：siRNA缀合物在CD-1小鼠组织分布试验Example 11: Tissue distribution test of siRNA conjugates in CD-1 mice
试验动物：CD-1小鼠，SPF级，雄性，30g左右，购买于斯贝福(北京)生物技术有限公司。Experimental animals: CD-1 mice, SPF grade, male, about 30 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.
给药剂量和方式：siRNA缀合物在3mg/kg(10mL/kg)的剂量下给药，随机分组后单次皮下注射给药，每个时间点3只动物，共24只小鼠。Dosage and administration method: siRNA conjugates were administered at a dose of 3 mg/kg (10 mL/kg) and given by a single subcutaneous injection after randomization, with 3 animals at each time point, for a total of 24 mice.
样品采集：Sample collection:
给药后24h：采集血浆、肝、肾、脾；24 h after administration: plasma, liver, kidney, and spleen were collected;
给药后72h：采集血浆、肝、肾、脾；72h after administration: plasma, liver, kidney, and spleen were collected;
给药后168h(1周)：采集血浆、肝、肾、脾、脑、心、肺、胃、小肠、肌肉、睾丸；168 h after administration (1 week): plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, and testis were collected;
给药后336h(2周)：采集血浆、肝、肾、脾；336h after administration (2 weeks): plasma, liver, kidney, and spleen were collected;
给药后672h(4周)：采集血浆、肝、肾、脾、脑、心、肺、胃、小肠、肌肉、睾丸；672h after administration (4 weeks): plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, and testis were collected;
给药后1008h(6周)：采集血浆、肝、肾、脾；1008 h after administration (6 weeks): plasma, liver, kidney, and spleen were collected;
给药后1344h(8周)：采集血浆、肝、肾、脾；1344h after administration (8 weeks): plasma, liver, kidney, and spleen were collected;
给药后1680h(10周)：采集血浆、肝、肾、脾、脑、心、肺、胃、小肠、肌肉、睾丸。1680h after administration (10 weeks): plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, and testis were collected.
样品检测与分析：采用LC-MS/MS方法检测各时间点血浆和组织样品中原形药物的浓度，采用 梯形面积法计算血浆及组织中的AUC。Sample detection and analysis: LC-MS/MS method was used to detect the concentration of the original drug in plasma and tissue samples at each time point. The AUC in plasma and tissues was calculated by the trapezoidal area method.
从该实验中可以得出，本公开的siRNA缀合物siRNA225-siRNA296、siRNA415-siRNA441、siRNA454-siRNA464主要富集于肝脏，在组织中保留时间较长，具有很好的稳定性。It can be concluded from this experiment that the siRNA conjugates siRNA225-siRNA296, siRNA415-siRNA441, and siRNA454-siRNA464 disclosed in the present invention are mainly enriched in the liver, have a long retention time in the tissue, and have good stability.
实施例12：siRNA缀合物单次皮下注射C57B/L小鼠给予MTD试验Example 12: MTD test of single subcutaneous injection of siRNA conjugate into C57B/L mice
试验动物：C57小鼠，SPF级，雄性，25g左右，购买于斯贝福(北京)生物技术有限公司。动物根据适应期最后1天的体重，采用体重随机区组的方法，具体剂量设计和分组如下表17所示：Experimental animals: C57 mice, SPF grade, male, about 25g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Animals were randomly divided into groups according to their body weight on the last day of the adaptation period. The specific dosage design and grouping are shown in Table 17 below:
表17
Table 17
检测指标：Detection indicators:
临床观察：给药日连续观察4小时，恢复期每天至少进行一次临床观察。Clinical observation: Continuous observation for 4 hours on the day of administration, and clinical observation at least once a day during the recovery period.
体重：对所有存活动物每周进行2次体重称量。Body weight: All surviving animals were weighed twice a week.
免疫毒性：MTD剂量组动物于D1给药后1h±2min，4h±5min，8h±10min，24h±20min交替采血，每个时间点采集3只/性别/组动物，检测细胞因子(IFN-γ、TNF-α、IL-2/6/8)。Immunotoxicity: Blood was collected from animals in the MTD dose group at 1h±2min, 4h±5min, 8h±10min, and 24h±20min after administration on D1. Three animals/sex/group were collected at each time point for detection of cytokines (IFN-γ, TNF-α, IL-2/6/8).
毒代动力学：MTD剂量组动物于D1给药前、给药后30min±2min，1h±2min，4h±5min，8h±10min，24h±20min交替采血，每个时间点采集3只/性别/组动物，检测血药浓度。Toxicokinetics: Blood samples were collected from animals in the MTD dose group at 30min±2min, 1h±2min, 4h±5min, 8h±10min, and 24h±20min before and after administration on D1. Three animals/sex/group were collected at each time point to detect blood drug concentration.
血液生化学：主试验组动物于D28剖检，卫星组动物于D7、D14、D21、D28分批次剖检，检测血液生化学。Blood biochemistry: The animals in the main test group were autopsied on D28, and the animals in the satellite group were autopsied in batches on D7, D14, D21, and D28 to test blood biochemistry.
组织分布：主试验组动物于D28剖检，卫星组动物于D7、D14、D21、D28分批次剖检，采集血、肝，检测组织药物浓度。Tissue distribution: The animals in the main test group were autopsied on D28, and the animals in the satellite group were autopsied in batches on D7, D14, D21, and D28. Blood and liver were collected to detect tissue drug concentrations.
组织病理学检查：主试验组动物于D28剖检，采集主要脏器(心、肝、脾、肺、肾、脑、肾上腺、胸腺、胃、子宫/睾丸、卵巢/附睾)以及发现异常的组织或脏器，取材并固定，进行组织病理学检查。Histopathological examination: The animals in the main experimental group were autopsied on D28, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and abnormal tissues or organs were collected and fixed for histopathological examination.
从该实验中可以得出，本公开的siRNA缀合物siRNA225-siRNA296、siRNA415-siRNA441、siRNA454-siRNA464毒性较低，具有优异的用药安全窗口。It can be concluded from this experiment that the siRNA conjugates siRNA225-siRNA296, siRNA415-siRNA441, and siRNA454-siRNA464 disclosed in the present invention have low toxicity and an excellent safety window for use.
本公开的上述实施例仅是为清楚地说明本公开所作的举例，而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本公开权利要求的保护范围之内。 The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims (46)

  1. 一种双链核糖核酸，所述双链核糖核酸包括正义链和反义链，所述正义链与所述反义链互补和/或基本上反向互补形成所述双链核糖核酸的双链区；A double-stranded RNA, comprising a sense strand and an antisense strand, wherein the sense strand is complementary and/or substantially reverse complementary to the antisense strand to form a double-stranded region of the double-stranded RNA;
    其中，所述正义链包含与靶标序列中至少15个连续核苷酸的差异不超过3个核苷酸的序列A，所述反义链包含与靶标序列中至少15个连续核苷酸的反向互补序列的差异不超过3个核苷酸的序列B；The sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence, and the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence;
    所述靶标序列选自如SEQ ID NO:1～15，641～648任一项所示的核苷酸序列。The target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NO: 1 to 15, 641 to 648.
  2. 根据权利要求1所述的双链核糖核酸，其中，所述靶标序列选自如SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列，所述正义链包含如SEQ ID NO:2,4～6,8～10,13～14，16～29,644,645,647,649～666任一项所示的核苷酸序列中至少15个连续核苷酸组成的序列A，所述反义链包含如SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中至少15个连续核苷酸组成的序列反向互补和/或基本上反向互补的序列B。The double-stranded ribonucleic acid according to claim 1, wherein the target sequence is selected from the nucleotide sequence as shown in any one of SEQ ID NO: 2, 4-6, 8-10, 13-14, 16-29, 644, 645, 647, 649-666, the positive strand comprises a sequence A consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NO: 2, 4-6, 8-10, 13-14, 16-29, 644, 645, 647, 649-666, and the antisense strand comprises a sequence B that is reverse complementary and/or substantially reverse complementary to a sequence consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NO: 2, 4-6, 8-10, 13-14, 16-29, 644, 645, 647, 649-666.
  3. 根据权利要求1或2所述的双链核糖核酸，其中，所述正义链由15-28个核苷酸组成，优选18-25个核苷酸，更优选18-23个核苷酸，更优选19、21或23个核苷酸。The double-stranded ribonucleic acid according to claim 1 or 2, wherein the sense strand consists of 15-28 nucleotides, preferably 18-25 nucleotides, more preferably 18-23 nucleotides, more preferably 19, 21 or 23 nucleotides.
  4. 根据权利要求3所述的双链核糖核酸，其中，所述正义链的核苷酸序列是与SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中的15-28个连续核苷酸组成的序列相比差异不超过1个核苷酸的序列A，优选18-25个连续核苷酸，更优选18-23个连续核苷酸，更优选19、21或23个核苷酸。The double-stranded ribonucleic acid according to claim 3, wherein the nucleotide sequence of the positive chain is a sequence A that differs by no more than 1 nucleotide compared with a sequence consisting of 15-28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NO: 2, 4-6, 8-10, 13-14, 16-29, 644, 645, 647, 649-666, preferably 18-25 consecutive nucleotides, more preferably 18-23 consecutive nucleotides, more preferably 19, 21 or 23 nucleotides.
  5. 根据权利要求1-4任一项所述的双链核糖核酸，其中，所述反义链由15-28个核苷酸组成，优选18-25个核苷酸，更优选18-23个核苷酸，更优选19、21或23个核苷酸。The double-stranded ribonucleic acid according to any one of claims 1 to 4, wherein the antisense strand consists of 15-28 nucleotides, preferably 18-25 nucleotides, more preferably 18-23 nucleotides, more preferably 19, 21 or 23 nucleotides.
  6. 根据权利要求5所述的双链核糖核酸，其中，所述反义链的核苷酸序列是与SEQ ID NO:2,4～6,8～10,13～14,16～29,644,645,647,649～666任一项所示的核苷酸序列中的15-28个连续核苷酸组成的序列的反向互补序列相比差异不超过1个核苷酸的序列B，优选18-25个连续核苷酸，更优选18-23个连续核苷酸，更优选19、21或23个核苷酸。The double-stranded ribonucleic acid according to claim 5, wherein the nucleotide sequence of the antisense strand is a sequence B having a difference of no more than 1 nucleotide compared to the reverse complementary sequence of a sequence consisting of 15-28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NO: 2, 4-6, 8-10, 13-14, 16-29, 644, 645, 647, 649-666, preferably 18-25 consecutive nucleotides, more preferably 18-23 consecutive nucleotides, more preferably 19, 21 or 23 nucleotides.
  7. 根据权利要求1-6任一项所述的双链核糖核酸，其中，所述双链区的长度为15-25个核苷酸，优选18-23个核苷酸，更优选18-21个核苷酸，更优选19、21或23个核苷酸。The double-stranded ribonucleic acid according to any one of claims 1 to 6, wherein the length of the double-stranded region is 15-25 nucleotides, preferably 18-23 nucleotides, more preferably 18-21 nucleotides, more preferably 19, 21 or 23 nucleotides.
  8. 根据权利要求1-7任一项所述的双链核糖核酸，其中，The double-stranded RNA according to any one of claims 1 to 7, wherein
    所述正义链与所述反义链互补形成所述双链区，且所述正义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，所述反义链的3’末端形成平末端；或者，The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end; or,
    所述正义链与所述反义链互补形成所述双链区，且所述反义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸，所述正义链的3’末端形成平末端；或者，The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end; or,
    所述正义链与所述反义链互补形成所述双链区，且所述正义链与所述反义链的3’末端均具有1-2个延伸出所述双链区的突出的核苷酸；或者，The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region; or,
    所述正义链与所述反义链互补形成所述双链区，且所述正义链与所述反义链的3’末端均形成平末端。The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.
