WO2024008114A1 - 用于抑制补体因子B表达的siRNA、其缀合物和药物组合物及其用途 - Google Patents

用于抑制补体因子B表达的siRNA、其缀合物和药物组合物及其用途 Download PDF

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WO2024008114A1
WO2024008114A1 PCT/CN2023/105855 CN2023105855W WO2024008114A1 WO 2024008114 A1 WO2024008114 A1 WO 2024008114A1 CN 2023105855 W CN2023105855 W CN 2023105855W WO 2024008114 A1 WO2024008114 A1 WO 2024008114A1
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nucleotide sequence
seq
nucleotide
nucleotides
sequence shown
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王书成
黄河
刘薇
王岩
林国良
产运霞
耿玉先
汪小君
荣梅
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北京福元医药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present application relates to siRNA, siRNA conjugates, pharmaceutical compositions containing the same, preparation methods and uses thereof capable of inhibiting complement factor B (CFB) gene expression.
  • CFB complement factor B
  • Complement is a group of heat-labile proteins that exist in the serum and tissue fluid of humans and animals, have enzymatic activity after activation, and can mediate immune responses and inflammatory responses. After the complement system is activated, it mediates a series of cellular reactions, such as cell lysis, opsonophagocytosis (antigen-antibody binding), inflammatory response, clearance of immune complexes, etc.
  • the complement system is mainly composed of more than 30 types of glycoproteins such as intrinsic components, regulatory proteins and receptors. These proteins exist as soluble proteins in the blood or as membrane-associated proteins.
  • the complement system is divided into three initiation pathways - the classical, lectin and alternative pathways, which converge at component C3 to produce an enzyme complex called C3 convertase, which cleaves C3 into C3a and C3b.
  • C3 convertase The binding of C3b to C3 convertase is mediated by complement factor B (CFB) and leads to the production of C5 convertase, which cleaves C5 into C5a and C5b, which initiates the membrane attack pathway, leading to the membrane attack complex (MAC)
  • CFB complement factor B
  • MAC membrane attack complex
  • Formation of the membrane attack complex (MAC) includes components C5b, C6, C7, C8 and C9.
  • Membrane attack complex (MAC) forms transmembrane channels and disrupts the phospholipid bilayer of target cells, leading to cell lysis.
  • Inappropriate activation of the complement system CFB is responsible for proliferation and/or initiating pathology in many diseases including, for example, primary membranous nephropathy, hemolytic uremic syndrome, C3 glomerulopathy, IgA Kidney disease, patterned nocturnal hemoglobinuria (PNH), age-related macular degeneration, and ophthalmic disease.
  • diseases including, for example, primary membranous nephropathy, hemolytic uremic syndrome, C3 glomerulopathy, IgA Kidney disease, patterned nocturnal hemoglobinuria (PNH), age-related macular degeneration, and ophthalmic disease.
  • PNH patterned nocturnal hemoglobinuria
  • CFB complement inhibitors are a promising treatment method. There are currently no related drugs on the market. The fastest progress is the small molecule CFB inhibitor developed by Novartis (for the treatment of IgA nephropathy), which is currently in clinical phase 3. In addition, Ionis Pharmaceuticals The antisense nucleic acid developed is also in clinical phase 2. The development of new complement CFB inhibitors has great clinical value and market prospects.
  • the present invention aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the CFB gene, thereby inhibiting the expression of the CFB gene in the liver. , to achieve the purpose of disease treatment.
  • RISC RNA-induced silencing complex
  • the present invention provides a siRNA capable of inhibiting the expression of complement factor B (CFB) gene.
  • the siRNA includes a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified.
  • Nucleotides, wherein the sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, and the nucleotide sequence Column I and said nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein said nucleotide sequence I and nucleotide sequence II are selected from the following sequences:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:217
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:218:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:219
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:220:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:221, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:222:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:223, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:224:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:225
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:226:
  • nucleotide sequence I is not SEQ ID NO:47, and the nucleotide sequence II is not SEQ ID NO:48;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:227
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:228:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:229
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:230:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:231, and the nucleotide sequence II contains the nucleotide sequence shown in SEQ ID NO:232:
  • nucleotide sequence I is not SEQ ID NO:85, and the nucleotide sequence II is not SEQ ID NO:86;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:233
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:234:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:235, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:236:
  • nucleotide sequence I is not SEQ ID NO: 127, and the nucleotide sequence II is not SEQ ID NO: 128;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:129, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:130:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:237, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:238:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:239
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:240:
  • nucleotide sequence I is not SEQ ID NO: 145, and the nucleotide sequence II is not SEQ ID NO: 146;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:241, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:242:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:243, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:244:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:245, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:246:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:247, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:248:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:2;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:3, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:4;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:28;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:29, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:30;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:31, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:32;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:51, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:52;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:87, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:88;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:93, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:94;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:95, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:96;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:98;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:99, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:100;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:103, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:104;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 131, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 132;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 133, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 134;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 135, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 136;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 137
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 138;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:201, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:202;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:207, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:208;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:209, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:210;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:211, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:212;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:213, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:214;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:215, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:216;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:401, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:402:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:403
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:404:
  • nucleotide sequence I is not SEQ ID NO:285, and the nucleotide sequence II is not SEQ ID NO:286;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:405
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:406:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:407
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:408:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:354, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:355:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:409
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:410:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 391, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 392;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 393, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 392;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 391, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 394;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 391, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 395;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 391, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 396;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:259, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:260;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:259, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:397;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:398, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:397;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:259, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:399;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:259, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:400;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO:250, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO:130.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two nuclei There are no more than 3 base mismatches between the nucleotide sequences; the substantial reverse complementarity refers to the presence of no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity refers to the absence of mismatches between two nucleotide sequences.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 9 nucleotides, wherein the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence III It is equal in length to the IV of the nucleotide sequence and is substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences. ;Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences; and/or,
  • the nucleotide sequence III is connected to the 3' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence II
  • the IV lengths are equal and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; There are no mismatches between the two nucleotide sequences.
  • the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI being 0 to 3 nucleotides in length , the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' end of the antisense strand. 'Protruding end.
  • the length of the nucleotide sequence V or VI is 2 nucleotides.
  • the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In a preferred embodiment, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.
  • the length of the double-stranded region is 15-30 nucleotide pairs; preferably, the length of the double-stranded region is 17-23 nucleotide pairs; more preferably, the length of the double-stranded region is It is 19-21 nucleotide pairs.
  • the sense or antisense strand has 15-30 nucleotides; preferably, the sense strand The strand or antisense strand has 19-25 nucleotides; more preferably the sense strand or antisense strand has 19-23 nucleotides.
  • At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide
  • at least one phosphate group is a phosphate group with a modifying group; preferably , the phosphate group containing a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.
  • the siRNA includes a sense strand that does not include a 3' overhanging nucleotide.
  • 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 5' phosphate derivative group.
  • the modified nucleotide is selected from the group consisting of 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleosides Acid, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-substituted amino Modified nucleotides, nucleotide analogs or a combination of any two or more thereof.
  • each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide or a non-fluoro modified nucleotide.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores In the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nuclei. glycosides.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide
  • the 2'-methoxy modified nucleotide refers to the 2'- of the ribosyl group Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA and GNA.
  • each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, GNA Modified nucleotides or a combination of any two or more thereof.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified of nucleotides.
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy modified nucleotides.
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand. Position 7, and the remaining positions are 2'-methoxy modified nucleotides.
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at position 1 of the antisense strand. Position 6, and the remaining positions are 2'-methoxy modified nucleotides.
  • At least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:
  • the siRNA is directed from the 5' end to the 3' end, and the sense strand contains a phosphorothioate group at a position as follows:
  • the sense strand contains phosphorothioate groups at the positions shown below:
  • the siRNA is directed from the 5' end to the 3' end and the antisense strand contains a phosphorothioate group at a position as follows:
  • each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, GNA Modified nucleotides or a combination of any two or more thereof.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, the 2'-fluoro modified nucleotide is located at the even position of the antisense strand, the remaining positions are the 2'-methoxy modified nucleotide, the 1st nucleotide and the 2nd core at the 5' end between nucleotides, between the 2nd and 3rd nucle
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups
  • Modified nucleotides, between the first and second nucleotides at the 5' end, and between the second and third nucleotides at the 5' end are phosphorothioates Base connection, remove the overhang at the 3'end; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleosides Acid, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, the 1st core at the
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups
  • Modified nucleotides, between the first and second nucleotides at the 5' end, and between the second and third nucleotides at the 5' end are phosphorothioates base connection, and the overhang is removed from the 3'end; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy-modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, 3 There is a phosphorothioate group connection between the first and second nucleotides at the 'end and between the second and third nucle
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, the first core at the 5' end Between the nucleotide and the second nucleotide, between the second and third nucleotides
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 5' end Between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides at the
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups
  • Modified nucleotides, between the first and second nucleotides at the 5' end, and between the second and third nucleotides at the 5' end are phosphorothioates Base connection, remove the overhang at the 3'end; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions It is a 2'-methoxy modified nucleotide, between the first and second nucleotides at the 5' end, and between the second and third nucleotides at the 5' end between the first and second nucleotides at the 3' end, and between the second and third nucleotides at the 3' end are phosphorothioate groups.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy base-modified nucleotide, between the first and second nucleotides at the
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, between the first and second nucleotides at the 5'
  • the invention provides a siRNA selected from Table 1, wherein the siRNA is not N-ER-FY010019, N-ER-FY010015, N-ER-FY010051, N-ER-FY010021, N- ER-FY010013, N-ER-FY010035, N-ER-FY010009, N-ER-FY-010099; preferably, the siRNA is selected from N-ER-FY010016, N-ER-FY010016M2, N-ER-FY010016M2D2, N-ER-FY010016M3, N-ER-FY010016M3D2, N-ER-FY010016M4, N-ER010016M5, N-ER010034, N-ER010034M3, N-ER010034M3D2, N-ER010034m2, N-R-FY010034M2 ER-FY010034M2D2, N-ER-FY01000100
  • the present invention also provides an siRNA conjugate, which contains the siRNA of the present invention and a conjugation group conjugated to the siRNA (as shown in the figure below, the double helix structure represents the siRNA, and the The conjugation group is attached to the 3' end of the sense strand of the siRNA):
  • X can be selected as O or S. In one embodiment, X is O.
  • the conjugation group includes a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker, and the targeting group are sequentially linked covalently or non-covalently.
  • the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the antisense strand forms a blunt end.
  • the 3' end of the chain has 1-3 protruding nucleotides extending out of the double-stranded region;
  • the sense strand and the antisense strand of siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand forms a blunt end. 'The ends form blunt ends.
  • the conjugating group is L96 of the formula:
  • the siRNA conjugate is a siRNA conjugate selected from Table 2.
  • the present invention also provides a pharmaceutical composition, which contains the siRNA of the present invention, or the siRNA conjugate of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention also provides a kit comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.
  • the present invention also provides the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.
  • the compound is used for preparing a medicament for inhibiting CFB gene expression.
  • the present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for preparing a medicament for preventing and/or treating diseases related to CFB gene overexpression.
  • the disease is selected from the group consisting of primary membranous nephropathy, hemolytic uremic syndrome, C3 glomerulopathy, IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, and Eye diseases.
  • the present invention also provides a method for inhibiting CFB gene expression, which includes contacting a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention with CFB-expressing cells or administering it to a patient in need subjects.
  • the present invention also provides methods for treating and/or preventing diseases related to CFB gene overexpression, including administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention in need. of subjects.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by this application show excellent CFB gene expression inhibitory activity in in vitro cell experiments, and have good potential to treat diseases related to CFB gene overexpression.
  • the siRNA and its conjugates disclosed in this application can reduce the expression of CFB mRNA in the liver, have low toxic and side effects, good plasma stability, and have good clinical application prospects.
  • the siRNA provided by this application shows good inhibitory effect on CFB gene in human liver cancer cell Hep3B.
  • the siRNA provided in this application has an inhibition rate as high as 92.41% at a concentration of 0.1 nM; and an inhibition rate as high as 98.78% at a concentration of 1 nM.
  • the siRNA provided by the present application has higher CFB gene inhibitory activity in Hep3B cells, for example, the IC 50 is as low as 0.030 nM.
  • the siRNA conjugate provided by the present application has higher CFB gene inhibitory activity in PHH cells.
  • the IC 50 when entering PHH cells through free uptake, the IC 50 can be as low as 0.282nM; when through transfection When stained into PHH cells, the IC 50 can be as low as 0.005nM.
  • G", “C”, “A”, “T” and “U” usually represent guanine, cytosine, adenine and thymine respectively. , the base of uracil, but it is also commonly known in the art that "G”, “C”, “A”, “T” and “U” each usually represent guanine, cytosine, adenine, respectively.
  • Thymine and uracil serve as the bases of nucleotides, which are represented in the deoxyribose nucleus It is a common approach in acid sequences and/or ribonucleic acid sequences, so in the context of this disclosure, the meanings represented by "G”, “C”, “A”, “T”, and “U” include the various possible possibilities mentioned above. situation.
  • Lowercase letters a, u, c, g represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro modified nucleotides; lowercase letter s represents the same letter as this letter
  • the two adjacent nucleotides to the left and right of s are connected by a phosphorothioate group; P1: indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide; (Underline + bold + italics): Indicates GNA modified nucleotides.
  • the "2'-fluoro-modified nucleotide” refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine.
  • “Non-fluorinated modified nucleotides” refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2’ position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with a non-fluorinated group.
  • nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art.
  • nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'-Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted One of the amino-modified nucleotides and 2'-deoxynucleotides.
  • 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. group.
  • the "C 1-6 " in "C 1-6 alkyl” refers to a linear, branched or cyclic arrangement containing 1, 2, 3, 4, 5 or 6 carbon atoms. group.
  • Alkoxy as used herein means an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein said alkyl group is as defined herein.
  • alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n- Pentyloxy etc.
  • Nucleotide analogue refers to a nucleotide that can replace a nucleotide in a nucleic acid, but whose structure is different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymus Pyrimidine deoxyribonucleotide group.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation.
  • 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 BNA, etc., where LNA is as shown in formula (1) shown, ENA is shown in formula (2), cET BNA is shown in formula (3).
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA).
  • UNA is represented by formula (4)
  • GNA is represented by formula (4). 5 shown.
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotides refer to compounds formed by changing the position of the base on the ribose ring in the nucleotide. For example, the base moves from the 1'-position to the 2'-position or 3'-position of the ribose ring.
  • the compound is shown in formula (6) or (7).
  • Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or the non-fluorine group as mentioned above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA, and GNA.
  • each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof.
