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WO2020147847A1 - Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation - Google Patents

Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation Download PDF

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WO2020147847A1
WO2020147847A1 PCT/CN2020/072813 CN2020072813W WO2020147847A1 WO 2020147847 A1 WO2020147847 A1 WO 2020147847A1 CN 2020072813 W CN2020072813 W CN 2020072813W WO 2020147847 A1 WO2020147847 A1 WO 2020147847A1
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nucleotide sequence
nucleotide
sirna
seq
nucleotides
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Chinese (zh)
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张鸿雁
高山
康代武
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Suzhou Ribo Life Science Co Ltd
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Suzhou Ribo Life Science Co Ltd
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Priority to CN202080009787.1A priority Critical patent/CN113330117B/zh
Priority to CN202410666760.XA priority patent/CN118562796A/zh
Publication of WO2020147847A1 publication Critical patent/WO2020147847A1/fr
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    • 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
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • 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
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21038—Coagulation factor XIIa (3.4.21.38)
    • 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 nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present disclosure relates to a nucleic acid capable of inhibiting the expression of factor XII gene and a composition and conjugate containing the nucleic acid.
  • the present disclosure also relates to preparation methods and uses of these nucleic acids, compositions and conjugates.
  • Hereditary angioedema is a rare disease characterized by repeated episodes of severe swelling. The most common swollen areas of the body are the limbs, face, intestines, and airways. The onset may be spontaneous, or it may be caused by physical trauma or stress. Laryngeal (airway) edema can be life-threatening because it can lead to death from suffocation.
  • Factor XII is a serine protease mainly expressed in the liver and found in the blood. It has a dual function in the endogenous blood coagulation pathway and the kinin-kallikrein system. The kinin-kallikrein system plays a role in inflammation, blood pressure control, blood clotting and pain.
  • the active form of factor XII also known as FXII, F12 or Hageman factor
  • Factor XII is one of the key targets for the treatment of HAE. By inhibiting the expression of factor XII, the occurrence of HAE can be effectively inhibited. Therefore, if gene expression can be silenced at the gene level and the production of factor XII can be blocked, it will undoubtedly be the most ideal treatment.
  • Small interfering RNA siRNA
  • RNAi RNA interference
  • siRNA sequences and modifications and their delivery systems are two key technologies in the development of small RNA drugs.
  • the present disclosure provides a siRNA conjugate having a structure represented by formula (308):
  • n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
  • Each of m1, m2 and m3 is independently an integer selected from 2-10;
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently H, or is selected from the group consisting of C 1 -C 10 alkyl, C 1 -C 10 haloalkyl and C 1 -C 10 alkoxy;
  • R 3 is a group of the structure shown in formula A59:
  • E 1 is OH, SH or BH 2 ;
  • Nu is siRNA, the siRNA has a sense strand and an antisense strand, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the sense strand contains a nucleotide sequence I,
  • the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence
  • the nucleotide sequence II is selected from the following group i)-v):
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences
  • nucleotide sequence II is the same as SEQ ID NO: 2
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 3 whose position corresponds to Z 1 , and the nucleotide sequence II Comprising the nucleotide Z 4 at the position corresponding to Z 2 , where Z 4 is the first nucleotide at the 5'end of the antisense strand;
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 7 whose position corresponds to Z 5 , and the nucleotide sequence II Comprising the nucleotide Z 8 at the position corresponding to Z 6 , said Z 8 being the first nucleotide at the 5'end of the antisense strand;
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 15 whose position corresponds to Z 13 , and the nucleotide sequence II Comprising the nucleotide Z 16 at the position corresponding to Z 14 , said Z 16 being the first nucleotide at the 5'end of the antisense strand;
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 19 at the position corresponding to Z 17 , and the nucleotide sequence II Comprising the nucleotide Z 20 at the position corresponding to Z 18 , said Z 20 being the first nucleotide at the 5'end of the antisense strand;
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences
  • nucleotide sequence II is the same as SEQ ID NO: 242
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 23 corresponding to the position Z 21 , and the nucleotide sequence II Comprising the nucleotide Z 24 at the position corresponding to Z 22 , said Z 24 being the first nucleotide at the 5'end of the antisense strand;
  • M 1 represents a targeting group.