  9. 根据权利要求1-8任一项所述的双链核糖核酸，其中，所述正义链与所述反义链选自如下组合：The double-stranded RNA according to any one of claims 1 to 8, wherein the sense strand and the antisense strand are selected from the following combinations:
    1)所述正义链包含如SEQ ID NO:30所示的核苷酸序列，所述反义链包含如SEQ ID NO:142所示的核苷酸序列；1) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:30, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:142;
    2)所述正义链包含如SEQ ID NO:31所示的核苷酸序列，所述反义链包含如SEQ ID NO:143所示的核苷酸序列；2) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:31, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:143;
    3)所述正义链包含如SEQ ID NO:32所示的核苷酸序列，所述反义链包含如SEQ ID NO:144所示的核苷酸序列；3) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:32, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:144;
    4)所述正义链包含如SEQ ID NO:33所示的核苷酸序列，所述反义链包含如SEQ ID NO:145所示的核苷酸序列； 4) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:33, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:145;
    5)所述正义链包含如SEQ ID NO:34所示的核苷酸序列，所述反义链包含如SEQ ID NO:146所示的核苷酸序列；5) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:34, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:146;
    6)所述正义链包含如SEQ ID NO:35所示的核苷酸序列，所述反义链包含如SEQ ID NO:147所示的核苷酸序列；6) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:35, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:147;
    7)所述正义链包含如SEQ ID NO:36所示的核苷酸序列，所述反义链包含如SEQ ID NO:148所示的核苷酸序列；7) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:36, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:148;
    8)所述正义链包含如SEQ ID NO:38所示的核苷酸序列，所述反义链包含如SEQ ID NO:150所示的核苷酸序列；8) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:38, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:150;
    9)所述正义链包含如SEQ ID NO:39所示的核苷酸序列，所述反义链包含如SEQ ID NO:151所示的核苷酸序列；9) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:39, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:151;
    10)所述正义链包含如SEQ ID NO:40所示的核苷酸序列，所述反义链包含如SEQ ID NO:152所示的核苷酸序列；10) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:40, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:152;
    11)所述正义链包含如SEQ ID NO:41所示的核苷酸序列，所述反义链包含如SEQ ID NO:153所示的核苷酸序列；11) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:41, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:153;
    12)所述正义链包含如SEQ ID NO:42所示的核苷酸序列，所述反义链包含如SEQ ID NO:154所示的核苷酸序列；12) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:42, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:154;
    13)所述正义链包含如SEQ ID NO:43所示的核苷酸序列，所述反义链包含如SEQ ID NO:155所示的核苷酸序列；13) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:43, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:155;
    14)所述正义链包含如SEQ ID NO:44所示的核苷酸序列，所述反义链包含如SEQ ID NO:156所示的核苷酸序列；14) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:44, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:156;
    15)所述正义链包含如SEQ ID NO:45所示的核苷酸序列，所述反义链包含如SEQ ID NO:157所示的核苷酸序列；15) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:45, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:157;
    16)所述正义链包含如SEQ ID NO:46所示的核苷酸序列，所述反义链包含如SEQ ID NO:158所示的核苷酸序列；16) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:46, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:158;
    17)所述正义链包含如SEQ ID NO:48所示的核苷酸序列，所述反义链包含如SEQ ID NO:160所示的核苷酸序列；17) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:48, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:160;
    18)所述正义链包含如SEQ ID NO:49所示的核苷酸序列，所述反义链包含如SEQ ID NO:161所示的核苷酸序列；18) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:49, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:161;
    19)所述正义链包含如SEQ ID NO:50所示的核苷酸序列，所述反义链包含如SEQ ID NO:162所示的核苷酸序列；19) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:50, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:162;
    20)所述正义链包含如SEQ ID NO:52所示的核苷酸序列，所述反义链包含如SEQ ID NO:164所示的核苷酸序列；20) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:52, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:164;
    21)所述正义链包含如SEQ ID NO:53所示的核苷酸序列，所述反义链包含如SEQ ID NO:165所示的核苷酸序列；21) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:53, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:165;
    22)所述正义链包含如SEQ ID NO:54所示的核苷酸序列，所述反义链包含如SEQ ID NO:166所示的核苷酸序列；22) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:54, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:166;
    23)所述正义链包含如SEQ ID NO:55所示的核苷酸序列，所述反义链包含如SEQ ID NO:167所示的核苷酸序列；23) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:55, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:167;
    24)所述正义链包含如SEQ ID NO:58所示的核苷酸序列，所述反义链包含如SEQ ID NO:170所示的核苷酸序列；24) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:58, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:170;
    25)所述正义链包含如SEQ ID NO:59所示的核苷酸序列，所述反义链包含如SEQ ID NO:171所示的核苷酸序列；25) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:59, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:171;
    26)所述正义链包含如SEQ ID NO:61所示的核苷酸序列，所述反义链包含如SEQ ID NO:173所示的核苷酸序列；26) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:61, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:173;
    27)所述正义链包含如SEQ ID NO:62所示的核苷酸序列，所述反义链包含如SEQ ID  NO:174所示的核苷酸序列；27) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 62, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:174;
    28)所述正义链包含如SEQ ID NO:63所示的核苷酸序列，所述反义链包含如SEQ ID NO:175所示的核苷酸序列；28) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:63, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:175;
    29)所述正义链包含如SEQ ID NO:64所示的核苷酸序列，所述反义链包含如SEQ ID NO:176所示的核苷酸序列；29) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:64, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:176;
    30)所述正义链包含如SEQ ID NO:65所示的核苷酸序列，所述反义链包含如SEQ ID NO:177所示的核苷酸序列；30) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:65, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:177;
    31)所述正义链包含如SEQ ID NO:66所示的核苷酸序列，所述反义链包含如SEQ ID NO:178所示的核苷酸序列；31) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:66, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:178;
    32)所述正义链包含如SEQ ID NO:67所示的核苷酸序列，所述反义链包含如SEQ ID NO:179所示的核苷酸序列；32) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:67, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:179;
    33)所述正义链包含如SEQ ID NO:68所示的核苷酸序列，所述反义链包含如SEQ ID NO:180所示的核苷酸序列；33) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:68, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:180;
    34)所述正义链包含如SEQ ID NO:69所示的核苷酸序列，所述反义链包含如SEQ ID NO:181所示的核苷酸序列；34) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:69, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:181;
    35)所述正义链包含如SEQ ID NO:70所示的核苷酸序列，所述反义链包含如SEQ ID NO:182所示的核苷酸序列；35) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:70, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:182;
    36)所述正义链包含如SEQ ID NO:72所示的核苷酸序列，所述反义链包含如SEQ ID NO:184所示的核苷酸序列；36) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:72, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:184;
    37)所述正义链包含如SEQ ID NO:73所示的核苷酸序列，所述反义链包含如SEQ ID NO:185所示的核苷酸序列；37) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:73, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:185;
    38)所述正义链包含如SEQ ID NO:74所示的核苷酸序列，所述反义链包含如SEQ ID NO:186所示的核苷酸序列；38) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:74, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:186;
    39)所述正义链包含如SEQ ID NO:75所示的核苷酸序列，所述反义链包含如SEQ ID NO:187所示的核苷酸序列；39) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:75, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:187;
    40)所述正义链包含如SEQ ID NO:76所示的核苷酸序列，所述反义链包含如SEQ ID NO:188所示的核苷酸序列；40) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:76, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:188;
    41)所述正义链包含如SEQ ID NO:77所示的核苷酸序列，所述反义链包含如SEQ ID NO:189所示的核苷酸序列；41) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:77, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:189;
    42)所述正义链包含如SEQ ID NO:78所示的核苷酸序列，所述反义链包含如SEQ ID NO:190所示的核苷酸序列；42) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:78, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:190;
    43)所述正义链包含如SEQ ID NO:81所示的核苷酸序列，所述反义链包含如SEQ ID NO:193所示的核苷酸序列；43) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:81, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:193;
    44)所述正义链包含如SEQ ID NO:85所示的核苷酸序列，所述反义链包含如SEQ ID NO:197所示的核苷酸序列；44) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:85, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:197;
    45)所述正义链包含如SEQ ID NO:87所示的核苷酸序列，所述反义链包含如SEQ ID NO:199所示的核苷酸序列；45) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:87, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:199;
    46)所述正义链包含如SEQ ID NO:88所示的核苷酸序列，所述反义链包含如SEQ ID NO:200所示的核苷酸序列；46) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:88, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:200;
    47)所述正义链包含如SEQ ID NO:89所示的核苷酸序列，所述反义链包含如SEQ ID NO:201所示的核苷酸序列；47) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:89, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:201;
    48)所述正义链包含如SEQ ID NO:90所示的核苷酸序列，所述反义链包含如SEQ ID NO:202所示的核苷酸序列；48) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:90, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:202;
    49)所述正义链包含如SEQ ID NO:92所示的核苷酸序列，所述反义链包含如SEQ ID NO:204所示的核苷酸序列； 49) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:92, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:204;
    50)所述正义链包含如SEQ ID NO:94所示的核苷酸序列，所述反义链包含如SEQ ID NO:206所示的核苷酸序列；50) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:94, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:206;
    51)所述正义链包含如SEQ ID NO:97所示的核苷酸序列，所述反义链包含如SEQ ID NO:209所示的核苷酸序列；51) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:97, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:209;
    52)所述正义链包含如SEQ ID NO:99所示的核苷酸序列，所述反义链包含如SEQ ID NO:211所示的核苷酸序列；52) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:99, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:211;
    53)所述正义链包含如SEQ ID NO:101所示的核苷酸序列，所述反义链包含如SEQ ID NO:213所示的核苷酸序列；53) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 101, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 213;
    54)所述正义链包含如SEQ ID NO:102所示的核苷酸序列，所述反义链包含如SEQ ID NO:214所示的核苷酸序列；54) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 102, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 214;
    55)所述正义链包含如SEQ ID NO:103所示的核苷酸序列，所述反义链包含如SEQ ID NO:215所示的核苷酸序列；55) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 103, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 215;
    56)所述正义链包含如SEQ ID NO:104所示的核苷酸序列，所述反义链包含如SEQ ID NO:216所示的核苷酸序列；56) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 104, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 216;
    57)所述正义链包含如SEQ ID NO:105所示的核苷酸序列，所述反义链包含如SEQ ID NO:217所示的核苷酸序列；57) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 105, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 217;
    58)所述正义链包含如SEQ ID NO:106所示的核苷酸序列，所述反义链包含如SEQ ID NO:218所示的核苷酸序列；58) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 106, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 218;
    59)所述正义链包含如SEQ ID NO:108所示的核苷酸序列，所述反义链包含如SEQ ID NO:220所示的核苷酸序列；59) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 108, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 220;
    60)所述正义链包含如SEQ ID NO:109所示的核苷酸序列，所述反义链包含如SEQ ID NO:221所示的核苷酸序列；60) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 109, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 221;
    61)所述正义链包含如SEQ ID NO:110所示的核苷酸序列，所述反义链包含如SEQ ID NO:222所示的核苷酸序列；61) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 110, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 222;
    62)所述正义链包含如SEQ ID NO:111所示的核苷酸序列，所述反义链包含如SEQ ID NO:223所示的核苷酸序列；62) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 111, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 223;
    63)所述正义链包含如SEQ ID NO:112所示的核苷酸序列，所述反义链包含如SEQ ID NO:224所示的核苷酸序列；63) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 112, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 224;
    64)所述正义链包含如SEQ ID NO:118所示的核苷酸序列，所述反义链包含如SEQ ID NO:230所示的核苷酸序列；64) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 118, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 230;
    65)所述正义链包含如SEQ ID NO:119所示的核苷酸序列，所述反义链包含如SEQ ID NO:231所示的核苷酸序列；65) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 119, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 231;
    66)所述正义链包含如SEQ ID NO:120所示的核苷酸序列，所述反义链包含如SEQ ID NO:232所示的核苷酸序列；66) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 120, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 232;
    67)所述正义链包含如SEQ ID NO:121所示的核苷酸序列，所述反义链包含如SEQ ID NO:233所示的核苷酸序列；67) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 121, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 233;
    68)所述正义链包含如SEQ ID NO:122所示的核苷酸序列，所述反义链包含如SEQ ID NO:234所示的核苷酸序列；68) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 122, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 234;
    69)所述正义链包含如SEQ ID NO:125所示的核苷酸序列，所述反义链包含如SEQ ID NO:237所示的核苷酸序列；69) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 125, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 237;
    70)所述正义链包含如SEQ ID NO:129所示的核苷酸序列，所述反义链包含如SEQ ID NO:241所示的核苷酸序列；70) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 129, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 241;
    71)所述正义链包含如SEQ ID NO:130所示的核苷酸序列，所述反义链包含如SEQ ID NO:242所示的核苷酸序列；71) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 130, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 242;
    72)所述正义链包含如SEQ ID NO:131所示的核苷酸序列，所述反义链包含如SEQ ID  NO:243所示的核苷酸序列；72) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 131, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:243;
    73)所述正义链包含如SEQ ID NO:134所示的核苷酸序列，所述反义链包含如SEQ ID NO:246所示的核苷酸序列；73) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 134, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 246;
    74)所述正义链包含如SEQ ID NO:135所示的核苷酸序列，所述反义链包含如SEQ ID NO:247所示的核苷酸序列；74) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 135, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 247;
    75)所述正义链包含如SEQ ID NO:136所示的核苷酸序列，所述反义链包含如SEQ ID NO:248所示的核苷酸序列；75) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 136, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 248;
    76)所述正义链包含如SEQ ID NO:137所示的核苷酸序列，所述反义链包含如SEQ ID NO:249所示的核苷酸序列；76) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 137, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 249;
    77)所述正义链包含如SEQ ID NO:139所示的核苷酸序列，所述反义链包含如SEQ ID NO:251所示的核苷酸序列；77) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 139, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 251;
    78)所述正义链包含如SEQ ID NO:140所示的核苷酸序列，所述反义链包含如SEQ ID NO:252所示的核苷酸序列；78) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 140, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 252;
    79)所述正义链包含如SEQ ID NO:254所示的核苷酸序列，所述反义链包含如SEQ ID NO:261所示的核苷酸序列；79) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 254, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 261;
    80)所述正义链包含如SEQ ID NO:255所示的核苷酸序列，所述反义链包含如SEQ ID NO:262所示的核苷酸序列；80) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 255, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 262;
    292)所述正义链包含如SEQ ID NO:432所示的核苷酸序列，所述反义链包含如SEQ ID NO:500所示的核苷酸序列；292) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:432, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:500;
    293)所述正义链包含如SEQ ID NO:434所示的核苷酸序列，所述反义链包含如SEQ ID NO:502所示的核苷酸序列；293) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:434, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:502;
    294)所述正义链包含如SEQ ID NO:435所示的核苷酸序列，所述反义链包含如SEQ ID NO:503所示的核苷酸序列；294) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:435, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:503;
    295)所述正义链包含如SEQ ID NO:438所示的核苷酸序列，所述反义链包含如SEQ ID NO:506所示的核苷酸序列；295) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:438, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:506;
    296)所述正义链包含如SEQ ID NO:440所示的核苷酸序列，所述反义链包含如SEQ ID NO:508所示的核苷酸序列；296) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:440, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:508;
    297)所述正义链包含如SEQ ID NO:442所示的核苷酸序列，所述反义链包含如SEQ ID NO:510所示的核苷酸序列；297) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:442, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:510;
    298)所述正义链包含如SEQ ID NO:444所示的核苷酸序列，所述反义链包含如SEQ ID NO:512所示的核苷酸序列；298) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:444, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:512;
    299)所述正义链包含如SEQ ID NO:446所示的核苷酸序列，所述反义链包含如SEQ ID NO:514所示的核苷酸序列；299) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:446, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:514;
    300)所述正义链包含如SEQ ID NO:449所示的核苷酸序列，所述反义链包含如SEQ ID NO:517所示的核苷酸序列；300) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:449, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:517;
    301)所述正义链包含如SEQ ID NO:450所示的核苷酸序列，所述反义链包含如SEQ ID NO:518所示的核苷酸序列；301) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:450, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:518;
    302)所述正义链包含如SEQ ID NO:451所示的核苷酸序列，所述反义链包含如SEQ ID NO:519所示的核苷酸序列；302) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:451, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:519;
    303)所述正义链包含如SEQ ID NO:453所示的核苷酸序列，所述反义链包含如SEQ ID NO:521所示的核苷酸序列；303) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:453, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:521;
    304)所述正义链包含如SEQ ID NO:455所示的核苷酸序列，所述反义链包含如SEQ ID NO:523所示的核苷酸序列；304) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:455, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:523;
    305)所述正义链包含如SEQ ID NO:458所示的核苷酸序列，所述反义链包含如SEQ ID NO:526所示的核苷酸序列； 305) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:458, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:526;
    306)所述正义链包含如SEQ ID NO:459所示的核苷酸序列，所述反义链包含如SEQ ID NO:527所示的核苷酸序列；306) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:459, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:527;
    307)所述正义链包含如SEQ ID NO:461所示的核苷酸序列，所述反义链包含如SEQ ID NO:529所示的核苷酸序列；307) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:461, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:529;
    308)所述正义链包含如SEQ ID NO:467所示的核苷酸序列，所述反义链包含如SEQ ID NO:535所示的核苷酸序列；308) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:467, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:535;
    309)所述正义链包含如SEQ ID NO:470所示的核苷酸序列，所述反义链包含如SEQ ID NO:538所示的核苷酸序列；309) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:470, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:538;
    310)所述正义链包含如SEQ ID NO:478所示的核苷酸序列，所述反义链包含如SEQ ID NO:546所示的核苷酸序列；310) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:478, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:546;
    311)所述正义链包含如SEQ ID NO:479所示的核苷酸序列，所述反义链包含如SEQ ID NO:547所示的核苷酸序列；311) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:479, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:547;
    312)所述正义链包含如SEQ ID NO:480所示的核苷酸序列，所述反义链包含如SEQ ID NO:548所示的核苷酸序列；312) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:480, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:548;
    313)所述正义链包含如SEQ ID NO:481所示的核苷酸序列，所述反义链包含如SEQ ID NO:549所示的核苷酸序列；313) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:481, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:549;
    314)所述正义链包含如SEQ ID NO:482所示的核苷酸序列，所述反义链包含如SEQ ID NO:550所示的核苷酸序列；314) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:482, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:550;
    315)所述正义链包含如SEQ ID NO:483所示的核苷酸序列，所述反义链包含如SEQ ID NO:551所示的核苷酸序列；315) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:483, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:551;
    316)所述正义链包含如SEQ ID NO:485所示的核苷酸序列，所述反义链包含如SEQ ID NO:553所示的核苷酸序列；316) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:485, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:553;
    317)所述正义链包含如SEQ ID NO:494所示的核苷酸序列，所述反义链包含如SEQ ID NO:562所示的核苷酸序列；317) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:494, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:562;
    318)所述正义链包含如SEQ ID NO:499所示的核苷酸序列，所述反义链包含如SEQ ID NO:567所示的核苷酸序列；318) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:499, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:567;
    优选地，所述正义链与所述反义链选自如下：Preferably, the sense strand and the antisense strand are selected from the following:
    307)所述正义链包含如SEQ ID NO:461所示的核苷酸序列，所述反义链包含如SEQ ID NO:529所示的核苷酸序列。307) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:461, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:529.