  • each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, above and below, the 2'-methoxy modified nucleoside Acid refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the "2'-methoxy modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the "phosphorothioate group” refers to a phosphorothioate group in which one oxygen atom in the phosphodiester bond of the phosphate group is replaced by a sulfur atom.
  • the "5'-phosphate nucleotide” refers to the structure of the following formula:
  • the expressions "complementary” and “reverse complementary” are used interchangeably and have The meaning is well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases on one strand are each paired with the bases on the other strand in a complementary manner.
  • the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) always pairs with the pyrimidine base Pairs with cytosine (G).
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “ means that there is no more than one base mismatch between the two nucleotide sequences; “complete reverse complementarity” means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide difference between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed between the former and the latter. For example, when one nucleotide base in the latter is A, and when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is regarded as one of the two nucleotide sequences. There are nucleotide differences at this position. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference is generated at that position.
  • an "overhang” refers to one or more unpaired nucleotides that protrude from the duplex structure of an siRNA when one 3' end of one strand of the siRNA 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 siRNA, ie, no nucleotide overhangs.
  • a “blunt-ended" siRNA is one that is double-stranded throughout its length, ie, has no nucleotide overhangs at either end of the molecule.
  • the nucleoside monomer refers to the siRNA or siRNA to be prepared according to Type and sequence of nucleotides in siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers used in solid-phase phosphoramidite synthesis.
  • Solid-phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in this application are all commercially available.
  • conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, “conjugate” is Refers to a compound formed by covalent connections between various chemical parts.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugates should be According to the context, it is understood as a general term for multiple siRNA conjugates or an siRNA conjugate represented by a certain chemical formula.
  • conjugation molecule should be understood as a specific compound that can be conjugated to siRNA through a reaction, ultimately forming the siRNA conjugate of the present application.
  • hydroxyl protecting groups can be used in this application. Generally speaking, protecting groups render a chemical functional group insensitive to specific reaction conditions and can be attached to and removed from that functional group in the molecule without materially damaging the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox).
  • DMT dimethoxytrityl
  • Azure 9-phenylxanthine-9-yl
  • Mox 9-(p-methoxyphenyl)xanthine-9-yl
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl) trityl) and TMTr (4,4',4′′-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any species of poultry.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any species of poultry.
  • treatment refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, although the subject may still be suffering from the underlying disorder.
  • prevention refers to a method of obtaining beneficial or desired results, including but not limited to preventive benefits.
  • a siRNA, siRNA conjugate or pharmaceutical composition may be administered to a subject at risk of developing a particular disease, or to a subject who reports one or more physiological symptoms of the disease, even if possible A diagnosis of the disease has not yet been made.
  • the present application relates to a siRNA capable of inhibiting CFB gene expression.
  • the siRNA of the present application contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base. Typically active, ie, functional, siRNAs are about 12-40 nucleotides in length, and in some embodiments are about 15-30 nucleotides in length.
  • the siRNA of the present application contains a sense strand and an antisense strand.
  • Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, and the The antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary.
  • double-stranded region In some embodiments, the double-stranded region is 15-30 nucleotide pairs in length. In other embodiments, the double-stranded region is 17-23 nucleotide pairs in length. In other embodiments, the double-stranded region is 19-21 nucleotide pairs in length. In yet other embodiments, the double-stranded region is 19 or 21 nucleotide pairs in length.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 9 nucleotides
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II
  • the nucleotide sequences IV are equal in length and are substantially reverse complementary or completely reverse complementary
  • the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences
  • Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 9 nucleotides
  • the nucleotide sequence III is connected to the 3' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence II
  • the nucleotide sequences IV are equal in length and are substantially reverse complementary or completely reverse complementary
  • the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences
  • Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 9 nucleotides
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II
  • the nucleotide sequence III and The nucleotide sequence IV is equal in length and substantially reverse complementary or completely reverse complementary
  • the nucleotide sequence III is connected to the 3' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the nucleoside
  • the 5' end of the acid sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two nucleosides There is no more than 1 base mismatch between
  • the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI being 0 to 3 nucleotides in length , the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' end of the antisense strand. protruding end.
  • the nucleotide sequence V or VI is 2 nucleotides in length.
  • the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In other embodiments, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.
  • the lengths of the sense strand and the antisense strand provided by the application are the same or different.
  • the sense strand or the antisense strand has 15-30 nucleotides.
  • the sense or antisense strand has 19-25 nucleotides.
  • the sense or antisense strand has 19-23 nucleotides.
  • the length ratio of the sense strand and the antisense strand can be 15/15, 16/16, 17/17, 18/18, 19/19, 19/20, 19/21, 19/22, 19/23, 20/19 ,20/20,20/21,20/22,20/23,21/19,21/20,21/21,21/22,21/23,22/19,22/20,22/21,22 /22, 22/23, 23/19, 23/20, 23/21, 23/22, 23/23, 24/24, 25/25, 26/26, 27/27, 28/28, 29/29 , 30/30, 22/24, 22/25, 22/26, 23/24, 23/25 or 23/26, etc.
  • the length ratio of the siRNA sense strand and antisense strand is 19/19, 21/21, 19/21, 21/23 or 23/23.
  • the siRNA of the present disclosure has better Cellular mRNA silencing activity.
  • siRNA obtained by one of the modification methods while improving blood stability, It also maintained inhibitory activity that was essentially equivalent to that of unmodified siRNA.
  • each nucleotide in the siRNA of the present invention is independently a modified or unmodified nucleotide.
  • each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleosides. These modifications on the acid and nucleotide groups will not cause the siRNA of the present invention to significantly weaken or lose the function of inhibiting CFB gene expression.
  • the siRNA of the present application contains at least 1 modified nucleotide.
  • modified nucleotide is used to refer to a nucleotide or nucleotide analogue in which the 2' hydroxyl group of the ribose group of a nucleotide is replaced by another group, or has a modified Modified bases of nucleotides.
  • the modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression.
  • modified nucleotides 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.
  • At least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present invention is a modified nucleotide, and/or at least one phosphate group has a modified group.
  • Phosphate group in other words, at least part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modifying group and/or ribosyl groups with modifying groups.
  • the phosphate group containing a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.
  • the siRNA includes a sense strand that does not include a 3' overhanging nucleotide; that is, the sense strand of the siRNA may have a 3' overhanging nucleotide, excluding the 3' overhanging nucleotide of the sense strand. Then a flat end is formed.
  • nucleosides are added to the 3' end of the sense strand.
  • Acid sequence V as overhanging nucleotide.
  • the nucleotide sequence formed is chemically modified to exclude the nucleotide sequence V.
  • the sense strand of siRNA forms a blunt end.
  • the sense strand when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sense strand is located at The protruding nucleotide at the 3' end is excluded as the nucleotide sequence of the sense strand, and accordingly, the sense strand of siRNA forms a blunt end.
  • the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate derivative group. In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group.
  • An exemplary 5' phosphate group structure is: The structures of the 5' phosphate derivative group include but are not limited to: wait.
  • Base represents a base, such as A, U, G, C or T.
  • R' is hydroxyl or 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'-amino modified nucleotides, 2 '-Substituted amino-modified nucleotide, 2'-deoxynucleotide.
  • 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'-a
  • Exemplary modified nucleotides have 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 replaced by R.
  • the hydroxyl group at the 2' position of these ribose groups can be replaced by various groups known to those skilled in the art, for example, 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy groups Modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified Nucleotide, 2'-substituted amino-modified nucleotide, 2'-deoxynucleotide.
  • the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O- CH3 ) modified nucleotide, and the like.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided by the invention is independently a 2'-fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide or a GNA modified nucleotide, the 2'-methoxy modified nucleotide being A nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides .
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified of nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at position 1 of the antisense strand. 6 position, the remaining positions are 2'-methoxy groups decorated nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand. Position 7, and the remaining positions are 2'-methoxy modified nucleotides.
  • each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, the 2'-methoxy modified nucleotide being a ribosyl Nucleotides formed by replacing the 2'-hydroxyl group with a methoxy group.
  • At least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:
  • the siRNA is directed from the 5' end to the 3' end, and the sense strand contains a phosphorothioate group at a position as shown below:
  • the sense strand contains phosphorothioate groups at the positions shown below:
  • the siRNA is directed from the 5' end to the 3' end, and the antisense strand contains a phosphorothioate group at a position as shown below:
  • the present application relates to a siRNA conjugate, which contains the above-mentioned siRNA and a conjugation group conjugated to the siRNA.
  • the sense strand and the antisense strand of the siRNA conjugate form the double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the siRNA conjugate.
  • the 3' end of the sense strand of the siRNA conjugate forms a blunt end and the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleosides extending out of the double-stranded region acid.
  • the 3' end of the sense strand of the siRNA conjugate is blunt-ended, and the 3' end of the antisense strand of the siRNA conjugate is blunt-ended.
  • the siRNA conjugate is obtained by conjugating siRNA with a conjugating group.
  • the sense strand of siRNA and the antisense strand are complementary to form a double-stranded region of siRNA, and the 3' end of the sense strand of siRNA forms a blunt end, and the conjugation group is conjugated to the 3' end of the sense strand with a blunt end. , forming siRNA conjugates.
  • the 3' end of the sense strand of siRNA has a protruding nucleotide extending out of the double-stranded region, and the protruding nucleotide located at the 3' end of the sense strand is excluded to form a structure with 3'
  • the blunt-ended sequence serves as the nucleotide sequence for connecting the conjugation group, and the conjugation group is connected to the 3' blunt end of the sense strand to form an siRNA conjugate.
  • nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and there is no overhanging nucleotide at the 3' end of the sense strand, add Nucleotide sequence V, as overhanging nucleotide.
  • the sequence with a 3' blunt end formed by excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugation group, and the conjugation is connected to the 3' blunt end of the sense strand. groups to form siRNA conjugates.
  • the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide that extends out of the double-stranded region, it will be located in the sense strand.
  • the sequence with a 3' blunt end formed after excluding the protruding nucleotides at the 3' end of the chain is used as the nucleotide sequence for connecting the conjugation group, and the conjugation group is connected to the 3' blunt end of the sense strand. Formation of siRNA conjugates.
  • the 3' end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the protruding - located at the 3' end of the sense strand will be
  • the blunt-end sequence of gsasugaaAfgCfCfAfgucucuga formed after removing the sgsu nucleotide is used as the nucleotide sequence for connecting the L96 conjugation group.
  • the sequence to form the siRNA conjugate is: the sense strand is gsasugaaAfgCfCfAfgucucugaL96, and the antisense strand is P1usCfsaGfaGfaCfuGfgCfuUfuCfaUfcsGfsa.
  • the conjugation group includes at least one pharmaceutically acceptable targeting group, or further includes a linker, and the siRNA, the linker and the targeting group are connected in sequence .
  • the number of targeting groups is 1-6. In some embodiments, the number of targeting groups is 2-4.
  • the siRNA molecule may be non-covalently or covalently conjugated to the conjugation group, eg, may be covalently conjugated to the conjugation group.
  • the conjugation site of siRNA and the conjugation group can be at the 3' end or 5' end of the sense strand of siRNA, or at the 5' end of the antisense strand, or in the internal sequence of siRNA. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3' end of the sense strand of the siRNA.
  • the conjugation group can be attached to the phosphate group, the 2'-hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugation group can also be connected to the 3'-position hydroxyl group, in which case the nucleotides are connected via a 2'-5' phosphodiester bond.
  • the conjugation group is usually attached to the phosphate group of the nucleotide; when the conjugation group is attached to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base.
  • the siRNA and the conjugation group can be connected through acid-labile or reducible chemical bonds. In the acidic environment of cellular endosomes, these chemical bonds can be degraded, thereby leaving the siRNA in a free state.
  • the conjugation group can be connected to the sense strand of siRNA to minimize the impact of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA delivery, such as various ligands described in WO2009082607A2, which is fully incorporated into this specification by reference.
  • the pharmaceutically acceptable targeting group can be selected from one or more ligands formed by the following targeting molecules or derivatives thereof: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-penetrating peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, polylactose, and mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells, such as asialoglycoprotein, asialoglycoside residues, lipoproteins (such as high-density Lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.
  • lipophilic molecules such as cholesterol, bile acids, Vitamins (such
  • each ligand is independently selected from a ligand capable of binding to a cell surface receptor.
  • at least one ligand is a ligand capable of binding to a hepatocyte surface receptor.
  • at least one ligand is a ligand capable of binding to a mammalian cell surface receptor.
  • at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor.
  • at least one ligand is a ligand capable of binding to liver surface asialoglycoprotein receptor (ASGPR).
  • ASGPR liver surface asialoglycoprotein receptor
  • the pharmaceutically acceptable targeting group can be any ligand that binds to asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.
  • each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).
  • the ligand is a sugar or sugar derivative.
  • At least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands can be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar.
  • each ligand is independently selected from the group consisting of polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives.
  • each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives substances, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.
  • each of the ligands may be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylfuranose, L-xylfuranose, D- Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-pyranose Galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, gluco
  • the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be a monovalent , divalent, trivalent, quadrivalent.
  • the monovalent, bivalent, trivalent and tetravalent terms mentioned here respectively refer to siRNA molecules and siRNA molecules containing
  • the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate be 1:1, 1:2, 1:3 or 1:4.
  • the pharmaceutically acceptable targeting group is N-acetylgalactosamine.
  • the N-acetylgalactosamine molecule when an siRNA described herein is conjugated to an N-acetylgalactosamine-containing conjugation group, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when an siRNA described herein is conjugated to an N-acetylgalactosamine-containing conjugation group, the N-acetylgalactosamine molecule is trivalent.
  • the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • suitable linker those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • the types of these linkers, targeting groups and the connection methods with siRNA can be found in the disclosure of WO2015006740A2, which is fully incorporated into this specification by reference.
  • the nucleoside monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement through the conventional solid-phase phosphoramidite method in this field.
  • Each connection of a nucleoside monomer involves four steps of deprotection, coupling, capping, oxidation or sulfation.
  • deprotection, coupling, capping, oxidation or sulfation when two nucleotides are connected using a phosphate ester, when the next nucleoside monomer is connected, it includes four steps of deprotection, coupling, capping, and oxidation.
  • two nucleotides are connected using phosphorothioate, when the next nucleoside monomer is connected, it includes four steps of protection, coupling, capping and sulfation.
  • siRNA of the present application can be as follows:
  • reaction temperature is 25°C
  • reaction time is 70 seconds
  • deprotection reagent is selected from dichloroacetic acid in dichloromethane solution (3% V/V)
  • deprotection reagent and protective group on solid phase carrier The molar ratio is 5:1.
  • the coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from a 0.25M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), and the nucleic acid connected to the solid-phase carrier
  • ETT 5-ethylthio-1H-tetrazole
  • the molar ratio of sequence to nucleoside monomer is 1:10.