  10. 根据权利要求1-8任一项所述的双链核糖核酸，其中，双链区的长度为19个或21个核苷酸，The double-stranded ribonucleic acid according to any one of claims 1 to 8, wherein the length of the double-stranded region is 19 or 21 nucleotides,
    所述反义链的核苷酸序列与靶标序列存在不超过3个的错配碱基，优选的，错配碱基为1个、2个或3个；The nucleotide sequence of the antisense strand has no more than 3 mismatched bases with the target sequence, preferably, the number of mismatched bases is 1, 2 or 3;
    所述正义链与靶标序列存在的差异不超过3个核苷酸，优选的，核苷酸个数为1个、2个或3个；The difference between the sense strand and the target sequence does not exceed 3 nucleotides, preferably, the number of nucleotides is 1, 2 or 3;
    所述正义链中包括与反义链互补的区域，正义链的核苷酸序列与反义链在靶标序列结合区域的序列完全相同；The sense strand includes a region complementary to the antisense strand, and the nucleotide sequence of the sense strand is completely identical to the sequence of the antisense strand in the target sequence binding region;
    任选地，所述正义链的3’末端为腺嘌呤核苷酸，是由靶标序列的鸟嘌呤核苷酸或胞嘧啶核苷酸或尿嘧啶核苷酸替换得到的腺嘌呤核苷酸；Optionally, the 3' end of the sense strand is an adenine nucleotide, which is an adenine nucleotide obtained by replacing a guanine nucleotide, a cytosine nucleotide, or a uracil nucleotide of the target sequence;
    任选地，所述正义链的3’末端倒数第三个核苷酸为腺嘌呤核苷酸，是由靶标序列的鸟嘌呤核苷酸或胞嘧啶核苷酸替换得到的腺嘌呤核苷酸；Optionally, the third to last nucleotide at the 3' end of the sense strand is an adenine nucleotide, which is an adenine nucleotide obtained by replacing a guanine nucleotide or a cytosine nucleotide of the target sequence;
    任选地，所述正义链的5’末端为胞嘧啶核苷酸，是由靶标序列的腺嘌呤核苷酸替换得到的胞嘧啶核苷酸；Optionally, the 5' end of the sense strand is a cytosine nucleotide, which is a cytosine nucleotide obtained by replacing the adenine nucleotide of the target sequence;
    任选地，所述正义链的3’末端倒数第三个核苷酸为尿嘧啶核苷酸，是由靶标序列的腺嘌呤核苷酸替换得到的尿嘧啶核苷酸； Optionally, the third to last nucleotide at the 3' end of the sense strand is a uracil nucleotide, which is obtained by replacing the adenine nucleotide of the target sequence;
    任选地，所述反义链的5’末端为腺嘌呤核苷酸，是由靶标序列的尿嘧啶核苷酸替换得到的腺嘌呤核苷酸，所述反义链的3’末端为鸟嘌呤核苷酸，是由靶标序列的胞嘧啶核苷酸替换得到的鸟嘌呤核苷酸；Optionally, the 5' end of the antisense strand is an adenine nucleotide, which is obtained by replacing the uracil nucleotide of the target sequence, and the 3' end of the antisense strand is a guanine nucleotide, which is obtained by replacing the cytosine nucleotide of the target sequence;
    所述正义链与所述反义链选自如下组合：The sense strand and the antisense strand are selected from the following combinations:
    81)所述正义链包含如SEQ ID NO:37所示的核苷酸序列，所述反义链包含如SEQ ID NO:149所示的核苷酸序列；81) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:37, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:149;
    82)所述正义链包含如SEQ ID NO:47所示的核苷酸序列，所述反义链包含如SEQ ID NO:159所示的核苷酸序列；82) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:47, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:159;
    83)所述正义链包含如SEQ ID NO:51所示的核苷酸序列，所述反义链包含如SEQ ID NO:163所示的核苷酸序列；83) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:51, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:163;
    84)所述正义链包含如SEQ ID NO:56所示的核苷酸序列，所述反义链包含如SEQ ID NO:168所示的核苷酸序列；84) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:56, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:168;
    85)所述正义链包含如SEQ ID NO:57所示的核苷酸序列，所述反义链包含如SEQ ID NO:169所示的核苷酸序列；85) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:57, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:169;
    86)所述正义链包含如SEQ ID NO:60所示的核苷酸序列，所述反义链包含如SEQ ID NO:172所示的核苷酸序列；86) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:60, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:172;
    87)所述正义链包含如SEQ ID NO:71所示的核苷酸序列，所述反义链包含如SEQ ID NO:183所示的核苷酸序列；87) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:71, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:183;
    88)所述正义链包含如SEQ ID NO:79所示的核苷酸序列，所述反义链包含如SEQ ID NO:191所示的核苷酸序列；88) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:79, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:191;
    89)所述正义链包含如SEQ ID NO:80所示的核苷酸序列，所述反义链包含如SEQ ID NO:192所示的核苷酸序列；89) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:80, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:192;
    90)所述正义链包含如SEQ ID NO:82所示的核苷酸序列，所述反义链包含如SEQ ID NO:194所示的核苷酸序列；90) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:82, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:194;
    91)所述正义链包含如SEQ ID NO:83所示的核苷酸序列，所述反义链包含如SEQ ID NO:195所示的核苷酸序列；91) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:83, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:195;
    92)所述正义链包含如SEQ ID NO:84所示的核苷酸序列，所述反义链包含如SEQ ID NO:196所示的核苷酸序列；92) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:84, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:196;
    93)所述正义链包含如SEQ ID NO:86所示的核苷酸序列，所述反义链包含如SEQ ID NO:198所示的核苷酸序列；93) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:86, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:198;
    94)所述正义链包含如SEQ ID NO:91所示的核苷酸序列，所述反义链包含如SEQ ID NO:203所示的核苷酸序列；94) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:91, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:203;
    95)所述正义链包含如SEQ ID NO:93所示的核苷酸序列，所述反义链包含如SEQ ID NO:205所示的核苷酸序列；95) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:93, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:205;
    96)所述正义链包含如SEQ ID NO:95所示的核苷酸序列，所述反义链包含如SEQ ID NO:207所示的核苷酸序列；96) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:95, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:207;
    97)所述正义链包含如SEQ ID NO:96所示的核苷酸序列，所述反义链包含如SEQ ID NO:208所示的核苷酸序列；97) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:96, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:208;
    98)所述正义链包含如SEQ ID NO:98所示的核苷酸序列，所述反义链包含如SEQ ID NO:210所示的核苷酸序列；98) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:98, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:210;
    99)所述正义链包含如SEQ ID NO:100所示的核苷酸序列，所述反义链包含如SEQ ID NO:212所示的核苷酸序列；99) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 100, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 212;
    100)所述正义链包含如SEQ ID NO:107所示的核苷酸序列，所述反义链包含如SEQ ID NO:219所示的核苷酸序列；100) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 107, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 219;
    101)所述正义链包含如SEQ ID NO:113所示的核苷酸序列，所述反义链包含如SEQ ID  NO:225所示的核苷酸序列；101) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 113, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:225;
    102)所述正义链包含如SEQ ID NO:114所示的核苷酸序列，所述反义链包含如SEQ ID NO:226所示的核苷酸序列；102) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 114, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 226;
    103)所述正义链包含如SEQ ID NO:115所示的核苷酸序列，所述反义链包含如SEQ ID NO:227所示的核苷酸序列；103) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 115, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 227;
    104)所述正义链包含如SEQ ID NO:116所示的核苷酸序列，所述反义链包含如SEQ ID NO:228所示的核苷酸序列；104) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 116, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 228;
    105)所述正义链包含如SEQ ID NO:117所示的核苷酸序列，所述反义链包含如SEQ ID NO:229所示的核苷酸序列；105) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 117, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 229;
    106)所述正义链包含如SEQ ID NO:123所示的核苷酸序列，所述反义链包含如SEQ ID NO:235所示的核苷酸序列；106) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 123, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 235;
    107)所述正义链包含如SEQ ID NO:124所示的核苷酸序列，所述反义链包含如SEQ ID NO:236所示的核苷酸序列；107) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 124, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 236;
    108)所述正义链包含如SEQ ID NO:126所示的核苷酸序列，所述反义链包含如SEQ ID NO:238所示的核苷酸序列；108) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 126, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 238;
    109)所述正义链包含如SEQ ID NO:127所示的核苷酸序列，所述反义链包含如SEQ ID NO:239所示的核苷酸序列；109) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 127, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 239;
    110)所述正义链包含如SEQ ID NO:128所示的核苷酸序列，所述反义链包含如SEQ ID NO:240所示的核苷酸序列；110) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 128, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 240;
    111)所述正义链包含如SEQ ID NO:132所示的核苷酸序列，所述反义链包含如SEQ ID NO:244所示的核苷酸序列；111) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 132, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 244;
    112)所述正义链包含如SEQ ID NO:133所示的核苷酸序列，所述反义链包含如SEQ ID NO:245所示的核苷酸序列；112) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 133, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 245;
    113)所述正义链包含如SEQ ID NO:138所示的核苷酸序列，所述反义链包含如SEQ ID NO:250所示的核苷酸序列；113) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 138, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 250;
    114)所述正义链包含如SEQ ID NO:141所示的核苷酸序列，所述反义链包含如SEQ ID NO:253所示的核苷酸序列；114) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 141, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 253;
    319)所述正义链包含如SEQ ID NO:443所示的核苷酸序列，所述反义链包含如SEQ ID NO:511所示的核苷酸序列；319) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:443, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:511;
    320)所述正义链包含如SEQ ID NO:445所示的核苷酸序列，所述反义链包含如SEQ ID NO:513所示的核苷酸序列；320) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:445, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:513;
    321)所述正义链包含如SEQ ID NO:447所示的核苷酸序列，所述反义链包含如SEQ ID NO:515所示的核苷酸序列；321) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:447, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:515;
    322)所述正义链包含如SEQ ID NO:448所示的核苷酸序列，所述反义链包含如SEQ ID NO:516所示的核苷酸序列；322) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:448, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:516;
    323)所述正义链包含如SEQ ID NO:452所示的核苷酸序列，所述反义链包含如SEQ ID NO:520所示的核苷酸序列；323) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:452, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:520;
    324)所述正义链包含如SEQ ID NO:454所示的核苷酸序列，所述反义链包含如SEQ ID NO:522所示的核苷酸序列；324) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:454, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:522;
    325)所述正义链包含如SEQ ID NO:456所示的核苷酸序列，所述反义链包含如SEQ ID NO:524所示的核苷酸序列；325) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:456, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:524;
    326)所述正义链包含如SEQ ID NO:457所示的核苷酸序列，所述反义链包含如SEQ ID NO:525所示的核苷酸序列；326) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:457, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:525;
    327)所述正义链包含如SEQ ID NO:460所示的核苷酸序列，所述反义链包含如SEQ ID NO:528所示的核苷酸序列； 327) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 460, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 528;
    328)所述正义链包含如SEQ ID NO:462所示的核苷酸序列，所述反义链包含如SEQ ID NO:530所示的核苷酸序列；328) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:462, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:530;
    329)所述正义链包含如SEQ ID NO:433所示的核苷酸序列，所述反义链包含如SEQ ID NO:501所示的核苷酸序列；329) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:433, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:501;
    330)所述正义链包含如SEQ ID NO:436所示的核苷酸序列，所述反义链包含如SEQ ID NO:504所示的核苷酸序列；330) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:436, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:504;
    331)所述正义链包含如SEQ ID NO:437所示的核苷酸序列，所述反义链包含如SEQ ID NO:505所示的核苷酸序列；331) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:437, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:505;
    332)所述正义链包含如SEQ ID NO:439所示的核苷酸序列，所述反义链包含如SEQ ID NO:507所示的核苷酸序列；332) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:439, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:507;
    333)所述正义链包含如SEQ ID NO:441所示的核苷酸序列，所述反义链包含如SEQ ID NO:509所示的核苷酸序列；333) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:441, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:509;
    334)所述正义链包含如SEQ ID NO:463所示的核苷酸序列，所述反义链包含如SEQ ID NO:531所示的核苷酸序列；334) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:463, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:531;
    335)所述正义链包含如SEQ ID NO:464所示的核苷酸序列，所述反义链包含如SEQ ID NO:532所示的核苷酸序列；335) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:464, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:532;
    336)所述正义链包含如SEQ ID NO:466所示的核苷酸序列，所述反义链包含如SEQ ID NO:534所示的核苷酸序列；336) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:466, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:534;
    337)所述正义链包含如SEQ ID NO:468所示的核苷酸序列，所述反义链包含如SEQ ID NO:536所示的核苷酸序列；337) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:468, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:536;
    338)所述正义链包含如SEQ ID NO:469所示的核苷酸序列，所述反义链包含如SEQ ID NO:537所示的核苷酸序列；338) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:469, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:537;
    339)所述正义链包含如SEQ ID NO:472所示的核苷酸序列，所述反义链包含如SEQ ID NO:540所示的核苷酸序列；339) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:472, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:540;
    340)所述正义链包含如SEQ ID NO:473所示的核苷酸序列，所述反义链包含如SEQ ID NO:541所示的核苷酸序列；340) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:473, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:541;
    341)所述正义链包含如SEQ ID NO:475所示的核苷酸序列，所述反义链包含如SEQ ID NO:543所示的核苷酸序列；341) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:475, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:543;
    342)所述正义链包含如SEQ ID NO:477所示的核苷酸序列，所述反义链包含如SEQ ID NO:545所示的核苷酸序列；342) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:477, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:545;
    343)所述正义链包含如SEQ ID NO:484所示的核苷酸序列，所述反义链包含如SEQ ID NO:552所示的核苷酸序列；343) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:484, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:552;
    344)所述正义链包含如SEQ ID NO:486所示的核苷酸序列，所述反义链包含如SEQ ID NO:554所示的核苷酸序列；344) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:486, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:554;
    345)所述正义链包含如SEQ ID NO:487所示的核苷酸序列，所述反义链包含如SEQ ID NO:555所示的核苷酸序列；345) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:487, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:555;
    346)所述正义链包含如SEQ ID NO:491所示的核苷酸序列，所述反义链包含如SEQ ID NO:559所示的核苷酸序列；346) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:491, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:559;
    347)所述正义链包含如SEQ ID NO:493所示的核苷酸序列，所述反义链包含如SEQ ID NO:561所示的核苷酸序列；347) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:493, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:561;
    348)所述正义链包含如SEQ ID NO:496所示的核苷酸序列，所述反义链包含如SEQ ID NO:564所示的核苷酸序列；348) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:496, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:564;
    349)所述正义链包含如SEQ ID NO:497所示的核苷酸序列，所述反义链包含如SEQ ID NO:565所示的核苷酸序列；349) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:497, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:565;
    350)所述正义链包含如SEQ ID NO:498所示的核苷酸序列，所述反义链包含如SEQ ID  NO:566所示的核苷酸序列；350) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 498, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:566;
    351)所述正义链包含如SEQ ID NO:465所示的核苷酸序列，所述反义链包含如SEQ ID NO:533所示的核苷酸序列；351) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:465, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:533;
    352)所述正义链包含如SEQ ID NO:471所示的核苷酸序列，所述反义链包含如SEQ ID NO:539所示的核苷酸序列；352) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:471, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:539;
    353)所述正义链包含如SEQ ID NO:474所示的核苷酸序列，所述反义链包含如SEQ ID NO:542所示的核苷酸序列；353) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:474, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:542;
    354)所述正义链包含如SEQ ID NO:476所示的核苷酸序列，所述反义链包含如SEQ ID NO:544所示的核苷酸序列；354) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:476, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:544;
    355)所述正义链包含如SEQ ID NO:488所示的核苷酸序列，所述反义链包含如SEQ ID NO:556所示的核苷酸序列；355) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:488, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:556;
    356)所述正义链包含如SEQ ID NO:489所示的核苷酸序列，所述反义链包含如SEQ ID NO:557所示的核苷酸序列；356) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:489, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:557;
    357)所述正义链包含如SEQ ID NO:490所示的核苷酸序列，所述反义链包含如SEQ ID NO:558所示的核苷酸序列；357) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:490, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:558;
    358)所述正义链包含如SEQ ID NO:492所示的核苷酸序列，所述反义链包含如SEQ ID NO:560所示的核苷酸序列；358) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:492, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:560;
    359)所述正义链包含如SEQ ID NO:495所示的核苷酸序列，所述反义链包含如SEQ ID NO:563所示的核苷酸序列；359) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:495, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:563;
    437)所述正义链包含如SEQ ID NO:667所示的核苷酸序列，所述反义链包含如SEQ ID NO:668所示的核苷酸序列；437) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 667, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 668;
    438)所述正义链包含如SEQ ID NO:667所示的核苷酸序列，所述反义链包含如SEQ ID NO:669所示的核苷酸序列；438) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 667, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 669;
    优选地，所述正义链与所述反义链选自如下组合：Preferably, the sense strand and the antisense strand are selected from the following combinations:
    90)所述正义链包含如SEQ ID NO:82所示的核苷酸序列，所述反义链包含如SEQ ID NO:194所示的核苷酸序列。90) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:82, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:194.
  11. 根据权利要求1-10任一项所述的双链核糖核酸，其中，所述正义链中每个核苷酸彼此独立地为修饰的核苷酸或未修饰的核苷酸，和/或，所述反义链中每个核苷酸彼此独立地为修饰的核苷酸或未修饰的核苷酸。The double-stranded ribonucleic acid according to any one of claims 1 to 10, wherein each nucleotide in the sense strand is independently a modified nucleotide or an unmodified nucleotide, and/or each nucleotide in the antisense strand is independently a modified nucleotide or an unmodified nucleotide.
  12. 根据权利要求1-11任一项所述的双链核糖核酸，其中，所述正义链中任意相连的两个核苷酸由磷酸二酯键或硫代磷酸二酯键连接，和/或，所述反义链中任意相连的两个核苷酸由磷酸二酯键或硫代磷酸二酯键连接。The double-stranded ribonucleic acid according to any one of claims 1 to 11, wherein any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond, and/or any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.
  13. 根据权利要求1-12任一项所述的双链核糖核酸，其中，所述正义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团，和/或，所述反义链的5’末端核苷酸连接5’磷酸基团或5’磷酸衍生基团。The double-stranded ribonucleic acid according to any one of claims 1 to 12, wherein the 5' terminal nucleotide of the sense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.
  14. 根据权利要求1-13任一项所述的双链核糖核酸，其中，所述双链核糖核酸为siRNA。The double-stranded ribonucleic acid according to any one of claims 1 to 13, wherein the double-stranded ribonucleic acid is siRNA.
  15. 根据权利要求1-14任一项所述的双链核糖核酸，其中，所述双链核糖核酸为用于抑制凝血因子XII基因表达的siRNA。The double-stranded ribonucleic acid according to any one of claims 1 to 14, wherein the double-stranded ribonucleic acid is siRNA for inhibiting the expression of coagulation factor XII gene.
  16. 一种双链核糖核酸修饰物，其为如权利要求1-15任一项所述的双链核糖核酸的修饰物，所述双链核糖核酸修饰物包含如下至少一种的化学修饰：A double-stranded RNA modified substance, which is a double-stranded RNA modified substance according to any one of claims 1 to 15, wherein the double-stranded RNA modified substance comprises at least one of the following chemical modifications:
    (1)正义链中至少一个核苷酸的修饰，(1) modification of at least one nucleotide in the sense strand,
    (2)正义链中至少一个位置处的磷酸二酯键的修饰，(2) modification of the phosphodiester bond at at least one position in the sense strand,
    (3)反义链中至少一个核苷酸的修饰，(3) modification of at least one nucleotide in the antisense strand,
    (4)反义链中至少一个位置处的磷酸二酯键的修饰； (4) modification of the phosphodiester bond at at least one position in the antisense strand;
    任选地，所述双链核糖核酸的正义链中序列A的3’末端连接由1-2个核苷酸组成的序列D，优选由1-2个胸腺嘧啶脱氧核糖核苷酸组成的序列D；和/或，所述双链核糖核酸的反义链中序列B的3’末端连接由1-2个核苷酸组成的序列E，优选由1-2个胸腺嘧啶脱氧核糖核苷酸组成的序列E；和/或，所述双链核糖核酸的正义链中序列A的3’末端排除1-2个核苷酸后形成序列A’；Optionally, the 3' end of sequence A in the sense strand of the double-stranded RNA is connected to a sequence D consisting of 1-2 nucleotides, preferably a sequence D consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence B in the antisense strand of the double-stranded RNA is connected to a sequence E consisting of 1-2 nucleotides, preferably a sequence E consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence A in the sense strand of the double-stranded RNA excludes 1-2 nucleotides to form sequence A';
    可选地，所述双链核糖核酸修饰物的正义链和反义链选自如下的序列组合：Optionally, the sense strand and antisense strand of the double-stranded RNA modification are selected from the following sequence combinations:
    所述正义链的核苷酸序列为序列A所示的序列，所述反义链的核苷酸序列为序列B所示的序列；The nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
    或者，所述正义链的核苷酸序列为序列A所示的序列，所述反义链的核苷酸序列为序列B连接序列E所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E;
    或者，所述正义链的核苷酸序列为序列A连接序列D所示的序列，所述反义链的核苷酸序列为序列B所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
    或者，所述正义链的核苷酸序列为序列A连接序列D所示的序列，所述反义链的核苷酸序列为序列B连接序列E所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown by sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown by sequence B connected to sequence E;
    或者，所述正义链的核苷酸序列为序列A’所示的序列，所述反义链的核苷酸序列为序列B所示的序列；Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;
    或者，所述正义链的核苷酸序列为序列A’所示的序列，所述反义链的核苷酸序列为序列B连接序列E所示的序列。Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A’, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.
  17. 根据权利要求16所述的双链核糖核酸修饰物，其中，所述核苷酸的修饰选自2’-氟代修饰、2’-烷氧基修饰、2’-取代的烷氧基修饰、2’-烷基修饰、2’-取代的烷基修饰、2’-脱氧修饰、核苷酸衍生物修饰或其中任意两种以上的组合。The double-stranded RNA modified substance according to claim 16, wherein the modification of the nucleotide is selected from 2'-fluoro modification, 2'-alkoxy modification, 2'-substituted alkoxy modification, 2'-alkyl modification, 2'-substituted alkyl modification, 2'-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.
  18. 根据权利要求16或17所述的双链核糖核酸修饰物，其中，所述核苷酸的修饰选自2’-F修饰、2’-O-CH3修饰、2’-O-CH2-CH2-O-CH3修饰、2’-O-CH2-CH＝CH2修饰、2’-CH2-CH2-CH＝CH2修饰、2’-脱氧修饰、核苷酸衍生物修饰或其中任意两种以上的组合。The double-stranded ribonucleic acid modified substance according to claim 16 or 17, wherein the modification of the nucleotide is selected from 2'-F modification, 2'-O-CH 3 modification, 2'-O-CH 2 -CH 2 -O-CH 3 modification, 2'-O-CH 2 -CH＝CH 2 modification, 2'-CH 2 -CH 2 -CH＝CH 2 modification, 2'-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.
  19. 根据权利要求17或18所述的双链核糖核酸修饰物，其中，所述核苷酸衍生物修饰中的核苷酸衍生物选自异核苷酸、LNA、ENA、cET、UNA或GNA。The double-stranded RNA modification according to claim 17 or 18, wherein the nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.
  20. 根据权利要求16-19任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述正义链中第7位、第9位、第10位和第11位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述正义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸。The double-stranded ribonucleic acid modified substance according to any one of claims 16 to 19, wherein, along the 5' end to the 3' end direction, the 7th, 9th, 10th and 11th ribonucleotides in the sense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the sense strand are 2'-O-CH 3 modified ribonucleotides.
  21. 根据权利要求16-20任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述正义链包含位于如下所示位置处的硫代磷酸二酯键：The double-stranded RNA modification according to any one of claims 16 to 20, wherein, from the 5' end to the 3' end, the sense strand comprises a phosphorothioate diester bond located at the following position:
    所述正义链5’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;
    所述正义链5’末端起始的第2个核苷酸与第3个核苷酸之间；Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand;
    所述正义链3’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand;
    所述正义链3’末端起始的第2个核苷酸与第3个核苷酸之间；Between the second nucleotide and the third nucleotide starting from the 3' end of the sense strand;
    或者，or,
    所述正义链包含位于如下所示位置处的硫代磷酸二酯键：The sense strand contains phosphorothioate diester bonds located at the positions shown below:
    所述正义链5’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;
    所述正义链5’末端起始的第2个核苷酸与第3个核苷酸之间。The positive strand is located between the second and third nucleotides starting from the 5' end.
  22. 根据权利要求16-21任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述反义链中任意奇数位置处的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，所述反义链中任意偶数位置处的核糖核苷酸为2’-F修饰的核糖核苷酸；The double-stranded ribonucleic acid modified substance according to any one of claims 16 to 21, wherein, from the 5' end to the 3' end, the ribonucleotide at any odd position in the antisense strand is a 2'-O-CH 3- modified ribonucleotide, and the ribonucleotide at any even position in the antisense strand is a 2'-F-modified ribonucleotide;
    或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸； Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
    或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第8位、第9位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
    或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中第7位的核糖核苷酸为核苷酸衍生物GNA修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
    或者，沿5’末端向3’末端方向，所述反义链中第2位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中第6位的核糖核苷酸为核苷酸衍生物GNA修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸；Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;
    或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，且所述反义链中第1位的核糖核苷酸为5’-反式乙烯基膦酸酯核苷酸；Alternatively, along the 5' end to the 3' end direction, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide;
    或者，沿5’末端向3’末端方向，所述反义链中第2位、第6位、第8位、第9位、第14位和第16位的核糖核苷酸为2’-F修饰的核糖核苷酸，所述反义链中其余位置的核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，且所述反义链中第1位的核糖核苷酸为5’-反式乙烯基膦酸酯核苷酸。Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides, and the ribonucleotide at position 1 in the antisense strand is a 5'-trans vinyl phosphonate nucleotide.
  23. 根据权利要求16-22任一项所述的双链核糖核酸修饰物，其中，沿5’末端向3’末端方向，所述反义链的5’末端的核苷酸连接5’磷酸基团或5’磷酸衍生基团。The double-stranded RNA modification according to any one of claims 16 to 22, wherein, in the direction from the 5' end to the 3' end, the nucleotide at the 5' end of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.
  24. 根据权利要求16-23任一项所述的双链核糖核酸修饰物，其中，所述反义链包含位于如下所示位置处的硫代磷酸二酯键：The double-stranded RNA modification according to any one of claims 16 to 23, wherein the antisense strand comprises a phosphorothioate diester bond located at the following position:
    所述反义链5’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand;
    所述反义链5’末端起始的第2个核苷酸与第3个核苷酸之间；Between the second nucleotide and the third nucleotide starting from the 5' end of the antisense strand;
    所述反义链3’末端起始的第1个核苷酸与第2个核苷酸之间；Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand;
    所述反义链3’末端起始的第2个核苷酸与第3个核苷酸之间。Between the second and third nucleotides starting from the 3' end of the antisense strand.
  25. 根据权利要求16-24任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物的正义链具有如(a1)-(a5)任一项所示的结构：The double-stranded RNA modified substance according to any one of claims 16 to 24, wherein the sense strand of the double-stranded RNA modified substance has a structure as shown in any one of (a 1 ) to (a 5 ):
    (a1)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-(s)-T-(s)-T-3’，(a 1 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (a2)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(a 2 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (a3)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(a 3 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
    (a4)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-3’，(a 4 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',
    (a5)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N10f-N11f-mN12-mN13-mN14-mN15-mN16-mN17-mN18-mN19-mN20-mN21-3’；(a 5 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -3';
    其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，wherein N 1 -N 23 are independently selected from ribonucleotides whose base is A, U, C or G,
    大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，The capital letter T represents a deoxyribonucleotide whose base is thymine.
    小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.
    小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.