  • the capping reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, and the capping reagent is selected from CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine/ Acetonitrile solution), the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride: N-methylimidazole: the molar ratio of the nucleic acid sequence connected to the solid phase carrier is 1:1:1.
  • the oxidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the oxidation reagent is selected from 0.05M iodine tetrahydrofuran solution, and the molar ratio of the oxidation reagent to the nucleic acid sequence connected to the solid-phase carrier in the coupling step is 30:1.
  • the sulfidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfide reagent is selected from hydrogenated xanthogen, and the molar ratio of the sulfide reagent to the nucleic acid sequence connected to the solid-phase carrier in the coupling step is 120:1.
  • the nucleic acid sequences connected to the solid phase carrier are cut, deprotected, purified, and desalted in sequence to obtain the siRNA sense strand and antisense strand. Finally, the two strands are heated and annealed to obtain the product.
  • Methods of cleavage, deprotection, purification, desalting and annealing are well known in the art. For example, by connecting The nucleotide sequence with the solid phase carrier is contacted with concentrated ammonia water for cleavage and deprotection; purification by chromatography; desalting by reversed-phase chromatography; by mixing the sense strand and the antisense strand in equal molar ratios under different stringent conditions and then gradually Cool down.
  • compound L96-A is obtained by reacting DMTr-L96 and succinic anhydride:
  • Preparation process Add DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine into dichloromethane, stir and react at 25°C for 24 hours, and then wash with 0.5M triethylamine phosphate. The aqueous phase of the reaction solution was washed three times with dichloromethane, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product; then the pure product L96-A was purified by column chromatography.
  • Preparation process Mix L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) in acetonitrile, stir at room temperature for 5 minutes to obtain a uniform solution , add aminomethyl resin (NH 2 -SPS, 100-200 mesh) to the reaction liquid, start the shaking reaction at 25°C, filter after 18 hours of reaction, and wash the filter cake with dichloromethane and acetonitrile in sequence to obtain the 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 the conjugated molecule.
  • the nucleoside monomer is connected to the conjugated molecule under the coupling reaction, and then the nucleoside monomer is connected to the conjugated molecule as described above.
  • the siRNA molecule synthesis method is used to synthesize the siRNA sense strand connected to the conjugate molecule, and the siRNA molecule synthesis method described above is used to synthesize the siRNA antisense strand, and annealed to generate the siRNA conjugate of the present application.
  • the present application provides a pharmaceutical composition containing the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier commonly used in the field of siRNA administration, such as but not limited to Lipid Nanoparticles (LNP), magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon nanotubes, mesoporous silicon, Calcium phosphate nanoparticles, polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine (poly(L-lysine), PLL), Chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly D-type or L-type lactic acid/glycolic acid copolymer (poly (D&L-lactic/glycolic acid)copolymer, PLGA), poly(2-aminoethyl ethylene phosphate), PPEEA, and poly(methacrylic acid-N,N-dimethylaminoethyl Este
  • siRNA and pharmaceutically acceptable carriers there are no special requirements on the contents of siRNA and pharmaceutically acceptable carriers, and they can be the conventional contents of each component.
  • the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg).
  • the content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation may be, but is not limited to, administered by subcutaneous, intramuscular or intravenous injection, may be administered to the lungs by spray, or may be administered to other organs and tissues (such as the liver) through the lungs by spray.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • siRNA molecule with the following sequence was synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.
  • the capital letters "G”, “C”, “A”, “T” and “U” each usually represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases respectively.
  • Lowercase letters a, u, c, g represent: 2'-methoxy modified nucleotides
  • Af, Gf, Cf, Uf represent: 2'-fluoro modified nucleotides
  • lowercase letters s represent the same
  • the two adjacent nucleotides on the left and right of the letter s are connected by a phosphorothioate group
  • P1 indicates that the adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide
  • siRNA conjugate with the following sequence was synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.:
  • L96 is:
  • Hep3B cells were purchased from ATCC, catalog number HB-8064;
  • RNAiMax transfection reagent purchased from Invitrogen, product number 13778-150;
  • Opti-medium reduced serum medium, purchased from Gibco, Cat. No. 31985-070;
  • EMEM culture medium ATCC, Cat. No. 30-2003;
  • Ultrapure distilled water (DNAse, RNAse, Free): purchased from Invitrogen, item number 10977-015;
  • TaqMan TM Fast Advanced Cells-to-CT TM Kit (with gDNase), purchased from Invitrogen, product number A35377;
  • PBS Phosphate Buffer Saline
  • Human ACTB probe primer was purchased from Invitrogen 4448491, Hs01060665_g1;
  • CFB TaqMan probe primers were purchased from Invitrogen 4351370, Hs00156060_m1.
  • ⁇ Ct ⁇ Ct (detection sample group)- ⁇ Ct (Mock group), where the Mock group represents the group in which siRNA is not added compared with the detection 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% Table 3 Inhibition rate of siRNA of the present invention
  • the siRNA of the present invention can significantly inhibit the expression of CFB gene at 0.1 nM.
  • the IC 50 of siRNA inhibiting CFB gene expression was measured, where the concentrations of siRNA used for transfection were 5nM, 2nM, 1nM, 0.5nM, 0.2nM, 0.1nM, 0.05nM, 0.01 respectively. nM, 0.005nM.
  • the Mock group indicates: compared with the test sample group, the group without siRNA was added.
  • 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%
  • 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 percent inhibition curve.
  • the siRNA of the present invention has a lower IC 50 and a higher CFB gene inhibitory activity in Hep3B cells.
  • PHH medium invitroGRO CP Meduim serum free BIOVIT, Cat. No.: S03316
  • Lipofectamine RNAiMax transfection reagent purchased from Invitrogen, product number: 13778-150;
  • RNA extraction kit 96 Kit purchased from QIAGEN, product number: QIAGEN-74182;
  • Reverse transcription kit FastQuant RT Kit (With gDNase), purchased from TianGen, product number: KR116-02;
  • siRNA conjugates (siRNA conjugate final concentrations are 10nM, 2.5nM, 0.63nM, 0.16nM, 0.04nM, 0.01nM, 0.0024nM and 0.0006nM, respectively, in duplicate) into PHH cells through transfection, the process is as follows : Take the frozen PHH cells, resuscitate, count, adjust the cells to 6 ⁇ 10 5 cells/ml, and use Lipofectamine RNAiMax transfection reagent to transfer the siRNA conjugate into the cells, and inoculate 96 cells at a density of 54,000 cells per well. In the well plate, add 100 ⁇ L of PPH medium to each well. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for RNA extraction. Use according to instructions 96Kit extracted total RNA.
  • siRNA conjugates enter PHH cells through free uptake, and the process is as follows: Take the frozen PHH cells, resuscitate, count, adjust the cells to 6 ⁇ 10 5 cells/ml, add siRNA conjugate at the same time, and inoculate them into a 96-well plate at a density of 54,000 cells per well, with 100 ⁇ l of culture medium per well. . Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for RNA extraction. Use according to instructions 96 Kit to extract total RNA.
  • the extracted total RNA was reverse transcribed into cDNA through a reverse transcription reaction.
  • Target cDNA will be detected by qPCR.
  • GAPDH cDNA will be used as an internal control for parallel testing.
  • the PCR reaction program is: 95°C for 10 minutes, then enter the cycle mode, 95°C for 15 seconds, then 60°C for 60 seconds, a total of 40 cycles.
  • ⁇ Ct ⁇ Ct (detection sample group) – ⁇ Ct (Mock group), where the Mock group represents the group in which siRNA is not added compared with the detection 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%.
  • 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.
  • siRNA conjugate of the present invention has high CFB gene inhibitory activity in PHH cells.
  • Hep3B cells were purchased from ATCC, catalog number HB-8064;
  • Lipofectamine RNAiMax transfection reagent purchased from Invitrogen, product number 13778-150;
  • Opti-medium reduced serum medium, purchased from Gibco, Cat. No. 31985-070;
  • EMEM culture medium ATCC, Cat. No. 30-2003;
  • Ultrapure distilled water (DNAse, RNAse, Free): purchased from Invitrogen, item number 10977-015;
  • TaqMan TM Fast Advanced Cells-to-CT TM Kit (with gDNase), purchased from Invitrogen, product number A35377;
  • PBS Phosphate Buffer Saline
  • Human ACTB probe primer was purchased from Invitrogen 4448491, Hs01060665_g1;
  • CFB TaqMan probe primers were purchased from Invitrogen 4351370, Hs00156060_m1.
  • the 1nM siRNA transfection dilution can be prepared similarly to the above.
  • ⁇ Ct ⁇ Ct (detection sample group)- ⁇ Ct (Mock group), where the Mock group represents the group in which siRNA is not added compared with the detection 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 siRNA of the present invention can significantly inhibit the expression of CFB gene at both 0.1 nM and 1 nM.
  • PHH medium invitroGRO CP Meduim serum free BIOVIT, Cat. No.: S03316;
  • Lipofectamine RNAiMax transfection reagent purchased from Invitrogen, product number: 13778-150;
  • RNA extraction kit 96 Kit purchased from QIAGEN, product number: QIAGEN-74182;
  • Reverse transcription kit FastQuant RT Kit (With gDNase), purchased from TianGen, product number: KR116-02;
  • siRNA conjugates (the final concentrations of siRNA conjugates are 5 nM and 0.1 nM, respectively, in duplicate wells) are transfected into PHH cells.
  • the process is as follows: take the frozen PHH cells, resuscitate, count, and adjust the cells to 6 ⁇ 10 5 cells/ml, and Lipofectamine RNAiMax transfection reagent was used to transfer the siRNA conjugate into the cells.
  • the cells were seeded into a 96-well plate at a density of 54,000 cells per well, and 100 ⁇ L of PPH medium was added to each well. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for RNA extraction. Use according to instructions 96 Kit to extract total RNA.
  • siRNA conjugates enter PHH cells through free uptake.
  • the process is as follows: take the frozen PHH cells, resuscitate, count, and adjust the cells to 6 ⁇ 10 5 cells/ml, and siRNA conjugate was added at the same time, and seeded into a 96-well plate at a density of 54,000 cells per well, with 100 ⁇ l of culture medium per well. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for RNA extraction. Use according to instructions 96 Kit to extract total RNA.
  • the extracted total RNA was reverse transcribed into cDNA through a reverse transcription reaction.
  • Target cDNA will be detected by qPCR.
  • GAPDH cDNA will be used as an internal control for parallel testing.
  • the PCR reaction program is: 95°C for 10 minutes, then enter the cycle mode, 95°C for 15 seconds, then 60°C for 60 seconds, a total of 40 cycles.
  • ⁇ Ct ⁇ Ct (detection sample group) – ⁇ Ct (Mock group), where the Mock group represents the group in which siRNA is not added compared with the detection 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%
  • mice C57BL/6-hCFB mice (provided by Shanghai Southern Model Biotechnology Co., Ltd.) aged 6-8 weeks entered the breeding facility. After adaptive feeding for 7 days, the mice were subcutaneously administered N at a single dose of 3 mg/kg. -ER-FY010016M2L96 and N-ER-FY010034M3L96 (6 mice per group). The serum hCFB protein expression was detected on the 7th, 14th, 21st, 28th, 35th and 42nd days after administration, and the inhibition rate of hCFB protein expression by the siRNA conjugate was obtained.
  • the siRNA conjugate of the present disclosure has high inhibitory activity on the hCFB gene in vivo and can reduce the level of hCFB protein for a long time, indicating that the siRNA conjugate of the present invention can better inhibit the hCFB protein level.
  • N-ER-FY010016M2L96 showed 70.4% inhibition of hCFB gene expression on day 7; on day 42, N-ER-FY010016M2L96 still inhibited hCFB gene expression by 55.8%.
  • N-ER-FY010034M3L96 showed 72.3% inhibition of hCFB gene expression.
  • N-ER-FY010034M3L96 still inhibited hCFB gene expression by 44.2%.
  • siRNA conjugate was administered at a dose of 3 mg/kg (10 mL/kg), randomized A single subcutaneous injection was administered after each group, with 6 mice in each group.
  • Sample collection Collect whole blood samples at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, and 48 hours after administration, a total of 10 points. The first three animals in each group were collected at 0.0833, 0.5, 2, 8, and 36 hours, and the last three animals were collected at 0.25, 1, 4, 24, and 48 hours. Whole blood was collected and plasma was centrifuged for detection and analysis.
  • siRNA conjugate of the present disclosure has a shorter half-life in plasma and is cleared faster.
  • siRNA conjugate was administered at a dose of 3 mg/kg (10 mL/kg), and administered as a single subcutaneous injection after randomization, with 3 animals at each time point, for a total of 24 mice.
  • Sample detection and analysis The LC-MS/MS method was used to detect the concentration of the prototype drug in plasma and tissue samples at each time point, and the trapezoidal area method was used to calculate the AUC in plasma and tissue.
  • the siRNA conjugate of the present disclosure is mainly enriched in the liver, has a long retention time in the tissue, and has good stability.
  • mice SPF grade, male, about 25 g, were purchased from Spefford (Beijing) Biotechnology Co., Ltd. The animals were randomly grouped according to their body weight on the last day of the adaptation period.
  • the specific dose design and grouping are as follows:
  • Clinical observation Observe continuously for 4 hours on the dosing day, and perform clinical observation at least once a day during the recovery period.
  • Body weight All surviving animals were weighed twice a week.
  • Immunotoxicity Animals in the MTD dose group were blood collected alternately at 1h ⁇ 2min, 4h ⁇ 5min, 8h ⁇ 10min, and 24h ⁇ 20min after D1 administration. 3 animals/sex/group were collected at each time point to detect cytokines (IFN- ⁇ ). , TNF- ⁇ , IL-2/6/8).
  • Toxicokinetics Animals in the MTD dose group were blood collected alternately before D1 administration and 30min ⁇ 2min, 1h ⁇ 2min, 4h ⁇ 5min, 8h ⁇ 10min, and 24h ⁇ 20min after D1 administration. 3 animals/sex/ were collected at each time point. Group animals and detect blood drug concentrations.
  • Blood biochemistry The animals in the main experimental group were necropsied at R28, and the animals in the satellite group were necropsied in batches at R7, R14, R21, and R28 to detect blood biochemistry.
  • Tissue distribution The animals in the main experimental group were necropsied at R28, and the animals in the satellite group were necropsied in batches at R7, R14, R21, and R28. Blood and liver were collected to detect tissue drug concentration.
  • Histopathological examination The animals in the main test group were necropsied at R28, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and abnormal tissues were collected Or organs were harvested and fixed for histopathological examination.
  • siRNA conjugate of the present disclosure has low toxicity and has an excellent drug safety window.