    -(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接。-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
  26. 根据权利要求16-25任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物的反义链具有如(b1)-(b27)任一项所示的结构：The double-stranded RNA modified substance according to any one of claims 16 to 25, wherein the antisense strand of the double-stranded RNA modified substance has a structure as shown in any one of (b 1 ) to (b 27 ):
    (b1)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-T-(s)-T-3’， (b 1 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-T-(s)-T-3',
    (b2)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N1 4f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 2 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 1 4 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (b3)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 3 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (b4)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 4 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
    (b5)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-(s)-N20f-(s)-mN21-3’，(b 5 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-N 20 f-(s)-mN 21 -3',
    (b6)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N1 4f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 6 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 1 4 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b7)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 7 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b8)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 8 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b9)5’-P1mN1-(s)-N2f-(s)-mN3-N4f-mN5-N6f-mN7-N8f-mN9-N10f-mN11-N12f-mN13-N14f-mN15-N16f-mN17-N18f-mN19-N20f-mN21-(s)-N22f-(s)-mN23-3’，(b 9 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -N 20 f-mN 21 -(s)-N 22 f-(s)- mN 23 -3',
    (b10)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 10 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
    (b11)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 11 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
    (b12)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 12 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-(GNA)N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3 ',
    (b13)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 13 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',
    (b14)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 14 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b15)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 15 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -(GNA)N 6 -N 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3' ,
    (b16)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 16 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (b17)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 17 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b18)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 18 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
    (b19)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 19 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (b20)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 20 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b21)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 21 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
    (b22)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 22 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (b23)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 23 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b24)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN10-mN11-mN12-mN13- N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’，(b 24 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 - N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',
    (b25)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-T-(s)-T-3’，(b 25 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，
    (b26)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-(s)-mN20-(s)-mN21-3’，(b 26 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',
    (b27)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN10-mN11-mN12-mN13-N14f-mN15-N16f-mN17-mN18-mN19-mN20-mN21-(s)-mN22-(s)-mN23-3’；(b 27 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';
    其中，N1-N23彼此独立地选自碱基为A、U、C或G的核糖核苷酸，wherein N 1 -N 23 are independently selected from ribonucleotides whose base is A, U, C or G,
    大写字母T表示碱基为胸腺嘧啶的脱氧核糖核苷酸，The capital letter T represents a deoxyribonucleotide whose base is thymine.
    小写字母m表示该字母m右侧相邻的一个核糖核苷酸为2’-O-CH3修饰的核糖核苷酸，The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.
    小写字母f表示该字母f左侧相邻的一个核糖核苷酸为2’-F修饰的核糖核苷酸，The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.
    P1表示该字母右侧相邻的一个核苷酸为5’-磷酸核苷酸，P1 means that the nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide.
    EVP表示该字母其右侧相邻的一个核苷酸为5’-反式乙烯基膦酸酯核苷酸，EVP means that the nucleotide adjacent to the right of this letter is a 5'-trans vinylphosphonate nucleotide.
    -(s)-表示前后相邻的两个核苷酸以硫代磷酸二酯键连接，-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
    (GNA)表示其右侧相邻的一个核糖核苷酸为存在GNA修饰的核糖核苷酸。(GNA) indicates that the adjacent ribonucleotide on the right is a ribonucleotide with GNA modification.
  27. 根据权利要求16-26任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物为siRNA修饰物。The double-stranded RNA modified substance according to any one of claims 16 to 26, wherein the double-stranded RNA modified substance is a siRNA modified substance.
  28. 根据权利要求16-27任一项所述的双链核糖核酸修饰物，其中，所述双链核糖核酸修饰物为用于抑制凝血因子XII基因表达的siRNA修饰物。The double-stranded RNA modified substance according to any one of claims 16 to 27, wherein the double-stranded RNA modified substance is a siRNA modified substance for inhibiting the expression of the coagulation factor XII gene.
  29. 根据权利要求16-28任一项所述的双链核糖核酸修饰物，其中，所述正义链与所述反义链选自如下组合：The double-stranded RNA modification according to any one of claims 16 to 28, wherein the sense strand and the antisense strand are selected from the following combinations:
    115)所述正义链包含如SEQ ID NO:268所示的核苷酸序列，所述反义链包含如SEQ ID NO:323所示的核苷酸序列；115) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 268, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 323;
    116)所述正义链包含如SEQ ID NO:269所示的核苷酸序列，所述反义链包含如SEQ ID NO:324所示的核苷酸序列；116) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 269, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 324;
    117)所述正义链包含如SEQ ID NO:270所示的核苷酸序列，所述反义链包含如SEQ ID NO:324所示的核苷酸序列；117) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 270, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 324;
    118)所述正义链包含如SEQ ID NO:269所示的核苷酸序列，所述反义链包含如SEQ ID NO:325所示的核苷酸序列；118) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 269, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 325;
    119)所述正义链包含如SEQ ID NO:269所示的核苷酸序列，所述反义链包含如SEQ ID NO:326所示的核苷酸序列；119) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 269, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 326;
    120)所述正义链包含如SEQ ID NO:269所示的核苷酸序列，所述反义链包含如SEQ ID NO:327所示的核苷酸序列；120) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 269, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 327;
    121)所述正义链包含如SEQ ID NO:271所示的核苷酸序列，所述反义链包含如SEQ ID NO:328所示的核苷酸序列；121) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 271, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 328;
    122)所述正义链包含如SEQ ID NO:272所示的核苷酸序列，所述反义链包含如SEQ ID NO:329所示的核苷酸序列；122) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 272, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 329;
    123)所述正义链包含如SEQ ID NO:273所示的核苷酸序列，所述反义链包含如SEQ ID NO:330所示的核苷酸序列；123) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 273, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 330;
    124)所述正义链包含如SEQ ID NO:274所示的核苷酸序列，所述反义链包含如SEQ ID NO:330所示的核苷酸序列；124) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 274, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 330;
    125)所述正义链包含如SEQ ID NO:273所示的核苷酸序列，所述反义链包含如SEQ ID NO:331所示的核苷酸序列；125) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 273, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 331;
    126)所述正义链包含如SEQ ID NO:273所示的核苷酸序列，所述反义链包含如SEQ ID NO:332所示的核苷酸序列； 126) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 273, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 332;
    127)所述正义链包含如SEQ ID NO:273所示的核苷酸序列，所述反义链包含如SEQ ID NO:333所示的核苷酸序列；127) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 273, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 333;
    128)所述正义链包含如SEQ ID NO:275所示的核苷酸序列，所述反义链包含如SEQ ID NO:334所示的核苷酸序列；128) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 275, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 334;
    129)所述正义链包含如SEQ ID NO:276所示的核苷酸序列，所述反义链包含如SEQ ID NO:334所示的核苷酸序列；129) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 276, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 334;
    130)所述正义链包含如SEQ ID NO:276所示的核苷酸序列，所述反义链包含如SEQ ID NO:335所示的核苷酸序列；130) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 276, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 335;
    131)所述正义链包含如SEQ ID NO:276所示的核苷酸序列，所述反义链包含如SEQ ID NO:336所示的核苷酸序列；131) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 276, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 336;
    132)所述正义链包含如SEQ ID NO:276所示的核苷酸序列，所述反义链包含如SEQ ID NO:337所示的核苷酸序列；132) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 276, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 337;
    133)所述正义链包含如SEQ ID NO:277所示的核苷酸序列，所述反义链包含如SEQ ID NO:338所示的核苷酸序列；133) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 277, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 338;
    134)所述正义链包含如SEQ ID NO:278所示的核苷酸序列，所述反义链包含如SEQ ID NO:339所示的核苷酸序列；134) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 278, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 339;
    135)所述正义链包含如SEQ ID NO:279所示的核苷酸序列，所述反义链包含如SEQ ID NO:339所示的核苷酸序列；135) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 279, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 339;
    136)所述正义链包含如SEQ ID NO:278所示的核苷酸序列，所述反义链包含如SEQ ID NO:340所示的核苷酸序列；136) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 278, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 340;
    137)所述正义链包含如SEQ ID NO:278所示的核苷酸序列，所述反义链包含如SEQ ID NO:341所示的核苷酸序列；137) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 278, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 341;
    138)所述正义链包含如SEQ ID NO:278所示的核苷酸序列，所述反义链包含如SEQ ID NO:342所示的核苷酸序列；138) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 278, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 342;
    139)所述正义链包含如SEQ ID NO:280所示的核苷酸序列，所述反义链包含如SEQ ID NO:343所示的核苷酸序列；139) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 280, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 343;
    140)所述正义链包含如SEQ ID NO:281所示的核苷酸序列，所述反义链包含如SEQ ID NO:344所示的核苷酸序列；140) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 281, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 344;
    141)所述正义链包含如SEQ ID NO:282所示的核苷酸序列，所述反义链包含如SEQ ID NO:345所示的核苷酸序列；141) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 282, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 345;
    142)所述正义链包含如SEQ ID NO:283所示的核苷酸序列，所述反义链包含如SEQ ID NO:346所示的核苷酸序列；142) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 283, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 346;
    143)所述正义链包含如SEQ ID NO:284所示的核苷酸序列，所述反义链包含如SEQ ID NO:346所示的核苷酸序列；143) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 284, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 346;
    144)所述正义链包含如SEQ ID NO:283所示的核苷酸序列，所述反义链包含如SEQ ID NO:347所示的核苷酸序列；144) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 283, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 347;
    145)所述正义链包含如SEQ ID NO:283所示的核苷酸序列，所述反义链包含如SEQ ID NO:348所示的核苷酸序列；145) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 283, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 348;
    146)所述正义链包含如SEQ ID NO:283所示的核苷酸序列，所述反义链包含如SEQ ID NO:349所示的核苷酸序列；146) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 283, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 349;
    147)所述正义链包含如SEQ ID NO:285所示的核苷酸序列，所述反义链包含如SEQ ID NO:350所示的核苷酸序列；147) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 285, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 350;
    148)所述正义链包含如SEQ ID NO:286所示的核苷酸序列，所述反义链包含如SEQ ID NO:351所示的核苷酸序列；148) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 286, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 351;
    149)所述正义链包含如SEQ ID NO:287所示的核苷酸序列，所述反义链包含如SEQ ID  NO:351所示的核苷酸序列；149) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 287, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:351;
    150)所述正义链包含如SEQ ID NO:286所示的核苷酸序列，所述反义链包含如SEQ ID NO:352所示的核苷酸序列；150) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 286, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 352;
    151)所述正义链包含如SEQ ID NO:286所示的核苷酸序列，所述反义链包含如SEQ ID NO:353所示的核苷酸序列；151) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 286, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 353;
    152)所述正义链包含如SEQ ID NO:286所示的核苷酸序列，所述反义链包含如SEQ ID NO:354所示的核苷酸序列；152) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 286, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 354;
    153)所述正义链包含如SEQ ID NO:288所示的核苷酸序列，所述反义链包含如SEQ ID NO:355所示的核苷酸序列；153) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 288, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 355;
    154)所述正义链包含如SEQ ID NO:289所示的核苷酸序列，所述反义链包含如SEQ ID NO:355所示的核苷酸序列；154) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 289, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 355;
    155)所述正义链包含如SEQ ID NO:288所示的核苷酸序列，所述反义链包含如SEQ ID NO:356所示的核苷酸序列；155) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 288, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 356;
    156)所述正义链包含如SEQ ID NO:288所示的核苷酸序列，所述反义链包含如SEQ ID NO:357所示的核苷酸序列；156) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 288, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 357;
    157)所述正义链包含如SEQ ID NO:288所示的核苷酸序列，所述反义链包含如SEQ ID NO:358所示的核苷酸序列；157) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 288, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 358;
    158)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:359所示的核苷酸序列；158) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 359;
    159)所述正义链包含如SEQ ID NO:291所示的核苷酸序列，所述反义链包含如SEQ ID NO:359所示的核苷酸序列；159) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 291, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 359;
    160)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:360所示的核苷酸序列；160) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 360;
    161)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:361所示的核苷酸序列；161) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 361;
    162)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:362所示的核苷酸序列；162) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 362;
    163)所述正义链包含如SEQ ID NO:292所示的核苷酸序列，所述反义链包含如SEQ ID NO:363所示的核苷酸序列；163) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 292, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 363;
    164)所述正义链包含如SEQ ID NO:293所示的核苷酸序列，所述反义链包含如SEQ ID NO:364所示的核苷酸序列；164) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 293, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 364;
    165)所述正义链包含如SEQ ID NO:294所示的核苷酸序列，所述反义链包含如SEQ ID NO:365所示的核苷酸序列；165) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 294, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 365;
    166)所述正义链包含如SEQ ID NO:295所示的核苷酸序列，所述反义链包含如SEQ ID NO:366所示的核苷酸序列；166) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 295, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 366;
    167)所述正义链包含如SEQ ID NO:296所示的核苷酸序列，所述反义链包含如SEQ ID NO:367所示的核苷酸序列；167) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 296, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 367;
    168)所述正义链包含如SEQ ID NO:297所示的核苷酸序列，所述反义链包含如SEQ ID NO:368所示的核苷酸序列；168) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 297, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 368;
    169)所述正义链包含如SEQ ID NO:298所示的核苷酸序列，所述反义链包含如SEQ ID NO:369所示的核苷酸序列；169) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 298, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 369;
    170)所述正义链包含如SEQ ID NO:299所示的核苷酸序列，所述反义链包含如SEQ ID NO:370所示的核苷酸序列；170) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 299, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 370;
    171)所述正义链包含如SEQ ID NO:300所示的核苷酸序列，所述反义链包含如SEQ ID NO:371所示的核苷酸序列； 171) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 300, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 371;
    172)所述正义链包含如SEQ ID NO:301所示的核苷酸序列，所述反义链包含如SEQ ID NO:372所示的核苷酸序列；172) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:301, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:372;
    173)所述正义链包含如SEQ ID NO:302所示的核苷酸序列，所述反义链包含如SEQ ID NO:373所示的核苷酸序列；173) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:302, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:373;
    174)所述正义链包含如SEQ ID NO:303所示的核苷酸序列，所述反义链包含如SEQ ID NO:374所示的核苷酸序列；174) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 303, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 374;
    175)所述正义链包含如SEQ ID NO:304所示的核苷酸序列，所述反义链包含如SEQ ID NO:374所示的核苷酸序列；175) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 304, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 374;
    176)所述正义链包含如SEQ ID NO:303所示的核苷酸序列，所述反义链包含如SEQ ID NO:375所示的核苷酸序列；176) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:303, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:375;
    177)所述正义链包含如SEQ ID NO:303所示的核苷酸序列，所述反义链包含如SEQ ID NO:376所示的核苷酸序列；177) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:303, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:376;
    178)所述正义链包含如SEQ ID NO:303所示的核苷酸序列，所述反义链包含如SEQ ID NO:377所示的核苷酸序列；178) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 303, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 377;
    179)所述正义链包含如SEQ ID NO:305所示的核苷酸序列，所述反义链包含如SEQ ID NO:378所示的核苷酸序列；179) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 305, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 378;
    180)所述正义链包含如SEQ ID NO:306所示的核苷酸序列，所述反义链包含如SEQ ID NO:378所示的核苷酸序列；180) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 306, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 378;
    181)所述正义链包含如SEQ ID NO:305所示的核苷酸序列，所述反义链包含如SEQ ID NO:379所示的核苷酸序列；181) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:305, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:379;
    182)所述正义链包含如SEQ ID NO:305所示的核苷酸序列，所述反义链包含如SEQ ID NO:380所示的核苷酸序列；182) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 305, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 380;
    183)所述正义链包含如SEQ ID NO:305所示的核苷酸序列，所述反义链包含如SEQ ID NO:381所示的核苷酸序列；183) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 305, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 381;
    184)所述正义链包含如SEQ ID NO:307所示的核苷酸序列，所述反义链包含如SEQ ID NO:382所示的核苷酸序列；184) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:307, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:382;
    185)所述正义链包含如SEQ ID NO:308所示的核苷酸序列，所述反义链包含如SEQ ID NO:383所示的核苷酸序列；185) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:308, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:383;
    186)所述正义链包含如SEQ ID NO:308所示的核苷酸序列，所述反义链包含如SEQ ID NO:384所示的核苷酸序列；186) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:308, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:384;
    187)所述正义链包含如SEQ ID NO:308所示的核苷酸序列，所述反义链包含如SEQ ID NO:385所示的核苷酸序列；187) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 308, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 385;
    188)所述正义链包含如SEQ ID NO:309所示的核苷酸序列，所述反义链包含如SEQ ID NO:386所示的核苷酸序列；188) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 309, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 386;
    189)所述正义链包含如SEQ ID NO:310所示的核苷酸序列，所述反义链包含如SEQ ID NO:387所示的核苷酸序列；189) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 310, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 387;
    190)所述正义链包含如SEQ ID NO:310所示的核苷酸序列，所述反义链包含如SEQ ID NO:388所示的核苷酸序列；190) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 310, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 388;
    191)所述正义链包含如SEQ ID NO:310所示的核苷酸序列，所述反义链包含如SEQ ID NO:389所示的核苷酸序列；191) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:310, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:389;
    192)所述正义链包含如SEQ ID NO:311所示的核苷酸序列，所述反义链包含如SEQ ID NO:390所示的核苷酸序列；192) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:311, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:390;
    193)所述正义链包含如SEQ ID NO:312所示的核苷酸序列，所述反义链包含如SEQ ID NO:391所示的核苷酸序列；193) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:312, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:391;
    194)所述正义链包含如SEQ ID NO:312所示的核苷酸序列，所述反义链包含如SEQ ID  NO:392所示的核苷酸序列；194) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 312, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:392;
    195)所述正义链包含如SEQ ID NO:312所示的核苷酸序列，所述反义链包含如SEQ ID NO:393所示的核苷酸序列；195) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:312, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:393;
    196)所述正义链包含如SEQ ID NO:313所示的核苷酸序列，所述反义链包含如SEQ ID NO:394所示的核苷酸序列；196) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:313, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:394;
    197)所述正义链包含如SEQ ID NO:314所示的核苷酸序列，所述反义链包含如SEQ ID NO:395所示的核苷酸序列；197) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:314, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:395;
    198)所述正义链包含如SEQ ID NO:314所示的核苷酸序列，所述反义链包含如SEQ ID NO:396所示的核苷酸序列；198) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:314, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:396;
    199)所述正义链包含如SEQ ID NO:314所示的核苷酸序列，所述反义链包含如SEQ ID NO:397所示的核苷酸序列；199) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:314, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:397;
    200)所述正义链包含如SEQ ID NO:315所示的核苷酸序列，所述反义链包含如SEQ ID NO:398所示的核苷酸序列；200) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:315, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:398;
    201)所述正义链包含如SEQ ID NO:316所示的核苷酸序列，所述反义链包含如SEQ ID NO:398所示的核苷酸序列；201) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:316, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:398;
    202)所述正义链包含如SEQ ID NO:315所示的核苷酸序列，所述反义链包含如SEQ ID NO:399所示的核苷酸序列；202) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:315, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:399;
    203)所述正义链包含如SEQ ID NO:315所示的核苷酸序列，所述反义链包含如SEQ ID NO:400所示的核苷酸序列；203) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:315, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:400;
    204)所述正义链包含如SEQ ID NO:315所示的核苷酸序列，所述反义链包含如SEQ ID NO:401所示的核苷酸序列；204) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:315, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:401;
    205)所述正义链包含如SEQ ID NO:317所示的核苷酸序列，所述反义链包含如SEQ ID NO:402所示的核苷酸序列；205) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:317, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:402;
    206)所述正义链包含如SEQ ID NO:318所示的核苷酸序列，所述反义链包含如SEQ ID NO:402所示的核苷酸序列；206) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:318, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:402;
    207)所述正义链包含如SEQ ID NO:317所示的核苷酸序列，所述反义链包含如SEQ ID NO:403所示的核苷酸序列；207) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:317, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:403;
    208)所述正义链包含如SEQ ID NO:317所示的核苷酸序列，所述反义链包含如SEQ ID NO:404所示的核苷酸序列；208) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:317, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:404;
    209)所述正义链包含如SEQ ID NO:317所示的核苷酸序列，所述反义链包含如SEQ ID NO:405所示的核苷酸序列；209) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:317, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:405;
    210)所述正义链包含如SEQ ID NO:319所示的核苷酸序列，所述反义链包含如SEQ ID NO:406所示的核苷酸序列；210) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:319, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:406;
    211)所述正义链包含如SEQ ID NO:320所示的核苷酸序列，所述反义链包含如SEQ ID NO:406所示的核苷酸序列；211) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:320, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:406;
    212)所述正义链包含如SEQ ID NO:319所示的核苷酸序列，所述反义链包含如SEQ ID NO:407所示的核苷酸序列；212) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:319, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:407;
    213)所述正义链包含如SEQ ID NO:319所示的核苷酸序列，所述反义链包含如SEQ ID NO:408所示的核苷酸序列；213) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:319, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:408;
    214)所述正义链包含如SEQ ID NO:319所示的核苷酸序列，所述反义链包含如SEQ ID NO:409所示的核苷酸序列；214) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:319, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:409;
    215)所述正义链包含如SEQ ID NO:321所示的核苷酸序列，所述反义链包含如SEQ ID NO:410所示的核苷酸序列；215) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:321, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:410;
    216)所述正义链包含如SEQ ID NO:322所示的核苷酸序列，所述反义链包含如SEQ ID NO:410所示的核苷酸序列； 216) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:322, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:410;
    217)所述正义链包含如SEQ ID NO:321所示的核苷酸序列，所述反义链包含如SEQ ID NO:411所示的核苷酸序列；217) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:321, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:411;
    218)所述正义链包含如SEQ ID NO:321所示的核苷酸序列，所述反义链包含如SEQ ID NO:412所示的核苷酸序列；218) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:321, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:412;
    219)所述正义链包含如SEQ ID NO:321所示的核苷酸序列，所述反义链包含如SEQ ID NO:413所示的核苷酸序列；219) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:321, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:413;
    360)所述正义链包含如SEQ ID NO:281所示的核苷酸序列，所述反义链包含如SEQ ID NO:583所示的核苷酸序列；360) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 281, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 583;
    361)所述正义链包含如SEQ ID NO:281所示的核苷酸序列，所述反义链包含如SEQ ID NO:584所示的核苷酸序列；361) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 281, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 584;
    362)所述正义链包含如SEQ ID NO:281所示的核苷酸序列，所述反义链包含如SEQ ID NO:585所示的核苷酸序列；362) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 281, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 585;
    363)所述正义链包含如SEQ ID NO:281所示的核苷酸序列，所述反义链包含如SEQ ID NO:586所示的核苷酸序列；363) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 281, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 586;
    364)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:587所示的核苷酸序列；364) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 587;
    365)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:588所示的核苷酸序列；365) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 588;
    366)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:589所示的核苷酸序列；366) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 589;
    367)所述正义链包含如SEQ ID NO:569所示的核苷酸序列，所述反义链包含如SEQ ID NO:590所示的核苷酸序列；367) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:569, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:590;
    368)所述正义链包含如SEQ ID NO:570所示的核苷酸序列，所述反义链包含如SEQ ID NO:591所示的核苷酸序列；368) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:570, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:591;
    369)所述正义链包含如SEQ ID NO:571所示的核苷酸序列，所述反义链包含如SEQ ID NO:592所示的核苷酸序列；369) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:571, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:592;
    370)所述正义链包含如SEQ ID NO:571所示的核苷酸序列，所述反义链包含如SEQ ID NO:593所示的核苷酸序列；370) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:571, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:593;
    371)所述正义链包含如SEQ ID NO:571所示的核苷酸序列，所述反义链包含如SEQ ID NO:594所示的核苷酸序列；371) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:571, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:594;
    372)所述正义链包含如SEQ ID NO:571所示的核苷酸序列，所述反义链包含如SEQ ID NO:595所示的核苷酸序列；372) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:571, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:595;
    373)所述正义链包含如SEQ ID NO:572所示的核苷酸序列，所述反义链包含如SEQ ID NO:596所示的核苷酸序列；373) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:572, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:596;
    374)所述正义链包含如SEQ ID NO:572所示的核苷酸序列，所述反义链包含如SEQ ID NO:597所示的核苷酸序列；374) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:572, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:597;
    375)所述正义链包含如SEQ ID NO:572所示的核苷酸序列，所述反义链包含如SEQ ID NO:598所示的核苷酸序列；375) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:572, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:598;
    376)所述正义链包含如SEQ ID NO:572所示的核苷酸序列，所述反义链包含如SEQ ID NO:599所示的核苷酸序列；376) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:572, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:599;
    377)所述正义链包含如SEQ ID NO:572所示的核苷酸序列，所述反义链包含如SEQ ID NO:600所示的核苷酸序列；377) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:572, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:600;
    378)所述正义链包含如SEQ ID NO:572所示的核苷酸序列，所述反义链包含如SEQ ID NO:601所示的核苷酸序列；378) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:572, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:601;
    379)所述正义链包含如SEQ ID NO:573所示的核苷酸序列，所述反义链包含如SEQ ID  NO:602所示的核苷酸序列；379) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 573, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:602;
    380)所述正义链包含如SEQ ID NO:574所示的核苷酸序列，所述反义链包含如SEQ ID NO:603所示的核苷酸序列；380) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:574, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:603;
    381)所述正义链包含如SEQ ID NO:575所示的核苷酸序列，所述反义链包含如SEQ ID NO:604所示的核苷酸序列；381) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:575, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:604;
    382)所述正义链包含如SEQ ID NO:576所示的核苷酸序列，所述反义链包含如SEQ ID NO:605所示的核苷酸序列；382) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:576, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:605;
    383)所述正义链包含如SEQ ID NO:577所示的核苷酸序列，所述反义链包含如SEQ ID NO:606所示的核苷酸序列；383) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:577, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:606;
    384)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:607所示的核苷酸序列；384) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:607;
    385)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:608所示的核苷酸序列；385) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:608;
    386)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:609所示的核苷酸序列；386) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:609;
    387)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:610所示的核苷酸序列；387) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:610;
    388)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:611所示的核苷酸序列；388) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:611;
    389)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:612所示的核苷酸序列；389) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:612;
    390)所述正义链包含如SEQ ID NO:579所示的核苷酸序列，所述反义链包含如SEQ ID NO:613所示的核苷酸序列；390) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:613;
    391)所述正义链包含如SEQ ID NO:579所示的核苷酸序列，所述反义链包含如SEQ ID NO:614所示的核苷酸序列；391) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:614;
    392)所述正义链包含如SEQ ID NO:579所示的核苷酸序列，所述反义链包含如SEQ ID NO:615所示的核苷酸序列；392) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:615;
    393)所述正义链包含如SEQ ID NO:579所示的核苷酸序列，所述反义链包含如SEQ ID NO:616所示的核苷酸序列；393) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:616;
    394)所述正义链包含如SEQ ID NO:579所示的核苷酸序列，所述反义链包含如SEQ ID NO:617所示的核苷酸序列；394) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:617;
    395)所述正义链包含如SEQ ID NO:579所示的核苷酸序列，所述反义链包含如SEQ ID NO:618所示的核苷酸序列；395) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:618;
    396)所述正义链包含如SEQ ID NO:580所示的核苷酸序列，所述反义链包含如SEQ ID NO:619所示的核苷酸序列；396) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:580, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:619;
    397)所述正义链包含如SEQ ID NO:580所示的核苷酸序列，所述反义链包含如SEQ ID NO:620所示的核苷酸序列；397) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:580, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:620;
    398)所述正义链包含如SEQ ID NO:580所示的核苷酸序列，所述反义链包含如SEQ ID NO:621所示的核苷酸序列；398) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:580, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:621;
    399)所述正义链包含如SEQ ID NO:580所示的核苷酸序列，所述反义链包含如SEQ ID NO:622所示的核苷酸序列；399) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:580, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:622;
    400)所述正义链包含如SEQ ID NO:580所示的核苷酸序列，所述反义链包含如SEQ ID NO:623所示的核苷酸序列；400) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:580, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:623;
    401)所述正义链包含如SEQ ID NO:580所示的核苷酸序列，所述反义链包含如SEQ ID NO:624所示的核苷酸序列； 401) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 580, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 624;
    402)所述正义链包含如SEQ ID NO:581所示的核苷酸序列，所述反义链包含如SEQ ID NO:625所示的核苷酸序列；402) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:581, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:625;
    403)所述正义链包含如SEQ ID NO:581所示的核苷酸序列，所述反义链包含如SEQ ID NO:626所示的核苷酸序列；403) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:581, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:626;
    404)所述正义链包含如SEQ ID NO:582所示的核苷酸序列，所述反义链包含如SEQ ID NO:627所示的核苷酸序列；404) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:582, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:627;
    405)所述正义链包含如SEQ ID NO:582所示的核苷酸序列，所述反义链包含如SEQ ID NO:628所示的核苷酸序列；405) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:582, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:628;
    406)所述正义链包含如SEQ ID NO:582所示的核苷酸序列，所述反义链包含如SEQ ID NO:629所示的核苷酸序列；406) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:582, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:629;
    407)所述正义链包含如SEQ ID NO:582所示的核苷酸序列，所述反义链包含如SEQ ID NO:630所示的核苷酸序列；407) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:582, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:630;
    408)所述正义链包含如SEQ ID NO:582所示的核苷酸序列，所述反义链包含如SEQ ID NO:631所示的核苷酸序列；408) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:582, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:631;
    409)所述正义链包含如SEQ ID NO:582所示的核苷酸序列，所述反义链包含如SEQ ID NO:632所示的核苷酸序列；409) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:582, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:632;
    439)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:672所示的核苷酸序列；439) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 672;
    440)所述正义链包含如SEQ ID NO:290所示的核苷酸序列，所述反义链包含如SEQ ID NO:673所示的核苷酸序列；440) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 290, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 673;
    441)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:674所示的核苷酸序列；441) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:674;
    442)所述正义链包含如SEQ ID NO:578所示的核苷酸序列，所述反义链包含如SEQ ID NO:675所示的核苷酸序列；442) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:578, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:675;
    443)所述正义链包含如SEQ ID NO:670所示的核苷酸序列，所述反义链包含如SEQ ID NO:676所示的核苷酸序列；443) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 670, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 676;
    444)所述正义链包含如SEQ ID NO:670所示的核苷酸序列，所述反义链包含如SEQ ID NO:677所示的核苷酸序列；444) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 670, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 677;
    445)所述正义链包含如SEQ ID NO:670所示的核苷酸序列，所述反义链包含如SEQ ID NO:678所示的核苷酸序列；445) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 670, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 678;
    446)所述正义链包含如SEQ ID NO:670所示的核苷酸序列，所述反义链包含如SEQ ID NO:679所示的核苷酸序列；446) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 670, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 679;
    447)所述正义链包含如SEQ ID NO:671所示的核苷酸序列，所述反义链包含如SEQ ID NO:680所示的核苷酸序列；447) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:671, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:680;
    448)所述正义链包含如SEQ ID NO:671所示的核苷酸序列，所述反义链包含如SEQ ID NO:681所示的核苷酸序列。448) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:671, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:681.