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Abstract

本发明涉及能够抑制补体因子B(CFB)基因表达的siRNA、siRNA缀合物、包含其的药物组合物、及其用途。所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，该siRNA含有正义链和反义链。所述siRNA及其缀合物和药物组合物可以有效治疗和/或预防与CFB基因过表达相关的疾病。

Description

用于抑制补体因子B表达的siRNA、其缀合物和药物组合物及其用途 技术领域
本申请涉及能够抑制补体因子B(CFB)基因表达的siRNA、siRNA缀合物、包含其的药物组合物、其制备方法和用途。
背景技术
补体(Complement,C)是存在于人和动物血清与组织液中的一组不耐热、经活化后具有酶活性、可介导免疫应答和炎症反应的蛋白质。补体系统(Complement system)被激活后，介导一系列的细胞反应，如细胞溶解、调理吞噬(抗原抗体结合)、炎症反应、清除免疫复合物等。补体系统主要由固有成分、调节蛋白及受体等30余种糖蛋白组成，这些蛋白质以血液中可溶性蛋白存在或以膜相关蛋白存在。将补体系统分成三个启动途径——经典、凝集素和替代途径，其在组分C3处会聚以产生称为C3转化酶的酶复合物，该酶复合物将C3裂解成C3a和C3b。C3b与C3转化酶通过补体因子B(CFB)介导结合，并导致C5转化酶的产生，C5转化酶将C5裂解成C5a和C5b，这启动了膜攻击途径，导致膜攻击复合物(MAC)的形成，该膜攻击复合物(MAC)包括组分C5b、C6、C7、C8和C9。膜攻击复合物(MAC)形成跨膜通道并破坏靶细胞的磷脂双层，导致细胞裂解。
补体系统CFB的不适当的激活是在许多疾病中的增殖和/或起始病变的原因，这些疾病包括例如，原发性膜性肾病、溶血性尿毒症综合征、C3肾小球病、IgA肾病、阵法性夜间血红蛋白尿(PNH)、年龄相关性黄斑病变和眼科疾病。
开发CFB补体抑制剂是一项有前景的治疗方法，目前无相关药物上市，进展最快的是诺华开发的小分子CFB抑制剂(用于治疗IgA肾病)现处于临床3期，另外Ionis制药公司开发的反义核酸也处于临床2期。开发新型补体CFB抑制剂拥有巨大的临床价值和市场前景。
本发明旨在提供siRNA、siRNA缀合物及其药物组合物，其可影响RNA诱导的沉默复合体(RISC)介导的CFB基因的RNA转录物的切割，从而可以抑制肝脏中CFB基因的表达，实现疾病治疗的目的。
发明内容
本发明提供了一种能够抑制补体因子B(CFB)基因表达的siRNA，所述siRNA包含正义链与反义链，其中所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中所述正义链含有核苷酸序列I，反义链含有核苷酸序列II，所述核苷酸序 列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中所述核苷酸序列I和核苷酸序列II选自以下序列：
(1)所述核苷酸序列I包含SEQ ID NO:217所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:218所示的核苷酸序列：
5’-CAAGACCAAAA-3’(SEQ ID NO:217)
5’-UUUUGGUCUUG-3’(SEQ ID NO:218)；
(2)所述核苷酸序列I包含SEQ ID NO:219所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:220所示的核苷酸序列：
5’-CUACAAUGUGAGUGAU-3’(SEQ ID NO:219)
5’-AUCACUCACAUUGUAG-3’(SEQ ID NO:220)；
(3)所述核苷酸序列I包含SEQ ID NO:221所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:222所示的核苷酸序列：
5’-UCUCUUUCCACUGCUAU-3’(SEQ ID NO:221)
5’-AUAGCAGUGGAAAGAGA-3’(SEQ ID NO:222)；
(4)所述核苷酸序列I包含SEQ ID NO:223所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:224所示的核苷酸序列：
5’-CAGACAGCAGUA-3’(SEQ ID NO:223)
5’-UACUGCUGUCUG-3’(SEQ ID NO:224)；
(5)所述核苷酸序列I包含SEQ ID NO:225所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:226所示的核苷酸序列：
5’-AGCAGCUCAAU-3’(SEQ ID NO:225)
5’-AUUGAGCUGCU-3’(SEQ ID NO:226)，
其中所述核苷酸序列I不为SEQ ID NO:47，所述核苷酸序列II不为SEQ ID NO:48；
(6)所述核苷酸序列I包含SEQ ID NO:227所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:228所示的核苷酸序列：
5’-GUGUACAGCAUGAU-3’(SEQ ID NO:227)
5’-AUCAUGCUGUACAC-3’(SEQ ID NO:228)；
(7)所述核苷酸序列I包含SEQ ID NO:229所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:230所示的核苷酸序列：
5’-CAUCAUCCUCAUGA-3’(SEQ ID NO:229)
5’-UCAUGAGGAUGAUG-3’(SEQ ID NO:230)；
(8)所述核苷酸序列I包含SEQ ID NO:231所示的核苷酸序列，且所述核苷酸序列 II包含SEQ ID NO:232所示的核苷酸序列：
5’-UCAUGACUGAUGGA-3’(SEQ ID NO:231)
5’-UCCAUCAGUCAUGA-3’(SEQ ID NO:232)，
其中所述核苷酸序列I不为SEQ ID NO:85，所述核苷酸序列II不为SEQ ID NO:86；
(9)所述核苷酸序列I包含SEQ ID NO:233所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:234所示的核苷酸序列：
5’-ACCUGGAAGAUGUUU-3’(SEQ ID NO:233)
5’-AAACAUCUUCCAGGU-3’(SEQ ID NO:234)；
(10)所述核苷酸序列I包含SEQ ID NO:235所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:236所示的核苷酸序列：
5’-GAUGAAAGCCAGUCU-3’(SEQ ID NO:235)
5’-AGACUGGCUUUCAUC-3’(SEQ ID NO:236)，
其中所述核苷酸序列I不为SEQ ID NO:127，所述核苷酸序列II不为SEQ IDNO:128；
(11)所述核苷酸序列I包含SEQ ID NO:129所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:130所示的核苷酸序列：
5’-gsasugaaAfgCfCfAfgucucugasTsT-3’(SEQ ID NO:129)
5’-P1usCfsaGfaGfaCfuGfgCfuUfuCfaUfcsTsT-3’(SEQ ID NO:130)；
(12)所述核苷酸序列I包含SEQ ID NO:237所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:238所示的核苷酸序列：
5’-CAACAUUAAUGGGAAAAA-3’(SEQ ID NO:237)
5’-UUUUUCCCAUUAAUGUUG-3’(SEQ ID NO:238)；
(13)所述核苷酸序列I包含SEQ ID NO:239所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:240所示的核苷酸序列：
5’-AUUUUAUGACUAUGA-3’(SEQ ID NO:239)
5’-UCAUAGUCAUAAAAU-3’(SEQ ID NO:240)，
其中所述核苷酸序列I不为SEQ ID NO:145，所述核苷酸序列II不为SEQ ID NO:146；
(14)所述核苷酸序列I包含SEQ ID NO:241所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:242所示的核苷酸序列：
5’-AUGACGUUGCCCUGAU-3’(SEQ ID NO:241)
5’-AUCAGGGCAACGUCAU-3’(SEQ ID NO:242)；
(15)所述核苷酸序列I包含SEQ ID NO:243所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:244所示的核苷酸序列：
5’-CAAGAAUAAGCUGAAAUA-3’(SEQ ID NO:243)
5’-UAUUUCAGCUUAUUCUUG-3’(SEQ ID NO:244)；
(16)所述核苷酸序列I包含SEQ ID NO:245所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:246所示的核苷酸序列：
5’-CACUUGCCAGCAA-3’(SEQ ID NO:245)
5’-UUGCUGGCAAGUG-3’(SEQ ID NO:246)；
(17)所述核苷酸序列I包含SEQ ID NO:247所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:248所示的核苷酸序列：
5’-GCUAUGACAAAGUCAA-3’(SEQ ID NO:247)
5’-UUGACUUUGUCAUAGC-3’(SEQ ID NO:248)；
(18)所述核苷酸序列I包含SEQ ID NO:1所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:2所示的核苷酸序列；
(19)所述核苷酸序列I包含SEQ ID NO:3所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:4所示的核苷酸序列；
(20)所述核苷酸序列I包含SEQ ID NO:27所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:28所示的核苷酸序列；
(21)所述核苷酸序列I包含SEQ ID NO:29所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:30所示的核苷酸序列；
(22)所述核苷酸序列I包含SEQ ID NO:31所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:32所示的核苷酸序列；
(23)所述核苷酸序列I包含SEQ ID NO:51所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:52所示的核苷酸序列；
(24)所述核苷酸序列I包含SEQ ID NO:87所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:88所示的核苷酸序列；
(25)所述核苷酸序列I包含SEQ ID NO:93所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:94所示的核苷酸序列；
(26)所述核苷酸序列I包含SEQ ID NO:95所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:96所示的核苷酸序列；
(27)所述核苷酸序列I包含SEQ ID NO:97所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:98所示的核苷酸序列；
(28)所述核苷酸序列I包含SEQ ID NO:99所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:100所示的核苷酸序列；
(29)所述核苷酸序列I包含SEQ ID NO:103所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:104所示的核苷酸序列；
(30)所述核苷酸序列I包含SEQ ID NO:131所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:132所示的核苷酸序列；
(31)所述核苷酸序列I包含SEQ ID NO:133所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:134所示的核苷酸序列；
(32)所述核苷酸序列I包含SEQ ID NO:135所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:136所示的核苷酸序列；
(33)所述核苷酸序列I包含SEQ ID NO:137所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:138所示的核苷酸序列；
(34)所述核苷酸序列I包含SEQ ID NO:201所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:202所示的核苷酸序列；
(35)所述核苷酸序列I包含SEQ ID NO:207所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:208所示的核苷酸序列；
(36)所述核苷酸序列I包含SEQ ID NO:209所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:210所示的核苷酸序列；
(37)所述核苷酸序列I包含SEQ ID NO:211所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:212所示的核苷酸序列；
(38)所述核苷酸序列I包含SEQ ID NO:213所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:214所示的核苷酸序列；
(39)所述核苷酸序列I包含SEQ ID NO:215所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:216所示的核苷酸序列；
(40)所述核苷酸序列I包含SEQ ID NO:401所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:402所示的核苷酸序列：
5’-CAAGAUAUGGUCU-3’(SEQ ID NO:401)
5’-AGACCAUAUCUUG-3’(SEQ ID NO:402)；
(41)所述核苷酸序列I包含SEQ ID NO:403所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:404所示的核苷酸序列：
5’-AUGGUCUAGUGACAUA-3’(SEQ ID NO:403)
5’-UAUGUCACUAGACCAU-3’(SEQ ID NO:404)，
其中所述核苷酸序列I不为SEQ ID NO:285，所述核苷酸序列II不为SEQ ID NO:286；
(42)所述核苷酸序列I包含SEQ ID NO:405所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:406所示的核苷酸序列：
5’-CUACAACAUUAAUG-3’(SEQ ID NO:405)
5’-CAUUAAUGUUGUAG-3’(SEQ ID NO:406)；
(43)所述核苷酸序列I包含SEQ ID NO:407所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:408所示的核苷酸序列：
5’-GUGAAGCCAAGAUAU-3’(SEQ ID NO:407)
5’-AUAUCUUGGCUUCAC-3’(SEQ ID NO:408)；
(44)所述核苷酸序列I包含SEQ ID NO:354所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:355所示的核苷酸序列：
5’-CUAGUGACAUAUGCCACAU-3’(SEQ ID NO:354)
5’-AUGUGGCAUAUGUCACUAG-3’(SEQ ID NO:355)；
(45)所述核苷酸序列I包含SEQ ID NO:409所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:410所示的核苷酸序列：
5’-GGAAAAAAGAAGCAGGAA-3’(SEQ ID NO:409)
5’-UUCCUGCUUCUUUUUUCC-3’(SEQ ID NO:410)；
(46)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:392所示的核苷酸序列；
(47)所述核苷酸序列I包含SEQ ID NO:393所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:392所示的核苷酸序列；
(48)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:394所示的核苷酸序列；
(49)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:395所示的核苷酸序列；
(50)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:396所示的核苷酸序列；
(51)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:260所示的核苷酸序列；
(52)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:397所示的核苷酸序列；
(53)所述核苷酸序列I包含SEQ ID NO:398所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:397所示的核苷酸序列；
(54)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:399所示的核苷酸序列；
(55)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:400所示的核苷酸序列；
(56)所述核苷酸序列I包含SEQ ID NO:250所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:130所示的核苷酸序列。
在一个实施方案中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
在一个实施方案中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为0-9个核苷酸，其中所述核苷酸序列III连接在核苷酸序列I的5'末端，核苷酸序列IV连接在核苷酸序列II的3'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配；和/或，
所述核苷酸序列III连接在核苷酸序列I的3'末端，核苷酸序列IV连接在核苷酸序列II的5'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
在一个实施方案中，所述正义链还含有核苷酸序列V和/或所述反义链还含有核苷酸序列VI，核苷酸序列V和VI的长度为0至3个核苷酸，核苷酸序列V连接在所述正义链的3'末端构成正义链的3'突出端，和/或核苷酸序列VI连接在所述反义链的3'末端构成反义链的3'突出端。在一个优选的实施方式中，所述核苷酸序列V或VI的长度为2个核苷酸。在一个优选的实施方案中，所述核苷酸序列V或VI为连续的两个胸腺嘧啶脱氧核糖核苷酸或连续的两个尿嘧啶核糖核苷酸。在一个优选的实施方案中，所述核苷酸序列V或VI与靶mRNA相应位置的核苷酸错配或互补。
在一个实施方案中，所述双链区的长度是15-30个核苷酸对；优选地，双链区的长度是17-23个核苷酸对；更优选地，双链区的长度是19-21个核苷酸对。
在另一个实施方案中，所述正义链或反义链具有15-30个核苷酸；优选地，正义 链或反义链具有19-25个核苷酸；更优选地，正义链或反义链具有19-23个核苷酸。
在一个实施方案中，所述正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；优选地，所述含有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。
在一个实施方案中，所述siRNA包括不包含3’突出端核苷酸的正义链。
在一个实施方案中，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。
在一个实施方案中，所述修饰的核苷酸选自2’-氟代修饰的核苷酸，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’-脱氧核苷酸，2’-氨基修饰的核苷酸，2’-取代的氨基修饰的核苷酸，核苷酸类似物或其中任意两种以上的组合。
在一个实施方案中，所述修饰的核苷酸选自2’-氟代修饰的核苷酸，2’-甲氧基修饰的核苷酸，2’-O-CH2-CH2-O-CH3修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸，2’-CH2-CH2-CH＝CH2修饰的核苷酸，2’-脱氧核苷酸，核苷酸类似物或其中任意两种以上的组合。
在一个实施方案中，所述正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸或非氟代修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为非氟代修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为非氟代修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为非氟代修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，其余位置为非氟代修饰的核苷酸。在一个实施方案中，每一个非氟代修饰的核苷酸均为2’-甲氧基修饰的核苷酸，所述2’-甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在一个实施方案中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2’位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种，核苷酸类似物选自异核苷酸、LNA、ENA、cET BNA、UNA和GNA中的一种。
在一个实施方案中，所述正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸，2’-甲氧基修饰的核苷酸，GNA修饰的核苷酸或其中任意两种以上的组合。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，GNA修饰的核苷酸位于反义链的第7位，其余位置为2’-甲氧基修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，GNA修饰的核苷酸位于反义链的第6位，其余位置为2’-甲氧基修饰的核苷酸。
在一个更优选的实施方案中，所述siRNA中以下核苷酸之间的连接中至少一个为硫代磷酸酯基连接：
所述正义链的5’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的5’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
所述正义链的3’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的3’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的5’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的5’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的3’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的3’末端起始的第2个核苷酸和第3个核苷酸之间的连接。
在一些实施方案中，所述siRNA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酯基：
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；
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；
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；
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；
或者，
所述正义链包含位于如下所示位置处的硫代磷酸酯基：
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；
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。
在一些实施方案中，所述siRNA沿5’末端向3’末端方向，反义链包含位于如下所示位置处的硫代磷酸酯基：
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；
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；
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；
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。
在一个实施方案中，所述正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸，2’-甲氧基修饰的核苷酸，GNA修饰的核苷酸或其中任意两种以上的组合。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接，3’末端除去突出端；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2 个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接，3’末端除去突出端；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接，3’末端除去突出端；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基 修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，GNA修饰的核苷酸位于反义链的第7位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，GNA修饰的核苷酸位于反义链的第6位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。
在一个具体实施方案中，本发明提供了选自表1的siRNA，其中所述siRNA不为N-ER-FY010019、N-ER-FY010015、N-ER-FY010051、N-ER-FY010021、N-ER-FY010013、N-ER-FY010035、N-ER-FY010009、N-ER-FY-010099；优选地，所述siRNA选自N-ER-FY010016、N-ER-FY010016M2、N-ER-FY010016M2D2、N-ER-FY010016M3、N-ER-FY010016M3D2、N-ER-FY010016M4、N-ER-FY010016M5、N-ER-FY010034、N-ER-FY010034M3、N-ER-FY010034M3D2、N-ER-FY010034M2、N-ER-FY010034M2D2、N-ER-FY010034M4、N-ER-FY010034M5、N-ER-FY010003、N-ER-FY010003M2、N-ER-FY010003M3、N-ER-FY010076、N-ER-FY010076M1、N-ER-FY010076MD2、N-ER-FY010076M2、N-ER-FY010076M3、N-ER-FY010037、N-ER-FY010037M2、N-ER-FY010037M3、N-ER-FY010052、N-ER-FY010052M2、N-ER-FY010052M3、N-ER-FY010114、N-ER-FY010114M2、N-ER-FY010114M3、N-ER-FY010115、N-ER-FY010115M2、N-ER-FY010115M3、N-ER-FY010121、N-ER-FY010121M2、N-ER-FY010121M3。
本发明还提供了一种siRNA缀合物，所述siRNA缀合物含有本发明的siRNA以及缀合至该siRNA的缀合基团(如下图所示，双螺旋结构表示所述siRNA，并且所述缀合基团连接至所述siRNA的正义链3’末端)：
上述缀合物结构中X可选择为O或S，在一个实施方案中，X为O。
在一个实施方案中，所述缀合基团包含药学上可接受的靶向基团和接头，并且所述siRNA、所述接头和所述靶向基团依次共价或非共价连接。
优选地，在所述siRNA缀合物中，siRNA的正义链与反义链互补形成所述siRNA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1-3个延伸出所述双链区的突出的核苷酸；
或者，
在所述siRNA缀合物中，siRNA的正义链与反义链互补形成所述siRNA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。
在一个实施方案中，所述缀合基团为下式的L96：
在一个具体实施方案中，所述siRNA缀合物为选自表2的siRNA缀合物。
本发明还提供了一种药物组合物，其包含本发明的siRNA，或本发明的siRNA缀合物，以及药学上可接受的载体。
本发明还提供了试剂盒，其包含本发明的siRNA，或本发明的siRNA缀合物，或本发明的药物组合物。
本发明还提供了本发明的siRNA，或本发明的siRNA缀合物，或本发明的药物组 合物用于制备抑制CFB基因表达的药剂的用途。
本发明还提供了本发明的siRNA，或本发明的siRNA缀合物，或本发明的药物组合物用于制备预防和/或治疗CFB基因过表达相关的疾病的药剂的用途。
在一个实施方案中，所述疾病选自原发性膜性肾病、溶血性尿毒症综合征、C3肾小球病、IgA肾病、阵发性夜间血红蛋白尿(PNH)、年龄相关性黄斑病变和眼科疾病。
本发明还提供了抑制CFB基因表达的方法，包括将治疗有效量的本发明的siRNA，或本发明的siRNA缀合物，或本发明的药物组合物与表达CFB的细胞接触或给予有需要的受试者。
本发明还提供了治疗和/或预防CFB基因过表达相关的疾病的方法，包括将治疗有效量的本发明的siRNA，或本发明的siRNA缀合物，或本发明的药物组合物给予有需要的受试者。
有益效果
本申请提供的siRNA、药物组合物和siRNA缀合物在体外细胞实验中显示出优异的CFB基因表达抑制活性，具有良好的治疗CFB基因过表达相关的疾病的潜力。例如，本申请公开的siRNA及其缀合物能够降低肝脏中CFB mRNA的表达，毒副作用低，血浆稳定性好，具有良好的临床应用前景。
本申请提供的siRNA在人肝癌细胞Hep3B中显示出对CFB基因良好的抑制效果。在一些具体实施方式中，本申请提供的siRNA在0.1nM的浓度下抑制率高达92.41％；在1nM的浓度下抑制率高达98.78％。
在一些具体实施方式中，本申请提供的siRNA在Hep3B细胞中有较高的CFB基因抑制活性，例如，IC50低至0.030nM。
在一些具体实施方式中，本申请提供的siRNA缀合物在PHH细胞中具有较高的CFB基因抑制活性，例如，当通过自由摄取进入PHH细胞时，IC50可低至0.282nM；当通过转染进入PHH细胞时，IC50可低至0.005nM。
具体实施方式
定义
在说明书通篇中，如无特别说明，在本技术领域中，“G”、“C”、“A”、“T”和“U”通常分别代表鸟嘌呤、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。小写字母a、u、c、g：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；(下划线+粗体+斜体)：表示GNA修饰的核苷酸。
在上文及下文中，所述“2’-氟代修饰的核苷酸”指核苷酸的核糖基2’位的羟基被氟取代形成的核苷酸。“非氟代修饰的核苷酸”指核苷酸的核糖基2’位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2’位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。这些核糖基2’位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自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’-氨基修饰的核苷酸、2’-取代的氨基修饰的核苷酸、2’-脱氧核苷酸中的一种。
“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。
“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。
“核苷酸类似物”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基团。如异核苷酸、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 BNA等，其中，LNA如式(1)所示，ENA如式(2)所示，cET BNA如式(3)所示。
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。
上述式(4)和式(5)中，R选自H、OH或烷氧基(O-烷基)。
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，碱基从核糖环的1’-位移动至2’-位或3’-位而形成的化合物，如式(6)或(7)所示。
上述式(6)-式(7)化合物中，Base表示碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET BNA、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸为为2’-甲氧基修饰的核苷酸、GNA修饰的核苷酸或其中任意两种以上的组合。在一些优选实施方式中，每一个非氟代修饰的核苷酸均为2’-甲氧基修饰的核苷酸，在上文和下文中，所述2’-甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
所述“2’-甲氧基修饰的核苷酸”指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。所述“硫代磷酸酯基”指磷酸酯基中的磷酸二酯键中的一个氧原子被硫原子取代而成的硫代磷酸酯基。所述“5’-磷酸核苷酸”指下式的结构：
在本说明书的上下文中，表述“互补”和“反向互补”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，一条链的碱基各自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。
在上文及下文中，如无特别说明，“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；“实质上反向互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两段核苷酸序列之间不存在碱基错配。
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
在上下文中，“突出端”是指当siRNA的一条链的一个3’末端延伸超出另一条链的5’末端时从该siRNA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siRNA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”siRNA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siRNA。
在本申请说明书上文及下文中，特别是在描述本申请的siRNA、药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，固相亚磷酰胺合成中使用的修饰或未修饰的核苷亚磷酰胺单体。固相亚磷酰胺合成为本领域技术人员所公知的RNA合成中所用的方法。本申请所用的核苷单体均可商购得到。
在本申请的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。siRNA缀合物应根 据上下文，理解为多个siRNA缀合物的总称或者某个化学式所示的siRNA缀合物。在本申请说明书的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本申请的siRNA缀合物的特定化合物。
在本申请中可以使用各种羟基保护基团。一般来说，保护基团使化学官能团对特定的反应条件不敏感，并且可以在分子中的该官能团上附加以及去除，而不实质上损害分子的其余部分。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本申请可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基黄嘌呤-9-基(Pixyl)和9-(对甲氧基苯基)黄嘌呤-9-基(Mox)。在一些实施方式中，本申请可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4’-二甲氧基三苯甲基)和TMTr(4,4’,4”-三甲氧基三苯甲基)。
如本说明书所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且所述描述包括事件或状况发生的情况和其中不发生的情况。
“受试者”一词，如本说明书所使用的，指任何动物，例如哺乳动物或有袋动物。本申请的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠、兔或任何种类的家禽。
如本说明书所使用的，“治疗”是指获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本说明书所使用的，“预防”是指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将siRNA、siRNA缀合物或药物组合物给予有罹患特定疾病风险的受试者，或给予报告疾病的一种或多种生理症状的受试者，即便可能该疾病的诊断尚未做出。
siRNA
本申请涉及一种能够抑制CFB基因表达的siRNA。本申请的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基。通常具有活性的，即功能性的siRNA的长度约为12-40个核苷酸，在一些实施方式中约为15-30个核苷酸。
本申请的siRNA含有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成 双链区。在一些实施方式中，该双链区的长度是15-30个核苷酸对。在另一些实施方式中，双链区的长度是17-23个核苷酸对。在另一些实施方式中，双链区的长度是19-21个核苷酸对。在又另一些实施方式中，双链区的长度是19或21个核苷酸对。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为0-9个核苷酸，所述核苷酸序列III连接在核苷酸序列I的5'末端，核苷酸序列IV连接在核苷酸序列II的3'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为0-9个核苷酸，所述核苷酸序列III连接在核苷酸序列I的3'末端，核苷酸序列IV连接在核苷酸序列II的5'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为0-9个核苷酸，所述核苷酸序列III连接在核苷酸序列I的5'末端，核苷酸序列IV连接在核苷酸序列II的3'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；和所述核苷酸序列III连接在核苷酸序列I的3'末端，核苷酸序列IV连接在核苷酸序列II的5'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，所述正义链还含有核苷酸序列V和/或所述反义链还含有核苷酸序列VI，核苷酸序列V和VI的长度为0至3个核苷酸，核苷酸序列V连接在所述正义链的3'末端构成正义链的3'突出端和/或核苷酸序列VI连接在所述反义链的3'末端构成反义链的3'突出端。在一些实施方式中，所述核苷酸序列V或VI的长度为2个核苷酸。在另一些实施方案中，所述核苷酸序列V或VI为连续的两个胸腺嘧啶脱氧核糖核苷酸或连续的两个尿嘧啶核糖核苷酸。在另一些实施方案中，所述核苷酸序列V或VI与靶mRNA相应位置的核苷酸错配或互补。
本申请提供的正义链和反义链的长度相同或不同，在一些实施方式中，正义链或反义链具有15-30个核苷酸。在另一些实施方式中，正义链或反义链具有19-25个核苷酸。在另一些实施方式中，正义链或反义链具有19-23个核苷酸。本申请提供的siRNA 正义链和反义链的长度比可以是15/15、16/16、17/17、18/18、19/19、19/20、19/21、19/22、19/23、20/19、20/20、20/21、20/22、20/23、21/19、21/20、21/21、21/22、21/23、22/19、22/20、22/21、22/22、22/23、23/19、23/20、23/21、23/22、23/23、24/24、25/25、26/26、27/27、28/28、29/29、30/30、22/24、22/25、22/26、23/24、23/25或23/26等等。在一些实施方式中，所述siRNA正义链和反义链的长度比为19/19、21/21、19/21、21/23或23/23，此时，本公开的siRNA具有更好的细胞mRNA沉默活性。
研究发现，不同修饰策略会对siRNA的稳定性、生物活性及细胞毒性等指标产生截然不同的影响。例如，CN102140458B中对siRNA的多种化学修饰策略进行了研究，证实了7种有效的修饰方式，与未经修饰的siRNA相比，其中一种修饰方式所得的siRNA在提高血液稳定性的同时，还保持了与未经修饰的siRNA基本相当的抑制活性。
本发明的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方式中，本发明的siRNA中的每个核苷酸均为未经修饰的核苷酸；在一些实施方式中，本发明的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本发明的siRNA抑制CFB基因表达的功能明显削弱或丧失。
在一些实施方式中，本申请的siRNA至少含有1个修饰的核苷酸。在本申请的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2'位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者具有经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致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。
在一些实施方式中，本发明提供的siRNA的所述正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。在一些实施方式中，所述含有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。
在一些实施方式中，所述siRNA包括不包含3’突出端核苷酸的正义链；即siRNA的正义链可以存在3’突出端核苷酸，将正义链的3’突出端核苷酸排除后形成平末端。
在一些实施方式中，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’末端添加核苷酸序列V，作为突出的核苷酸。然后，当核苷酸序列V连接正义链的3'末端形成的核苷酸序列在完成化学修饰后，排除核苷酸序列V，相应地，siRNA的正义链形成平末端。
在一些实施方式中，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’末端具有延伸出双链区的突出的核苷酸时，将正义链中位于3’末端的突出的核苷酸排除后作为正义链的核苷酸序列，相应地，siRNA的正义链形成平末端。
在一些实施方式中，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。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。
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：
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’-氨基修饰的核苷酸，2’-取代的氨基修饰的核苷酸，2’-脱氧核苷酸。
示例性的修饰的核苷酸具有如下所示结构：
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。这些核糖基团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’-氨基修饰的核苷酸，2’-取代的氨基修饰的核苷酸，2’-脱氧核苷酸。
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。
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。
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。
在一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方式中，本发明提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸或非氟代修饰的核苷酸。在一些实施方式中，每一个非氟代修饰的核苷酸为2’-甲氧基修饰的核苷酸或GNA修饰的核苷酸，所述2’-甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
在一些实施方式中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为非氟代修饰的核苷酸。在一些实施方式中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为非氟代修饰的核苷酸。在一些实施方式中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为非氟代修饰的核苷酸。在一个优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，其余位置为非氟代修饰的核苷酸。在一些优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸。在一些优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸。在一些优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸。在一些优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，GNA修饰的核苷酸位于反义链的第6位，其余位置为2’-甲氧基修 饰的核苷酸。在一些优选的实施方案中，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，GNA修饰的核苷酸位于反义链的第7位，其余位置为2’-甲氧基修饰的核苷酸。在一些更优选的实施方案中，每一个非氟代修饰的核苷酸均为2’-甲氧基修饰的核苷酸，所述2’-甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
在一些实施方式中，所述siRNA中以下核苷酸之间的连接中至少一个为硫代磷酸酯基连接：
所述正义链的5’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的5’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
所述正义链的3’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的3’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的5’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的5’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的3’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的3’末端起始的第2个核苷酸和第3个核苷酸之间的连接。
在一些实施方式中，所述siRNA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酯基：
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；
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；
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；
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；
或者，
所述正义链包含位于如下所示位置处的硫代磷酸酯基：
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；
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。
在一些实施方式中，所述siRNA沿5’末端向3’末端方向，反义链包含位于如下所示位置处的硫代磷酸酯基：
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；
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；
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；
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。
siRNA缀合物
本申请涉及一种siRNA缀合物，所述siRNA缀合物含有上述siRNA以及缀合连接至该siRNA的缀合基团。
在本申请中，siRNA缀合物的正义链与反义链形成siRNA缀合物的双链区，并且，在siRNA缀合物的正义链的3’末端形成平末端。在一些实施方案中，siRNA缀合物的正义链的3’末端形成平末端，siRNA缀合物的反义链的3’末端具有1-3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，siRNA缀合物的正义链的3’末端形成平末端，siRNA缀合物的反义链的3’末端形成平末端。
在一些优选地实施方案中，siRNA缀合物由siRNA与缀合基团缀合连接得到。其中，siRNA的正义链与反义链互补形成siRNA的双链区，并且，siRNA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siRNA缀合物。
在一些优选的实施方案中，siRNA的正义链的3’末端具有延伸出双链区的突出的核苷酸，将位于正义链中3’末端的突出的核苷酸排除后形成的具有3’平末端的序列作为用于连接缀合基团的核苷酸序列，在正义链的3’平末端连接缀合基团，形成siRNA缀合物。
在一些更优选实施方式中，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’末端添加核苷酸序列V，作为突出的核苷酸。将位于正义链中3’末端的突出的核苷酸排除后形成的具有3’平末端的序列作为用于连接缀合基团的核苷酸序列，在正义链的3’平末端连接缀合基团，形成siRNA缀合物。
在一些更优选实施方式中，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’末端具有延伸出双链区的突出的核苷酸时，将位于正义链中3’末端的突出的核苷酸排除后形成的具有3’平末端的序列作为用于连接缀合基团的核苷酸序列，在正义链的3’平末端连接缀合基团，形成siRNA缀合物。
示例性地，序列如N-ER-FY010078M1所示的siRNA，该siRNA的正义链的3’末端具有延伸出双链区的突出的核苷酸，将位于正义链中3’末端的突出的-sgsu核苷酸去掉后形成的gsasugaaAfgCfCfAfgucucuga平末端序列作为用于连接L96缀合基团的核苷酸序列，因此，形成siRNA缀合物的序列为：正义链为gsasugaaAfgCfCfAfgucucugaL96，反义链为P1usCfsaGfaGfaCfuGfgCfuUfuCfaUfcsGfsa。
一般来说，所述缀合基团包含药学上可接受的至少一个靶向基团，或者进一步还包含接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一些 实施方式中，所述靶向基团为1-6个。在一些实施方式中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。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'末端，还可以在siRNA的内部序列中。在一些实施方式中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3'末端。
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.