  30. 一种双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物包括如权利要求1-15任一项所述的双链核糖核酸，或如权利要求16-29任一项所述的双链核糖核酸修饰物；以及，缀合连接于所述双链核糖核酸或所述双链核糖核酸修饰物的缀合基团。A double-stranded RNA conjugate, wherein the double-stranded RNA conjugate comprises the double-stranded RNA as described in any one of claims 1 to 15, or the double-stranded RNA modification as described in any one of claims 16 to 29; and a conjugated group conjugated to the double-stranded RNA or the double-stranded RNA modification.
  31. 根据权利要求30所述的双链核糖核酸缀合物，其中，所述缀合基团具有如下所示结构：
    The double-stranded RNA conjugate according to claim 30, wherein the conjugated group has the following structure:
  32. 根据权利要求30或31所述的双链核糖核酸缀合物，其中，所述缀合基团连接于正义链的3’末端。The double-stranded ribonucleic acid conjugate according to claim 30 or 31, wherein the conjugated group is connected to the 3' end of the sense strand.
  33. 根据权利要求32所述的双链核糖核酸缀合物，其中，所述缀合基团通过磷酸二酯键与正义链的3’末端缀合连接；The double-stranded RNA conjugate according to claim 32, wherein the conjugated group is conjugated to the 3' end of the sense strand via a phosphodiester bond;
    优选地，所述双链核糖核酸缀合物的正义链与反义链互补形成所述双链核糖核酸缀合物的双链区，且所述正义链的3’末端形成平末端，所述反义链的3’末端具有1-2个延伸出所述双链区的突出的核苷酸；Preferably, the sense strand and the antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region;
    或者，or,
    所述双链核糖核酸缀合物的正义链与反义链互补形成所述双链核糖核酸缀合物的双链区，且所述正义链的3’末端形成平末端，所述反义链的3’末端形成平末端。The sense strand and antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.
  34. 根据权利要求30-33任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物具有如下所示结构：
    The double-stranded ribonucleic acid conjugate according to any one of claims 30 to 33, wherein the double-stranded ribonucleic acid conjugate has the following structure:
    其中，双螺旋结构为双链核糖核酸或双链核糖核酸修饰物。The double helix structure is double-stranded RNA or a modified double-stranded RNA.
  35. 根据权利要求30-34任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物为siRNA缀合物。The double-stranded ribonucleic acid conjugate according to any one of claims 30 to 34, wherein the double-stranded ribonucleic acid conjugate is a siRNA conjugate.
  36. 根据权利要求30-35任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物是用于抑制凝血因子XII基因表达的siRNA缀合物。The double-stranded RNA conjugate according to any one of claims 30 to 35, wherein the double-stranded RNA conjugate is a siRNA conjugate for inhibiting the expression of the coagulation factor XII gene.
  37. 根据权利要求30-36任一项所述的双链核糖核酸缀合物，其中，所述双链核糖核酸缀合物由表1所示的任意一种siRNA与缀合基团连接形成，或者，所述双链核糖核酸缀合物由表1-1所示的任意一种siRNA与缀合基团连接形成，或者，所述双链核糖核酸缀合物由表2所示的任意一种siRNA修饰物与缀合基团连接形成；The double-stranded RNA conjugate according to any one of claims 30 to 36, wherein the double-stranded RNA conjugate is formed by connecting any one of the siRNAs shown in Table 1 to a conjugated group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNAs shown in Table 1-1 to a conjugated group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNA modifications shown in Table 2 to a conjugated group;
    优选地，所述双链核糖核酸缀合物中，其中，所述正义链与所述反义链选自如下组合：Preferably, in the double-stranded RNA conjugate, the sense strand and the antisense strand are selected from the following combinations:
    220)所述正义链包含如SEQ ID NO:414所示的核苷酸序列，所述反义链包含如SEQ ID NO:330所示的核苷酸序列；220) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:414, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:330;
    221)所述正义链包含如SEQ ID NO:414所示的核苷酸序列，所述反义链包含如SEQ ID NO:331所示的核苷酸序列； 221) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:414, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:331;
    222)所述正义链包含如SEQ ID NO:414所示的核苷酸序列，所述反义链包含如SEQ ID NO:332所示的核苷酸序列；222) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:414, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:332;
    223)所述正义链包含如SEQ ID NO:414所示的核苷酸序列，所述反义链包含如SEQ ID NO:333所示的核苷酸序列；223) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:414, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:333;
    224)所述正义链包含如SEQ ID NO:415所示的核苷酸序列，所述反义链包含如SEQ ID NO:339所示的核苷酸序列；224) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:415, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:339;
    225)所述正义链包含如SEQ ID NO:415所示的核苷酸序列，所述反义链包含如SEQ ID NO:340所示的核苷酸序列；225) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:415, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:340;
    226)所述正义链包含如SEQ ID NO:415所示的核苷酸序列，所述反义链包含如SEQ ID NO:341所示的核苷酸序列；226) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:415, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:341;
    227)所述正义链包含如SEQ ID NO:415所示的核苷酸序列，所述反义链包含如SEQ ID NO:342所示的核苷酸序列；227) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:415, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:342;
    228)所述正义链包含如SEQ ID NO:416所示的核苷酸序列，所述反义链包含如SEQ ID NO:346所示的核苷酸序列；228) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:416, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:346;
    229)所述正义链包含如SEQ ID NO:416所示的核苷酸序列，所述反义链包含如SEQ ID NO:347所示的核苷酸序列；229) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:416, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:347;
    230)所述正义链包含如SEQ ID NO:416所示的核苷酸序列，所述反义链包含如SEQ ID NO:348所示的核苷酸序列；230) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:416, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:348;
    231)所述正义链包含如SEQ ID NO:416所示的核苷酸序列，所述反义链包含如SEQ ID NO:349所示的核苷酸序列；231) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:416, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:349;
    232)所述正义链包含如SEQ ID NO:417所示的核苷酸序列，所述反义链包含如SEQ ID NO:351所示的核苷酸序列；232) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:417, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:351;
    233)所述正义链包含如SEQ ID NO:417所示的核苷酸序列，所述反义链包含如SEQ ID NO:352所示的核苷酸序列；233) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:417, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:352;
    234)所述正义链包含如SEQ ID NO:417所示的核苷酸序列，所述反义链包含如SEQ ID NO:353所示的核苷酸序列；234) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:417, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:353;
    235)所述正义链包含如SEQ ID NO:417所示的核苷酸序列，所述反义链包含如SEQ ID NO:354所示的核苷酸序列；235) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:417, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:354;
    236)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:359所示的核苷酸序列；236) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:359;
    237)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:360所示的核苷酸序列；237) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:360;
    238)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:361所示的核苷酸序列；238) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:361;
    239)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:362所示的核苷酸序列；239) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:362;
    240)所述正义链包含如SEQ ID NO:419所示的核苷酸序列，所述反义链包含如SEQ ID NO:410所示的核苷酸序列；240) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:419, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:410;
    241)所述正义链包含如SEQ ID NO:419所示的核苷酸序列，所述反义链包含如SEQ ID NO:411所示的核苷酸序列；241) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:419, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:411;
    242)所述正义链包含如SEQ ID NO:419所示的核苷酸序列，所述反义链包含如SEQ ID NO:412所示的核苷酸序列；242) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:419, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:412;
    243)所述正义链包含如SEQ ID NO:419所示的核苷酸序列，所述反义链包含如SEQ ID NO:413所示的核苷酸序列；243) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:419, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:413;
    244)所述正义链包含如SEQ ID NO:420所示的核苷酸序列，所述反义链包含如SEQ ID  NO:324所示的核苷酸序列；244) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 420, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:324;
    245)所述正义链包含如SEQ ID NO:420所示的核苷酸序列，所述反义链包含如SEQ ID NO:325所示的核苷酸序列；245) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:420, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:325;
    246)所述正义链包含如SEQ ID NO:420所示的核苷酸序列，所述反义链包含如SEQ ID NO:326所示的核苷酸序列；246) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:420, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:326;
    247)所述正义链包含如SEQ ID NO:420所示的核苷酸序列，所述反义链包含如SEQ ID NO:327所示的核苷酸序列；247) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:420, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:327;
    248)所述正义链包含如SEQ ID NO:421所示的核苷酸序列，所述反义链包含如SEQ ID NO:374所示的核苷酸序列；248) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:421, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:374;
    249)所述正义链包含如SEQ ID NO:421所示的核苷酸序列，所述反义链包含如SEQ ID NO:375所示的核苷酸序列；249) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:421, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:375;
    250)所述正义链包含如SEQ ID NO:421所示的核苷酸序列，所述反义链包含如SEQ ID NO:376所示的核苷酸序列；250) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:421, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:376;
    251)所述正义链包含如SEQ ID NO:421所示的核苷酸序列，所述反义链包含如SEQ ID NO:377所示的核苷酸序列；251) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:421, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:377;
    252)所述正义链包含如SEQ ID NO:422所示的核苷酸序列，所述反义链包含如SEQ ID NO:378所示的核苷酸序列；252) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:422, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:378;
    253)所述正义链包含如SEQ ID NO:422所示的核苷酸序列，所述反义链包含如SEQ ID NO:379所示的核苷酸序列；253) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:422, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:379;
    254)所述正义链包含如SEQ ID NO:422所示的核苷酸序列，所述反义链包含如SEQ ID NO:380所示的核苷酸序列；254) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:422, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:380;
    255)所述正义链包含如SEQ ID NO:422所示的核苷酸序列，所述反义链包含如SEQ ID NO:381所示的核苷酸序列；255) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:422, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:381;
    256)所述正义链包含如SEQ ID NO:423所示的核苷酸序列，所述反义链包含如SEQ ID NO:402所示的核苷酸序列；256) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:423, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:402;
    257)所述正义链包含如SEQ ID NO:423所示的核苷酸序列，所述反义链包含如SEQ ID NO:403所示的核苷酸序列；257) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:423, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:403;
    258)所述正义链包含如SEQ ID NO:423所示的核苷酸序列，所述反义链包含如SEQ ID NO:404所示的核苷酸序列；258) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:423, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:404;
    259)所述正义链包含如SEQ ID NO:423所示的核苷酸序列，所述反义链包含如SEQ ID NO:405所示的核苷酸序列；259) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:423, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:405;
    260)所述正义链包含如SEQ ID NO:424所示的核苷酸序列，所述反义链包含如SEQ ID NO:406所示的核苷酸序列；260) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:424, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:406;
    261)所述正义链包含如SEQ ID NO:424所示的核苷酸序列，所述反义链包含如SEQ ID NO:407所示的核苷酸序列；261) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:424, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:407;
    262)所述正义链包含如SEQ ID NO:424所示的核苷酸序列，所述反义链包含如SEQ ID NO:408所示的核苷酸序列；262) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:424, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:408;
    263)所述正义链包含如SEQ ID NO:424所示的核苷酸序列，所述反义链包含如SEQ ID NO:409所示的核苷酸序列；263) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:424, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:409;
    264)所述正义链包含如SEQ ID NO:425所示的核苷酸序列，所述反义链包含如SEQ ID NO:382所示的核苷酸序列；264) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:425, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:382;
    265)所述正义链包含如SEQ ID NO:425所示的核苷酸序列，所述反义链包含如SEQ ID NO:383所示的核苷酸序列；265) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:425, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:383;
    266)所述正义链包含如SEQ ID NO:425所示的核苷酸序列，所述反义链包含如SEQ ID NO:384所示的核苷酸序列； 266) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:425, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:384;
    267)所述正义链包含如SEQ ID NO:425所示的核苷酸序列，所述反义链包含如SEQ ID NO:385所示的核苷酸序列；267) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:425, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:385;
    268)所述正义链包含如SEQ ID NO:426所示的核苷酸序列，所述反义链包含如SEQ ID NO:386所示的核苷酸序列；268) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:426, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:386;
    269)所述正义链包含如SEQ ID NO:426所示的核苷酸序列，所述反义链包含如SEQ ID NO:387所示的核苷酸序列；269) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:426, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:387;
    270)所述正义链包含如SEQ ID NO:426所示的核苷酸序列，所述反义链包含如SEQ ID NO:388所示的核苷酸序列；270) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:426, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:388;
    271)所述正义链包含如SEQ ID NO:426所示的核苷酸序列，所述反义链包含如SEQ ID NO:389所示的核苷酸序列；271) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:426, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:389;
    272)所述正义链包含如SEQ ID NO:427所示的核苷酸序列，所述反义链包含如SEQ ID NO:390所示的核苷酸序列；272) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:427, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:390;
    273)所述正义链包含如SEQ ID NO:427所示的核苷酸序列，所述反义链包含如SEQ ID NO:391所示的核苷酸序列；273) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:427, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:391;
    274)所述正义链包含如SEQ ID NO:427所示的核苷酸序列，所述反义链包含如SEQ ID NO:392所示的核苷酸序列；274) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:427, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:392;
    275)所述正义链包含如SEQ ID NO:427所示的核苷酸序列，所述反义链包含如SEQ ID NO:393所示的核苷酸序列；275) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:427, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:393;
    276)所述正义链包含如SEQ ID NO:428所示的核苷酸序列，所述反义链包含如SEQ ID NO:394所示的核苷酸序列；276) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:428, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:394;
    277)所述正义链包含如SEQ ID NO:428所示的核苷酸序列，所述反义链包含如SEQ ID NO:395所示的核苷酸序列；277) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:428, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:395;
    278)所述正义链包含如SEQ ID NO:428所示的核苷酸序列，所述反义链包含如SEQ ID NO:396所示的核苷酸序列；278) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:428, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:396;
    279)所述正义链包含如SEQ ID NO:428所示的核苷酸序列，所述反义链包含如SEQ ID NO:397所示的核苷酸序列；279) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:428, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:397;