在一些实施方式中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。
在一些实施方式中，所述药学上可接受的靶向基团可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式全文纳入本说明书。
在一些实施方式中，所述药学上可接受的靶向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。
在一些实施方式中，所述的每个配体独立地选自一个能够与细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与哺乳动物细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与人肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与肝表面去唾液酸糖蛋白受体(ASGPR)结合的配体。这些配体 的种类为本领域技术人员所公知，其作用一般是与靶细胞表面的特异性受体相结合，介导与配体连接的siRNA递送至靶细胞。
在一些实施方式中，所述药学上可接受的靶向基团可以是与哺乳动物肝细胞表面上的去唾液酸糖蛋白受体(ASGPR)结合的任意一种配体。在一些实施方式中，每个配体独立地为去唾液酸糖蛋白，例如去唾液酸血清类粘蛋白(asialoorosomucoid，ASOR)或去唾液酸胎球蛋白(asialofetuin，ASF)。在一些实施方式中，所述配体为糖或糖的衍生物。
在一些实施方式中，至少一个配体是糖。在一些实施方式中，每个配体均是糖。在一些实施方式中，至少一个配体是单糖、多糖、修饰的单糖、修饰的多糖或糖衍生物。在一些实施方式中，至少一个所述配体可以是单糖，双糖或三糖。在一些实施方式中，至少有一个配体是修饰的糖。在一些实施方式中，每一个配体均为修饰的糖。在一些实施方式中，每个配体均独立地选自多糖、修饰的多糖、单糖、修饰的单糖、多糖衍生物或单糖衍生物。在一些实施方式中，每一个或至少一个配体选自于由以下糖所组成的组：葡萄糖及其衍生物、甘露聚糖及其衍生物、半乳糖及其衍生物、木糖及其衍生物、核糖及其衍生物、岩藻糖及其衍生物、乳糖及其衍生物、麦芽糖及其衍生物，阿拉伯糖及其衍生物、果糖及其衍生物和唾液酸。
在一些实施方式中，每个所述配体可独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳糖胺、N-异丁酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖或L-4-硫代核糖。所述配体的其它选择可参见例如CN105378082A的记载，以引用的方式全文纳入本说明书。
在一些实施方式中，所述siRNA缀合物中药学上可接受的靶向基团可以是半乳糖或N-乙酰半乳糖胺，其中，半乳糖或N-乙酰半乳糖胺分子可以是一价、二价、三价、四价。应当理解的是，这里所述的一价、二价、三价、四价分别指siRNA分子与含有 作为靶向基团的半乳糖或N-乙酰半乳糖胺分子的缀合基团形成siRNA缀合物后，该siRNA缀合物中siRNA分子与半乳糖或N-乙酰半乳糖胺分子的摩尔比为1:1、1:2、1:3或1:4。在一些实施方式中，所述药学上可接受的靶向基团是N-乙酰半乳糖胺。在一些实施方式中，当本申请所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价或四价。在一些实施方式中，当本申请所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价。
靶向基团可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些接头、靶向基团的种类以及与siRNA的连接方式，可参见WO2015006740A2的公开内容，以引用的方式全文纳入本说明书。
siRNA的合成方法
通过本领域常规的固相亚磷酰胺法，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，包括保护、偶联、盖帽、硫化四步反应。
例如，本申请的siRNA的合成条件可以如下：
脱保护的条件包括：反应温度为25℃，反应时间为70秒，脱保护试剂选自二氯乙酸的二氯甲烷溶液(3％V/V)，脱保护试剂与固相载体上的保护基的摩尔比为5:1。
偶联反应条件包括：反应温度为25℃，反应时间为600秒，偶联试剂选自5-乙硫基-1H-四氮唑(ETT)的0.25M乙腈溶液，固相载体上连接的核酸序列与核苷单体的摩尔比为1:10。
盖帽反应条件包括：反应温度为25℃，反应时间为15秒，盖帽试剂选自摩尔比为1:1的CapA(10％乙酸酐乙腈溶液)和CapB(10％的N-甲基咪唑吡啶/乙腈溶液)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的摩尔比为1:1:1。
氧化反应条件包括：反应温度为25℃，反应时间为15秒，氧化试剂选自0.05M碘四氢呋喃溶液，氧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30:1。
硫化反应条件包括：反应温度为25℃，反应时间为300秒，硫化试剂选自氢化黄原素，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。
在将所有核苷单体连接之后，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，得到siRNA正义链和反义链，最后将两条链进行加热退火得到产品。
切割、脱保护、纯化、脱盐和退火的方法是本领域中公知的。例如，通过将连接 有固相载体的核苷酸序列与浓氨水接触进行切割和脱保护；通过色谱法进行纯化；通过反相色谱进行脱盐；通过在不同严格条件下等摩尔比混合正义链和反义链后逐渐降温冷却。
siRNA缀合物合成方法
第一步，通过将DMTr-L96和丁二酸酐反应，得到化合物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。
第二步，将L96-A与NH2-SPS反应得到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(NH2-SPS，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。
药物组合物
本申请提供了一种药物组合物，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。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于 脂质纳米颗粒(Lipid Nanoparticle，LNP)、磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，可以是各组分常规的含量。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏、或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。在一些实施方式中，所述药物组合物用于静脉注射给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。
以下实施例用于进一步说明本发明，但不对本发明进行任何限制。
实施例
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。
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。
实施例1 siRNA的制备
由天霖生物科技(上海)有限公司合成了具有以下序列的siRNA分子。
表1 siRNA及其序列








其中，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；小写字母a、u、c、g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；(下划线+粗体+斜体)：表示GNA修饰的核苷酸。
由天霖生物科技(上海)有限公司合成了具有以下序列的siRNA缀合物：
表2 siRNA缀合物及其序列：


其中，L96为：
实施例2 siRNA抑制CFB基因表达
Hep3B细胞，购自ATCC，货号HB-8064；
Lipofectaine RNAiMax转染试剂，购自Invitrogen，货号13778-150；
Opti-medium：减血清培养基，购自Gibco，货号31985-070；
EMEM培养基：ATCC，货号30-2003；
超纯蒸馏水(DNAse,RNAse,Free)：购自Invitrogen，货号10977-015；
TaqManTMFast Advanced Cells-to-CTTMKit(with gDNase)，购自Invitrogen，货号A35377；
Phosphate Buffer Saline(PBS)，购自Gibco，货号10010-023；
Human ACTB探针引物购自Invitrogen 4448491,Hs01060665_g1；
CFB TaqMan探针引物购自Invitrogen 4351370,Hs00156060_m1。
实验步骤：
1.将人肝癌细胞Hep3B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。
2.将待测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 siRNA母液。
3.配制0.1nM的siRNA转染稀释液
(1)10μM siRNA贮备液配制：取100μM上述siRNA母液2μl，加入18μl超纯蒸馏水，得到浓度为10μM siRNA贮备液；
(2)0.1nM siRNA转染稀释液配制：取10μM siRNA贮备液2μl，加入18μl超纯蒸馏水，得到浓度为1μM siRNA稀释液；取1μM siRNA稀释液2μl，加入18μl超纯蒸馏水，得到浓度为0.1μM siRNA稀释液；再取0.1μM siRNA稀释液2μl，加入98μl超纯蒸馏水，得到浓度为2nM siRNA稀释液；再加入等体积的Lipofectaine RNAiMax转染试剂稀释液(将Lipofectaine RNAiMax转染试剂3μl，加入97μl Opti-medium，得到Lipofectaine RNAiMax转染试剂稀释液)得到1nM siRNA稀释液；取上述1nM siRNA稀释液10μl加入96孔板中所转染培养的Hep3B细胞，得siRNA终浓度为0.1nM。
4.转染后培养细胞48小时，设置三个复孔。
5.利用TaqManTMFast Advanced Cells-to-CTTMKit(with gDNase)试剂盒提取RNA：
a)吸掉每孔中的旧培养基，加入100μl PBS缓冲液洗涤两次；
b)吸掉PBS缓冲液，每孔中加入50μl裂解液；
c)室温下以500rpm震荡孵育5分钟；
d)每孔加入5μl终止液，室温下以500rpm震荡2分钟。
6.对RNA进行逆转录至cDNA，按照以下步骤进行：
a)如下表所述制备逆转录反应混合物并充分混匀，在整个操作过程中，所有试剂都放置在冰上：
b)如下所述进行逆转录程序
c)将逆转录产物储存在-20℃以进行实时PCR分析。
7.进行实时PCR分析
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：
b)如下所述进行qPCR程序
8.结果分析
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；
b)使用以下公式计算基因的相对表达量：
ΔCt＝Ct(CFB基因)–Ct(ACTB)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)，其中Mock组表示和检测样品组相比，未加入siRNA的组；
相对于Mock组的mRNA表达＝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％ 表3本发明的siRNA的抑制率

从表3可以看出，本发明的siRNA在0.1nM下均可显著抑制CFB基因的表达。
实施例3 siRNA抑制CFB基因表达的IC50测定
按照与实施例2相似的方法，进行siRNA抑制CFB基因表达的IC50测定，其中用于转染的siRNA的浓度分别为5nM、2nM、1nM、0.5nM、0.2nM、0.1nM、0.05nM、0.01nM、0.005nM。
结果分析
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；
b)使用以下公式计算基因的相对表达量：
ΔCt＝Ct(CPB2基因)–Ct(ACTB)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)
相对于Mock组的mRNA表达＝2-ΔΔCt。
Mock组表示：和检测样品组相比，未加入siRNA的组。
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％
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”功能模块，来拟合量效曲线，从而得出各个siRNA的IC50值。
拟合公式为：Y＝Bottom+(Top–Bottom)/(1+10^((LogIC50–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。
实验结果如表4所示。
表4 siRNA的IC50(nM)
可见，本发明的siRNA具有较低的IC50，在Hep3B细胞中有较高的CFB基因抑制活性。
实施例4 siRNA缀合物抑制CFB基因表达的IC50测定
材料：
人原代肝细胞PHH细胞，由药明康德提供；
PHH培养基：invitroGRO CP Meduim serum free BIOVIT，货号：S03316
Lipofectamine RNAiMax转染试剂，购自Invitrogen，货号：13778-150；
RNA提取试剂盒96 Kit，购自QIAGEN，货号：QIAGEN-74182；
逆转录试剂盒FastQuant RT Kit(With gDNase)，购自TianGen，货号：KR116-02；
FastStart Universal Probe master，购自Roche，货号：04914058001；
TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1)；
TaqMan Gene Expression Assay(CFB Thermo,Assay ID-Hs00156060_m1)。
siRNA缀合物(siRNA缀合物终浓度分别为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Lipofectamine 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，每孔加入PPH培养基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RNA提取。根据说明书使用96Kit提取总RNA。
siRNA缀合物(siRNA缀合物终浓度分别为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RNA提取。根据说明书使用96 Kit提取总RNA。
采用与实施例2中相似的方法，通过逆转录反应将提取的总RNA逆转录为cDNA。靶标cDNA将通过qPCR进行检测。GAPDH cDNA将作为内部对照进行平行检测。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。
结果分析：
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；
b)使用以下公式计算基因的相对表达量：
ΔCt＝Ct(CFB基因)–Ct(GAPDH)
ΔΔCt＝ΔCt(检测样品组)–ΔCt(Mock组)，其中Mock组表示和检测样品组相比，未加入siRNA的组；
相对于Mock组的mRNA表达＝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％。
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”功能模块，来拟合量效曲线，从而得出各个siRNA缀合物的IC50值。
拟合公式为：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-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。
实验结果如表5所示。
表5 siRNA缀合物抑制CFB基因表达的IC50值
可见，本发明的siRNA缀合物在PHH细胞中具有较高的CFB基因抑制活性。
实施例5 siRNA抑制CFB基因表达
Hep3B细胞，购自ATCC，货号HB-8064；
Lipofectamine RNAiMax转染试剂，购自Invitrogen，货号13778-150；
Opti-medium：减血清培养基，购自Gibco，货号31985-070；
EMEM培养基：ATCC，货号30-2003；
超纯蒸馏水(DNAse,RNAse,Free)：购自Invitrogen，货号10977-015；
TaqManTMFast Advanced Cells-to-CTTMKit(with gDNase)，购自Invitrogen，货号A35377；
Phosphate Buffer Saline(PBS)，购自Gibco，货号10010-023；
Human ACTB探针引物购自Invitrogen 4448491,Hs01060665_g1；
CFB TaqMan探针引物购自Invitrogen 4351370,Hs00156060_m1。
1.将人肝癌细胞Hep3B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。
2.将待测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 siRNA母液。
3.配制0.1nM的siRNA转染稀释液和1nM的siRNA转染稀释液
(1)10μM siRNA贮备液配制：取100μM上述siRNA母液2μl，加入18μl超纯蒸馏水，得到浓度为10μM siRNA贮备液；
(2)0.1nM siRNA转染稀释液配制：
取10μM siRNA贮备液2μl，加入18μl超纯蒸馏水，得到浓度为1μM siRNA稀释液；取1μM siRNA稀释液2μl，加入18μl超纯蒸馏水，得到浓度为0.1μM siRNA稀释液；再 取0.1μM siRNA稀释液2μl，加入98μl超纯蒸馏水，得到浓度为2nM siRNA稀释液；再加入等体积的Lipofectaine RNAiMax转染试剂稀释液(将Lipofectaine RNAiMax转染试剂3ul，加入97ul Opti-medium，得到Lipofectamine RNAiMax转染试剂稀释液)得到1nM siRNA稀释液；取上述1nM siRNA稀释液10μl加入96孔板中所转染培养的Hep3B细胞，得siRNA终浓度为0.1nM。
1nM的siRNA转染稀释液可按照上述类似操作配制。
4.转染后培养细胞48小时，设置三个复孔。
5.与实施例2中的“5”的实验步骤和方法相同。
6.与实施例2中的“6”的实验步骤和方法相同。
7.进行实时PCR分析
与实施例2中的“7”的实验步骤和方法相同。
8.结果分析
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；
b)使用以下公式计算基因的相对表达量：
ΔCt＝Ct(CFB基因)–Ct(ACTB)
ΔΔCt＝ΔCt(检测样品组)-ΔCt(Mock组)，其中Mock组表示和检测样品组相比，未加入siRNA的组；
相对于Mock组的mRNA表达＝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％
表6本发明的siRNA的抑制率



从表6可以看出，本发明的siRNA在0.1nM和1nM下均可显著抑制CFB基因的表达。
实施例6 siRNA缀合物抑制CFB基因表达的抑制率
材料：
人原代肝细胞PHH细胞，由药明康德提供；
PHH培养基：invitroGRO CP Meduim serum free BIOVIT，货号：S03316；
Lipofectamine RNAiMax转染试剂，购自Invitrogen，货号：13778-150；
RNA提取试剂盒96 Kit，购自QIAGEN，货号：QIAGEN-74182；
逆转录试剂盒FastQuant RT Kit(With gDNase)，购自TianGen，货号：KR116-02；
FastStart Universal Probe master，购自Roche，货号：04914058001；
TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1)；
TaqMan Gene Expression Assay(CFB Thermo,Assay ID-Hs00156060_m1)。
siRNA缀合物(siRNA缀合物终浓度分别为5nM和0.1nM，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Lipofectamine 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，每孔加入PPH培养基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RNA提取。根据说明书使用96 Kit提取总RNA。
siRNA缀合物(siRNA缀合物终浓度分别为10nM和1nM，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RNA提取。根据说明书使用96 Kit提取总RNA。
采用与实施例2中相似的方法，通过逆转录反应将提取的总RNA逆转录为cDNA。靶标cDNA将通过qPCR进行检测。GAPDH cDNA将作为内部对照进行平行检测。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。
结果分析：
a)使用Quant Studio 7软件采用默认设置，自动计算Ct值；
b)使用以下公式计算基因的相对表达量：
ΔCt＝Ct(CFB基因)–Ct(GAPDH)
ΔΔCt＝ΔCt(检测样品组)–ΔCt(Mock组)，其中Mock组表示和检测样品组相比，未加入siRNA的组；
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％
表7 siRNA缀合物抑制CFB基因表达的抑制率

实施例7 siRNA缀合物在人源化小鼠中对人CFB(hCFB)基因表达的抑制作用
6-8周龄的C57BL/6-hCFB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3mg/kg的单一剂量对小鼠分别皮下给药N-ER-FY010016M2L96和N-ER-FY010034M3L96(每组6只小鼠)。给药后第7日、第14日、第21日、第28日、第35日和第42日检测血清hCFB蛋白表达量，由此得出siRNA缀合物对hCFB蛋白表达的抑制率。
表8 siRNA缀合物对hCFB蛋白抑制率
从表8可以看出，本公开的siRNA缀合物在体内对hCFB基因具有较高的抑制活性，能够长时间降低hCFB蛋白水平，说明本发明的siRNA缀合物能较好地抑制hCFB蛋白的生成。具体而言，在单次皮下施用后，在第7天时，N-ER-FY010016M2L96对hCFB基因表达显示了70.4％的抑制；在42天时，N-ER-FY010016M2L96对hCFB基因表达仍能有55.8％的抑制；在第7天时，N-ER-FY010034M3L96对hCFB基因表达显示了72.3％的抑制，在42天时，N-ER-FY010034M3L96对hCFB基因表达仍能有44.2％的抑制。
实施例8 siRNA缀合物在CD-1小鼠中的血浆动力学研究
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。
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。
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。
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。
从该实验中可以得出，本公开的siRNA缀合物在血浆中半衰期较短，清除较快。
实施例9 siRNA缀合物在CD-1小鼠组织分布试验
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。
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。
样品采集：
给药后24h：采集血浆、肝、肾、脾；给药后72h：采集血浆、肝、肾、脾；
给药后168h(1周)：采集血浆、肝、肾、脾、脑、心、肺、胃、小肠、肌肉、睾丸；
给药后336h(2周)：采集血浆、肝、肾、脾；
给药后672h(4周)：采集血浆、肝、肾、脾、脑、心、肺、胃、小肠、肌肉、睾丸；给药后1008h(6周)：采集血浆、肝、肾、脾；
给药后1344h(8周)：采集血浆、肝、肾、脾；
给药后1680h(10周)：采集血浆、肝、肾、脾、脑、心、肺、胃、小肠、肌肉、睾丸。
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。
从该实验中可以得出，本公开的siRNA缀合物主要富集于肝脏，在组织中保留时间较长，具有很好的稳定性。
实施例10 siRNA缀合物单次皮下注射C57B/L小鼠给予MTD试验
C57B/L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具体如下：
检测指标：
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
体重：对所有存活动物每周进行2次体重称量。
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)。
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。
血液生化学：主试验组动物于R28剖检，卫星组动物于R7、R14、R21、R28分批次剖检，检测血液生化学。
组织分布：主试验组动物于R28剖检，卫星组动物于R7、R14、R21、R28分批次剖检，采集血、肝，检测组织药物浓度。
组织病理学检查：主试验组动物于R28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。
从该实验中可以得出，本公开的siRNA缀合物毒性较低，具有优异的用药安全窗口。

Claims (29)

  1. 一种能够抑制补体因子B(CFB)基因表达的siRNA，所述siRNA包含正义链与反义链，其中所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中所述正义链含有核苷酸序列I，反义链含有核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中所述核苷酸序列I和核苷酸序列II选自以下序列：
    (1)所述核苷酸序列I包含SEQ ID NO:217所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:218所示的核苷酸序列：
    5’-CAAGACCAAAA-3’(SEQ ID NO:217)
    5’-UUUUGGUCUUG-3’(SEQ ID NO:218)；
    (2)所述核苷酸序列I包含SEQ ID NO:219所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:220所示的核苷酸序列：
    5’-CUACAAUGUGAGUGAU-3’(SEQ ID NO:219)
    5’-AUCACUCACAUUGUAG-3’(SEQ ID NO:220)；
    (3)所述核苷酸序列I包含SEQ ID NO:221所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:222所示的核苷酸序列：
    5’-UCUCUUUCCACUGCUAU-3’(SEQ ID NO:221)
    5’-AUAGCAGUGGAAAGAGA-3’(SEQ ID NO:222)；
    (4)所述核苷酸序列I包含SEQ ID NO:223所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:224所示的核苷酸序列：
    5’-CAGACAGCAGUA-3’(SEQ ID NO:223)
    5’-UACUGCUGUCUG-3’(SEQ ID NO:224)；
    (5)所述核苷酸序列I包含SEQ ID NO:225所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:226所示的核苷酸序列：
    5’-AGCAGCUCAAU-3’(SEQ ID NO:225)
    5’-AUUGAGCUGCU-3’(SEQ ID NO:226)，
    其中所述核苷酸序列I不为SEQ ID NO:47，所述核苷酸序列II不为SEQ ID NO:48；
    (6)所述核苷酸序列I包含SEQ ID NO:227所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:228所示的核苷酸序列：
    5’-GUGUACAGCAUGAU-3’(SEQ ID NO:227)
    5’-AUCAUGCUGUACAC-3’(SEQ ID NO:228)；
    (7)所述核苷酸序列I包含SEQ ID NO:229所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:230所示的核苷酸序列：
    5’-CAUCAUCCUCAUGA-3’(SEQ ID NO:229)
    5’-UCAUGAGGAUGAUG-3’(SEQ ID NO:230)；
    (8)所述核苷酸序列I包含SEQ ID NO:231所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:232所示的核苷酸序列：
    5’-UCAUGACUGAUGGA-3’(SEQ ID NO:231)
    5’-UCCAUCAGUCAUGA-3’(SEQ ID NO:232)，
    其中所述核苷酸序列I不为SEQ ID NO:85，所述核苷酸序列II不为SEQ ID NO:86；
    (9)所述核苷酸序列I包含SEQ ID NO:233所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:234所示的核苷酸序列：
    5’-ACCUGGAAGAUGUUU-3’(SEQ ID NO:233)
    5’-AAACAUCUUCCAGGU-3’(SEQ ID NO:234)；
    (10)所述核苷酸序列I包含SEQ ID NO:235所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:236所示的核苷酸序列：
    5’-GAUGAAAGCCAGUCU-3’(SEQ ID NO:235)
    5’-AGACUGGCUUUCAUC-3’(SEQ ID NO:236)，
    其中所述核苷酸序列I不为SEQ ID NO:127，所述核苷酸序列II不为SEQ ID NO:128；
    (11)所述核苷酸序列I包含SEQ ID NO:129所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:130所示的核苷酸序列：
    5’-gsasugaaAfgCfCfAfgucucugasTsT-3’(SEQ ID NO:129)
    5’-P1usCfsaGfaGfaCfuGfgCfuUfuCfaUfcsTsT-3’(SEQ ID NO:130)；
    (12)所述核苷酸序列I包含SEQ ID NO:237所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:238所示的核苷酸序列：
    5’-CAACAUUAAUGGGAAAAA-3’(SEQ ID NO:237)
    5’-UUUUUCCCAUUAAUGUUG-3’(SEQ ID NO:238)；
    (13)所述核苷酸序列I包含SEQ ID NO:239所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:240所示的核苷酸序列：
    5’-AUUUUAUGACUAUGA-3’(SEQ ID NO:239)
    5’-UCAUAGUCAUAAAAU-3’(SEQ ID NO:240)，
    其中所述核苷酸序列I不为SEQ ID NO:145，所述核苷酸序列II不为SEQ ID  NO:146；
    (14)所述核苷酸序列I包含SEQ ID NO:241所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:242所示的核苷酸序列：
    5’-AUGACGUUGCCCUGAU-3’(SEQ ID NO:241)
    5’-AUCAGGGCAACGUCAU-3’(SEQ ID NO:242)；
    (15)所述核苷酸序列I包含SEQ ID NO:243所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:244所示的核苷酸序列：
    5’-CAAGAAUAAGCUGAAAUA-3’(SEQ ID NO:243)
    5’-UAUUUCAGCUUAUUCUUG-3’(SEQ ID NO:244)；
    (16)所述核苷酸序列I包含SEQ ID NO:245所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:246所示的核苷酸序列：
    5’-CACUUGCCAGCAA-3’(SEQ ID NO:245)
    5’-UUGCUGGCAAGUG-3’(SEQ ID NO:246)；
    (17)所述核苷酸序列I包含SEQ ID NO:247所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:248所示的核苷酸序列：
    5’-GCUAUGACAAAGUCAA-3’(SEQ ID NO:247)
    5’-UUGACUUUGUCAUAGC-3’(SEQ ID NO:248)；
    (18)所述核苷酸序列I包含SEQ ID NO:1所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:2所示的核苷酸序列；
    (19)所述核苷酸序列I包含SEQ ID NO:3所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:4所示的核苷酸序列；
    (20)所述核苷酸序列I包含SEQ ID NO:27所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:28所示的核苷酸序列；
    (21)所述核苷酸序列I包含SEQ ID NO:29所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:30所示的核苷酸序列；
    (22)所述核苷酸序列I包含SEQ ID NO:31所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:32所示的核苷酸序列；
    (23)所述核苷酸序列I包含SEQ ID NO:51所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:52所示的核苷酸序列；
    (24)所述核苷酸序列I包含SEQ ID NO:87所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:88所示的核苷酸序列；
    (25)所述核苷酸序列I包含SEQ ID NO:93所示的核苷酸序列，且所述核苷酸序列 II包含SEQ ID NO:94所示的核苷酸序列；
    (26)所述核苷酸序列I包含SEQ ID NO:95所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:96所示的核苷酸序列；
    (27)所述核苷酸序列I包含SEQ ID NO:97所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:98所示的核苷酸序列；
    (28)所述核苷酸序列I包含SEQ ID NO:99所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:100所示的核苷酸序列；
    (29)所述核苷酸序列I包含SEQ ID NO:103所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:104所示的核苷酸序列；
    (30)所述核苷酸序列I包含SEQ ID NO:131所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:132所示的核苷酸序列；
    (31)所述核苷酸序列I包含SEQ ID NO:133所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:134所示的核苷酸序列；
    (32)所述核苷酸序列I包含SEQ ID NO:135所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:136所示的核苷酸序列；
    (33)所述核苷酸序列I包含SEQ ID NO:137所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:138所示的核苷酸序列；
    (34)所述核苷酸序列I包含SEQ ID NO:201所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:202所示的核苷酸序列；
    (35)所述核苷酸序列I包含SEQ ID NO:207所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:208所示的核苷酸序列；
    (36)所述核苷酸序列I包含SEQ ID NO:209所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:210所示的核苷酸序列；
    (37)所述核苷酸序列I包含SEQ ID NO:211所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:212所示的核苷酸序列；
    (38)所述核苷酸序列I包含SEQ ID NO:213所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:214所示的核苷酸序列；
    (39)所述核苷酸序列I包含SEQ ID NO:215所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:216所示的核苷酸序列；
    (40)所述核苷酸序列I包含SEQ ID NO:401所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:402所示的核苷酸序列：
    5’-CAAGAUAUGGUCU-3’(SEQ ID NO:401)
    5’-AGACCAUAUCUUG-3’(SEQ ID NO:402)；