    280)所述正义链包含如SEQ ID NO:429所示的核苷酸序列，所述反义链包含如SEQ ID NO:334所示的核苷酸序列；280) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:429, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:334;
    281)所述正义链包含如SEQ ID NO:429所示的核苷酸序列，所述反义链包含如SEQ ID NO:335所示的核苷酸序列；281) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:429, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:335;
    282)所述正义链包含如SEQ ID NO:429所示的核苷酸序列，所述反义链包含如SEQ ID NO:336所示的核苷酸序列；282) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:429, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:336;
    283)所述正义链包含如SEQ ID NO:429所示的核苷酸序列，所述反义链包含如SEQ ID NO:337所示的核苷酸序列；283) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:429, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:337;
    284)所述正义链包含如SEQ ID NO:430所示的核苷酸序列，所述反义链包含如SEQ ID NO:355所示的核苷酸序列；284) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:430, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:355;
    285)所述正义链包含如SEQ ID NO:430所示的核苷酸序列，所述反义链包含如SEQ ID NO:356所示的核苷酸序列；285) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:430, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:356;
    286)所述正义链包含如SEQ ID NO:430所示的核苷酸序列，所述反义链包含如SEQ ID NO:357所示的核苷酸序列；286) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:430, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:357;
    287)所述正义链包含如SEQ ID NO:430所示的核苷酸序列，所述反义链包含如SEQ ID NO:358所示的核苷酸序列；287) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:430, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:358;
    288)所述正义链包含如SEQ ID NO:431所示的核苷酸序列，所述反义链包含如SEQ ID NO:398所示的核苷酸序列；288) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:431, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:398;
    289)所述正义链包含如SEQ ID NO:431所示的核苷酸序列，所述反义链包含如SEQ ID  NO:399所示的核苷酸序列；289) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 431, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO:399;
    290)所述正义链包含如SEQ ID NO:431所示的核苷酸序列，所述反义链包含如SEQ ID NO:400所示的核苷酸序列；290) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:431, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:400;
    291)所述正义链包含如SEQ ID NO:431所示的核苷酸序列，所述反义链包含如SEQ ID NO:401所示的核苷酸序列；291) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:431, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:401;
    410)所述正义链包含如SEQ ID NO:633所示的核苷酸序列，所述反义链包含如SEQ ID NO:592所示的核苷酸序列；410) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:633, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:592;
    411)所述正义链包含如SEQ ID NO:633所示的核苷酸序列，所述反义链包含如SEQ ID NO:593所示的核苷酸序列；411) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:633, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:593;
    412)所述正义链包含如SEQ ID NO:634所示的核苷酸序列，所述反义链包含如SEQ ID NO:619所示的核苷酸序列；412) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:634, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:619;
    413)所述正义链包含如SEQ ID NO:634所示的核苷酸序列，所述反义链包含如SEQ ID NO:620所示的核苷酸序列；413) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:634, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:620;
    414)所述正义链包含如SEQ ID NO:635所示的核苷酸序列，所述反义链包含如SEQ ID NO:625所示的核苷酸序列；414) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:635, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:625;
    415)所述正义链包含如SEQ ID NO:635所示的核苷酸序列，所述反义链包含如SEQ ID NO:626所示的核苷酸序列；415) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:635, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:626;
    416)所述正义链包含如SEQ ID NO:636所示的核苷酸序列，所述反义链包含如SEQ ID NO:627所示的核苷酸序列；416) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:636, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:627;
    417)所述正义链包含如SEQ ID NO:636所示的核苷酸序列，所述反义链包含如SEQ ID NO:628所示的核苷酸序列；417) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:636, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:628;
    418)所述正义链包含如SEQ ID NO:637所示的核苷酸序列，所述反义链包含如SEQ ID NO:583所示的核苷酸序列；418) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:637, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:583;
    419)所述正义链包含如SEQ ID NO:637所示的核苷酸序列，所述反义链包含如SEQ ID NO:584所示的核苷酸序列；419) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:637, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:584;
    420)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:588所示的核苷酸序列；420) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:588;
    421)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:589所示的核苷酸序列；421) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:589;
    422)所述正义链包含如SEQ ID NO:638所示的核苷酸序列，所述反义链包含如SEQ ID NO:607所示的核苷酸序列；422) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:638, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:607;
    423)所述正义链包含如SEQ ID NO:638所示的核苷酸序列，所述反义链包含如SEQ ID NO:608所示的核苷酸序列；423) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 638, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 608;
    424)所述正义链包含如SEQ ID NO:638所示的核苷酸序列，所述反义链包含如SEQ ID NO:611所示的核苷酸序列；424) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:638, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:611;
    425)所述正义链包含如SEQ ID NO:638所示的核苷酸序列，所述反义链包含如SEQ ID NO:612所示的核苷酸序列；425) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:638, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:612;
    426)所述正义链包含如SEQ ID NO:639所示的核苷酸序列，所述反义链包含如SEQ ID NO:596所示的核苷酸序列；426) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:639, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:596;
    427)所述正义链包含如SEQ ID NO:639所示的核苷酸序列，所述反义链包含如SEQ ID NO:597所示的核苷酸序列；427) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:639, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:597;
    428)所述正义链包含如SEQ ID NO:639所示的核苷酸序列，所述反义链包含如SEQ ID NO:600所示的核苷酸序列；428) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 639, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 600;
    429)所述正义链包含如SEQ ID NO:639所示的核苷酸序列，所述反义链包含如SEQ ID NO:601所示的核苷酸序列； 429) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 639, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 601;
    430)所述正义链包含如SEQ ID NO:640所示的核苷酸序列，所述反义链包含如SEQ ID NO:613所示的核苷酸序列；430) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 640, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 613;
    431)所述正义链包含如SEQ ID NO:640所示的核苷酸序列，所述反义链包含如SEQ ID NO:614所示的核苷酸序列；431) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:640, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:614;
    432)所述正义链包含如SEQ ID NO:640所示的核苷酸序列，所述反义链包含如SEQ ID NO:617所示的核苷酸序列；432) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:640, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:617;
    433)所述正义链包含如SEQ ID NO:640所示的核苷酸序列，所述反义链包含如SEQ ID NO:618所示的核苷酸序列；433) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:640, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:618;
    434)所述正义链包含如SEQ ID NO:634所示的核苷酸序列，所述反义链包含如SEQ ID NO:623所示的核苷酸序列；434) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 634, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 623;
    435)所述正义链包含如SEQ ID NO:634所示的核苷酸序列，所述反义链包含如SEQ ID NO:624所示的核苷酸序列；435) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 634, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 624;
    436)所述正义链包含如SEQ ID NO:636所示的核苷酸序列，所述反义链包含如SEQ ID NO:631所示的核苷酸序列；436) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:636, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:631;
    449)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:672所示的核苷酸序列；449) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:672;
    450)所述正义链包含如SEQ ID NO:418所示的核苷酸序列，所述反义链包含如SEQ ID NO:673所示的核苷酸序列；450) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:418, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:673;
    451)所述正义链包含如SEQ ID NO:638所示的核苷酸序列，所述反义链包含如SEQ ID NO:674所示的核苷酸序列；451) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:638, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:674;
    452)所述正义链包含如SEQ ID NO:638所示的核苷酸序列，所述反义链包含如SEQ ID NO:675所示的核苷酸序列；452) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:638, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:675;
    453)所述正义链包含如SEQ ID NO:568所示的核苷酸序列，所述反义链包含如SEQ ID NO:676所示的核苷酸序列；453) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:568, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:676;
    454)所述正义链包含如SEQ ID NO:568所示的核苷酸序列，所述反义链包含如SEQ ID NO:677所示的核苷酸序列；454) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:568, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:677;
    455)所述正义链包含如SEQ ID NO:568所示的核苷酸序列，所述反义链包含如SEQ ID NO:678所示的核苷酸序列；455) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:568, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:678;
    456)所述正义链包含如SEQ ID NO:568所示的核苷酸序列，所述反义链包含如SEQ ID NO:679所示的核苷酸序列；456) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:568, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:679;
    457)所述正义链包含如SEQ ID NO:428所示的核苷酸序列，所述反义链包含如SEQ ID NO:680所示的核苷酸序列；457) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:428, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:680;
    458)所述正义链包含如SEQ ID NO:428所示的核苷酸序列，所述反义链包含如SEQ ID NO:681所示的核苷酸序列；458) the sense strand comprises the nucleotide sequence shown in SEQ ID NO:428, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:681;
    459)所述正义链包含如SEQ ID NO:636所示的核苷酸序列，所述反义链包含如SEQ ID NO:632所示的核苷酸序列。459) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:636, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:632.
  38. 一种药物组合物，其中，所述药物组合物包括如下至少一项：如权利要求1-15任一项所述的双链核糖核酸，如权利要求16-29任一项所述的双链核糖核酸修饰物，如权利要求30-37任一项所述的双链核糖核酸缀合物。A pharmaceutical composition, wherein the pharmaceutical composition comprises at least one of the following: the double-stranded ribonucleic acid as described in any one of claims 1 to 15, the double-stranded ribonucleic acid modification as described in any one of claims 16 to 29, and the double-stranded ribonucleic acid conjugate as described in any one of claims 30 to 37.
  39. 根据权利要求38所述的药物组合物，其中，所述药物组合物还包括一种或多种药学上可接受的载体。The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.
  40. 根据权利要求1-15任一项所述的双链核糖核酸，根据权利要求16-29任一项所述的双链核糖核酸修饰物，根据权利要求30-37任一项所述的双链核糖核酸缀合物，或根据权利要求38-39任一项所述的药物组合物在如下至少一项中的用途：Use of the double-stranded RNA according to any one of claims 1 to 15, the double-stranded RNA modification according to any one of claims 16 to 29, the double-stranded RNA conjugate according to any one of claims 30 to 37, or the pharmaceutical composition according to any one of claims 38 to 39 in at least one of the following:
    (1)抑制凝血因子XII基因表达，或制备用于抑制凝血因子XII基因表达的药物； (1) Inhibiting the expression of coagulation factor XII gene, or preparing a drug for inhibiting the expression of coagulation factor XII gene;
    (2)用于预防或治疗与凝血因子XII基因异常表达相关的疾病，或制备用于预防或治疗与凝血因子XII基因异常表达相关的疾病的药物；(2) for preventing or treating diseases related to abnormal expression of coagulation factor XII gene, or for preparing drugs for preventing or treating diseases related to abnormal expression of coagulation factor XII gene;
    (3)用于治疗患有将受益于凝血因子XII基因表达降低的疾病的受试者，或制备用于治疗患有将受益于凝血因子XII基因表达降低的疾病的受试者的药物。(3) Use for treating a subject suffering from a disease that would benefit from reduced expression of the coagulation factor XII gene, or for preparing a medicament for treating a subject suffering from a disease that would benefit from reduced expression of the coagulation factor XII gene.
  41. 根据权利要求40所述的用途，其中，所述与凝血因子XII基因异常表达相关的疾病选自如下疾病组成的组：The use according to claim 40, wherein the disease associated with abnormal expression of coagulation factor XII gene is selected from the group consisting of the following diseases:
    多发性硬化症、动脉粥样硬化、阿尔兹海默症、遗传性血管水肿、脓毒症、深静脉血栓形成、社区获得性肺炎、新冠肺炎的血栓性炎症、显微镜下型多血管炎、神经性炎症、脑血管血栓、静脉血栓栓塞、动脉血栓形成、类风湿性关节炎及结肠炎以及其他尚未鉴定的相关病症、病理或综合征。Multiple sclerosis, atherosclerosis, Alzheimer's disease, hereditary angioedema, sepsis, deep vein thrombosis, community-acquired pneumonia, thrombotic inflammation of COVID-19, microscopic polyangiitis, neuroinflammation, cerebrovascular thrombosis, venous thromboembolism, arterial thrombosis, rheumatoid arthritis and colitis, and other related conditions, pathologies or syndromes not yet identified.
  42. 一种用于抑制细胞内凝血因子XII基因表达的方法，其中，所述方法包括将所述细胞与根据权利要求1-15任一项所述的双链核糖核酸，根据权利要求16-29任一项所述的双链核糖核酸修饰物，根据权利要求30-37任一项所述的双链核糖核酸缀合物，或根据权利要求38-39任一项所述的药物组合物接触。A method for inhibiting the expression of intracellular coagulation factor XII gene, wherein the method comprises contacting the cell with the double-stranded ribonucleic acid according to any one of claims 1 to 15, the double-stranded ribonucleic acid modification according to any one of claims 16 to 29, the double-stranded ribonucleic acid conjugate according to any one of claims 30 to 37, or the pharmaceutical composition according to any one of claims 38 to 39.
  43. 根据权利要求42所述的方法，其中，所述细胞为体内细胞或体外细胞。The method according to claim 42, wherein the cell is an in vivo cell or an in vitro cell.
  44. 根据权利要求42或43所述的方法，其中，所述细胞在受试者体内。The method of claim 42 or 43, wherein the cell is in a subject.
  45. 根据权利要求44所述的方法，其中，所述受试者为哺乳动物，优选为人。The method according to claim 44, wherein the subject is a mammal, preferably a human.
  46. 根据权利要求44或45所述的方法，其中，所述受试者具有如下至少一种特性：The method according to claim 44 or 45, wherein the subject has at least one of the following characteristics:
    体内凝血因子XII基因异常表达，更具体地为FXII基因异常高表达；Abnormal expression of coagulation factor XII gene in vivo, more specifically abnormally high expression of FXII gene;
    患有与凝血因子XII基因异常表达相关的疾病；Suffering from a disease associated with abnormal expression of the coagulation factor XII gene;
    患有将受益于凝血因子XII基因表达降低的疾病。 Having a disease that would benefit from decreased expression of the factor XII gene.
PCT/CN2024/074466 2023-02-14 2024-01-29 Double-stranded ribonucleic acid used to inhibit expression of fxii gene, and modifier, conjugate and use thereof WO2024169577A1 (en)

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Citations (4)

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US20190024088A1 (en) * 2016-01-08 2019-01-24 Alnylam Phaarmaceuticals, Inc. Polynucleotide agents targeting factor xii (hageman factor) (f12) and methods of use thereof
CN110234326A (en) * 2017-01-30 2019-09-13 箭头药业股份有限公司 Composition and method for inhibiting factor XII gene expression

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