    (41)所述核苷酸序列I包含SEQ ID NO:403所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:404所示的核苷酸序列：
    5’-AUGGUCUAGUGACAUA-3’(SEQ ID NO:403)
    5’-UAUGUCACUAGACCAU-3’(SEQ ID NO:404)，
    其中所述核苷酸序列I不为SEQ ID NO:285，所述核苷酸序列II不为SEQ ID NO:286；
    (42)所述核苷酸序列I包含SEQ ID NO:405所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:406所示的核苷酸序列：
    5’-CUACAACAUUAAUG-3’(SEQ ID NO:405)
    5’-CAUUAAUGUUGUAG-3’(SEQ ID NO:406)；
    (43)所述核苷酸序列I包含SEQ ID NO:407所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:408所示的核苷酸序列：
    5’-GUGAAGCCAAGAUAU-3’(SEQ ID NO:407)
    5’-AUAUCUUGGCUUCAC-3’(SEQ ID NO:408)；
    (44)所述核苷酸序列I包含SEQ ID NO:354所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:355所示的核苷酸序列：
    5’-CUAGUGACAUAUGCCACAU-3’(SEQ ID NO:354)
    5’-AUGUGGCAUAUGUCACUAG-3’(SEQ ID NO:355)；
    (45)所述核苷酸序列I包含SEQ ID NO:409所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:410所示的核苷酸序列：
    5’-GGAAAAAAGAAGCAGGAA-3’(SEQ ID NO:409)
    5’-UUCCUGCUUCUUUUUUCC-3’(SEQ ID NO:410)；
    (46)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:392所示的核苷酸序列；
    (47)所述核苷酸序列I包含SEQ ID NO:393所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:392所示的核苷酸序列；
    (48)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:394所示的核苷酸序列；
    (49)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:395所示的核苷酸序列；
    (50)所述核苷酸序列I包含SEQ ID NO:391所示的核苷酸序列，且所述核苷酸序列 II包含SEQ ID NO:396所示的核苷酸序列；
    (51)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:260所示的核苷酸序列；
    (52)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:397所示的核苷酸序列；
    (53)所述核苷酸序列I包含SEQ ID NO:398所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:397所示的核苷酸序列；
    (54)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:399所示的核苷酸序列；
    (55)所述核苷酸序列I包含SEQ ID NO:259所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:400所示的核苷酸序列；
    (56)所述核苷酸序列I包含SEQ ID NO:250所示的核苷酸序列，且所述核苷酸序列II包含SEQ ID NO:130所示的核苷酸序列。
  2. 根据权利要求1所述的siRNA，其中所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补；所述基本上反向互补是指两个核苷酸序列之间存在不多于3个的碱基错配；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
  3. 根据权利要求1或2所述的siRNA，其中所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度各自独立地为0-9个核苷酸，其中所述核苷酸序列III连接在核苷酸序列I的5'末端，核苷酸序列IV连接在核苷酸序列II的3'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配；和/或，所述核苷酸序列III连接在核苷酸序列I的3'末端，核苷酸序列IV连接在核苷酸序列II的5'末端，所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或完全反向互补；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；完全反向互补是指两个核苷酸序列之间没有错配。
  4. 根据权利要求1-3中任一项所述的siRNA，所述正义链还含有核苷酸序列V和/或所述反义链还含有核苷酸序列VI，核苷酸序列V和VI的长度为0至3个核苷酸， 核苷酸序列V连接在所述正义链的3'末端构成正义链的3'突出端和/或核苷酸序列VI连接在所述反义链的3'末端构成反义链的3'突出端；优选地，所述核苷酸序列V或VI的长度为2个核苷酸；更优选地，所述核苷酸序列V或VI为连续的两个胸腺嘧啶脱氧核糖核苷酸或连续的两个尿嘧啶核糖核苷酸；
    或者，所述核苷酸序列V或VI与靶mRNA相应位置的核苷酸错配或互补。
  5. 根据权利要求1-4中任一项所述的siRNA，其中该双链区的长度是15-30个核苷酸对；优选地，双链区的长度是17-23个核苷酸对；更优选地，双链区的长度是19-21个核苷酸对。
  6. 根据权利要求1-5中任一项所述的siRNA，其中正义链或反义链具有15-30个核苷酸；优选地，正义链或反义链具有19-25个核苷酸；更优选地，正义链或反义链具有19-23个核苷酸。
  7. 根据权利要求1-6中任一项所述的siRNA，其中所述正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；优选地，所述含有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；和/或，所述siRNA包括不包含3’突出端核苷酸的正义链。
  8. 根据权利要求1-7中任一项所述的siRNA，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。
  9. 根据权利要求1-8中任一项所述的siRNA，其中所述修饰的核苷酸选自2’-氟代修饰的核苷酸，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’-脱氧核苷酸，2’-氨基修饰的核苷酸，2’-取代的氨基修饰的核苷酸，核苷酸类似物或其中任意两种以上的组合。
  10. 根据权利要求1-9中任一项所述的siRNA，其中，所述修饰的核苷酸选自2’-氟代修饰的核苷酸，2’-甲氧基修饰的核苷酸，2’-O-CH2-CH2-O-CH3修饰的核苷酸，2’-O-CH2-CH＝CH2修饰的核苷酸，2’-CH2-CH2-CH＝CH2修饰的核苷酸，2’-脱氧核苷酸， 核苷酸类似物或其中任意两种以上的组合。
  11. 根据权利要求1-10中任一项所述的siRNA，其中所述正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸或非氟代修饰的核苷酸；
    优选地，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为非氟代修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为非氟代修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为非氟代修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为非氟代修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，其余位置为非氟代修饰的核苷酸；
    进一步优选地，每一个非氟代修饰的核苷酸均为2’-甲氧基修饰的核苷酸，所述2’-甲氧基修饰的核苷酸指核糖基的2’-羟基被甲氧基取代而形成的核苷酸。
  12. 根据权利要求11所述的siRNA，其中每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种，所述核苷酸类似物选自异核苷酸、LNA、ENA、cET BNA、UNA和GNA中的一种。
  13. 根据权利要求1-12中任一项所述的siRNA，其中所述正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸、2’-甲氧基修饰的核苷酸、GNA修饰的核苷酸或其中任意两种以上的组合；
    优选地，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，GNA修饰的核苷酸位于反义链的第6位，其余位置为2’-甲氧基修饰的核苷酸；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，GNA修饰的核苷酸位于反义链的第7位，其余位置为2’-甲氧基修饰的核苷酸。
  14. 根据权利要求1-13中任一项所述的siRNA，其中所述siRNA中以下核苷酸之间的连接中至少一个为硫代磷酸酯基连接：
    所述正义链的5’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
    所述正义链的5’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
    所述正义链的3’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
    所述正义链的3’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
    所述反义链的5’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
    所述反义链的5’末端起始的第2个核苷酸和第3个核苷酸之间的连接；
    所述反义链的3’末端起始的第1个核苷酸和第2个核苷酸之间的连接；
    所述反义链的3’末端起始的第2个核苷酸和第3个核苷酸之间的连接。
  15. 根据权利要求1-14中任一项所述的siRNA，按照5’到3’的方向，所述正义链包含位于如下所示位置处的硫代磷酸酯基：
    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；
    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；
    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；
    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；
    或者，
    所述正义链包含位于如下所示位置处的硫代磷酸酯基：
    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；
    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。
  16. 根据权利要求1-15中任一项所述的siRNA，按照5’到3’的方向，所述反义链包含位于如下所示位置处的硫代磷酸酯基：
    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；
    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；
    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；
    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。
  17. 根据权利要求1-16中任一项所述的siRNA，其中所述正义链和所述反义链中的每一个核苷酸独立地为2’-氟代修饰的核苷酸，2’-甲氧基修饰的核苷酸，GNA修饰的核苷酸或其中任意两种以上的组合；
    优选地，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接，3’末端除去突出端；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的偶数位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接，3’末端除去突出端；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的 第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接，3’末端除去突出端；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、8、9、14和16位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、6、14和16位，GNA修饰的核苷酸位于反义链的第7位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个 核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；
    或者，按照5’到3’的方向，2’-氟代修饰的核苷酸位于正义链的第7、9、10和11位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接；按照5’到3’的方向，2’-氟代修饰的核苷酸位于反义链的第2、14和16位，GNA修饰的核苷酸位于反义链的第6位，其余位置为2’-甲氧基修饰的核苷酸，5’末端的第1个核苷酸和第2个核苷酸之间，5’末端的第2个核苷酸和第3个核苷酸之间，3’末端的第1个核苷酸和第2个核苷酸之间，3’末端的第2个核苷酸和第3个核苷酸之间为硫代磷酸酯基连接。
  18. 根据权利要求1所述的siRNA，其选自表1的siRNA，其中所述siRNA不为N-ER-FY010019、N-ER-FY010015、N-ER-FY010051、N-ER-FY010021、N-ER-FY010013、N-ER-FY010035、N-ER-FY010009、N-ER-FY-010099；优选地，所述siRNA选自N-ER-FY010016、N-ER-FY010016M2、N-ER-FY010016M2D2、N-ER-FY010016M3、N-ER-FY010016M3D2、N-ER-FY010016M4、N-ER-FY010016M5、N-ER-FY010034、N-ER-FY010034M3、N-ER-FY010034M3D2、N-ER-FY010034M2、N-ER-FY010034M2D2、N-ER-FY010034M4、N-ER-FY010034M5、N-ER-FY010003、N-ER-FY010003M2、N-ER-FY010003M3、N-ER-FY010076、N-ER-FY010076M1、N-ER-FY010076MD2、N-ER-FY010076M2、N-ER-FY010076M3、N-ER-FY010037、N-ER-FY010037M2、N-ER-FY010037M3、N-ER-FY010052、N-ER-FY010052M2、N-ER-FY010052M3、N-ER-FY010114、N-ER-FY010114M2、N-ER-FY010114M3、N-ER-FY010115、N-ER-FY010115M2、N-ER-FY010115M3、N-ER-FY010121、N-ER-FY010121M2、N-ER-FY010121M3。
  19. 一种siRNA缀合物，所述siRNA缀合物含有权利要求1-18中任一项所述的siRNA以及缀合至该siRNA的缀合基团。
  20. 根据权利要求19所述的siRNA缀合物，其中所述缀合基团包含药学上可接受的靶向基团和接头，并且所述siRNA、所述接头和所述靶向基团依次共价或非共价连接；
    优选地，在所述siRNA缀合物中，siRNA的正义链与反义链互补形成所述siRNA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1-3个延伸出所述双链区的突出的核苷酸；
    或者，
    在所述siRNA缀合物中，siRNA的正义链与反义链互补形成所述siRNA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。
  21. 根据权利要求20所述的siRNA缀合物，其中所述缀合基团为下式的L96：
  22. 根据权利要求19-21中任一项所述的siRNA缀合物，其中所述siRNA缀合物为选自表2的siRNA缀合物。
  23. 一种药物组合物，其包含权利要求1-18中任一项所述的siRNA，或权利要求19-22中任一项所述的siRNA缀合物，以及药学上可接受的载体。
  24. 一种试剂盒，其包含权利要求1-18中任一项所述的siRNA，或权利要求19-22中任一项所述的siRNA缀合物，或权利要求23所述的药物组合物。
  25. 权利要求1-18中任一项所述的siRNA，或权利要求19-22中任一项所述的siRNA缀合物，或权利要求23所述的药物组合物用于制备抑制CFB基因表达的药剂的用途。
  26. 权利要求1-18中任一项所述的siRNA，或权利要求19-22中任一项所述的siRNA缀合物，或权利要求23所述的药物组合物用于制备预防和/或治疗CFB基因过表达相关的疾病的药剂的用途。
  27. 根据权利要求26所述的用途，所述疾病选自原发性膜性肾病、溶血性尿毒症 综合征、C3肾小球病、IgA肾病、阵发性夜间血红蛋白尿(PNH)、年龄相关性黄斑病变和眼科疾病。
  28. 一种抑制CFB基因表达的方法，包括将治疗有效量的权利要求1-18中任一项所述的siRNA，或权利要求19-22中任一项所述的siRNA缀合物，或权利要求23所述的药物组合物与表达CFB的细胞接触或给予有需要的受试者。
  29. 一种治疗和/或预防CFB基因过表达相关的疾病的方法，包括将治疗有效量的权利要求1-18中任一项所述的siRNA，或权利要求19-22中任一项所述的siRNA缀合物，或权利要求23所述的药物组合物给予有需要的受试者。
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WO2018117253A1 (ja) * 2016-12-23 2018-06-28 協和発酵キリン株式会社 補体ｂ因子の発現を抑制する核酸
WO2021222549A1 (en) * 2020-04-30 2021-11-04 Alnylam Pharmaceuticals, Inc. Complement factor b (cfb) irna compositions and methods of use thereof
CN116769780A (zh) * 2022-07-08 2023-09-19 北京福元医药股份有限公司 用于抑制补体因子B表达的siRNA、其缀合物和药物组合物及其用途

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