  • the present disclosure provides a siRNA comprising a sense strand and an antisense strand, and each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified core Nucleotides or non-fluorinated modified nucleotides; the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide Sequence II is at least partially reverse complementary to form a double-stranded region.
  • the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, in the direction from the 5'end to the 3'end, In the sense strand, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides According to the direction from the 5'end to the 3'end, in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluoro-modified Nucleotides, the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides, and,
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences
  • nucleotide sequence II is the same as SEQ ID NO: 2
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 3 whose position corresponds to Z 1 , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z 4 of Z 2 , where Z 4 is the first nucleotide at the 5'end of the antisense strand; or,
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 7 whose position corresponds to Z 5 , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z 8 of Z 6 , where Z 8 is the first nucleotide at the 5'end of the antisense strand; or,
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 15 whose position corresponds to Z 13 , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z 16 of Z 14 , where Z 16 is the first nucleotide at the 5'end of the antisense strand; or,
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 19 at the position corresponding to Z 17 , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z 20 of Z 18 , said Z 20 being the first nucleotide at the 5'end of the antisense strand; or,
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences
  • nucleotide sequence II is the same as SEQ ID NO: 242
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences.
  • the nucleotide sequence I includes the nucleotide Z 23 corresponding to the position Z 21 , and the nucleotide sequence II comprising a position corresponding to nucleotide Z 22 Z 24, Z 24 is the antisense strand of the 5 'end of the first nucleotide.
  • the present disclosure provides a pharmaceutical composition that contains the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides an siRNA conjugate comprising the siRNA provided in the present disclosure and a conjugating group conjugated to the siRNA.
  • the present disclosure provides that the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are used in the preparation of a medicine for the treatment and/or prevention of hereditary angioedema HAE and/or thrombosis the use of.
  • the present disclosure provides a method for treating and/or preventing HAE and/or thrombosis, the method comprising conjugating an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA of the present disclosure
  • the drug is administered to subjects with HAE.
  • the present disclosure provides a method for inhibiting FXII gene expression in liver cells, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver. Cell contact.
  • the present disclosure provides a kit that contains the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
  • the siRNA, the composition containing the siRNA, and the siRNA conjugate provided in the present disclosure have good stability, high gene suppression activity, and/or can significantly treat or relieve HAE symptoms.
  • the siRNA, the composition containing the siRNA, or the siRNA conjugate provided in the present disclosure show excellent target gene inhibitory activity in in vitro cell experiments.
  • the siRNA provided in the present disclosure shows an inhibition rate of FXII mRNA expression as high as 78.70% in human liver primary cells.
  • the siRNA provided in the present disclosure shows an inhibition rate of up to 70.09% of FXII mRNA expression in C57 mouse liver primary cells.
  • the siRNA conjugates of the present disclosure show up to 98.7% inhibition rate of FXII mRNA expression in C57 mice.
  • the siRNA, composition containing the siRNA, or siRNA conjugate provided in the present disclosure does not show obvious off-target effects.
  • Off-target effects can be, for example, suppression of normal expression of genes other than target genes. It is believed that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the target gene effect, the off-target effect is not significant.
  • siRNA, pharmaceutical composition and siRNA conjugate provided in the present disclosure can inhibit the expression of FXII gene, effectively treat and/or prevent HAE symptoms, and have good application prospects.
  • Fig. 1 and Fig. 2 are scatter plots of FXII mRNA expression in liver tissue of C57 mice (relative value with GAPDH as internal reference) after administration of PBS and different doses of each conjugate, respectively.
  • FXII mRNA refers to the sequence shown in Genbank registration number NM_000505.3.
  • target gene used in the present disclosure refers to a gene that expresses the aforementioned FXII mRNA
  • target mRNA refers to the aforementioned FXII mRNA.
  • capital letters C, G, U, A represent the base composition of nucleotides; lowercase letter m represents that the adjacent nucleotide to the left of the letter m is a methoxy group Modified nucleotides; a lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a fluorinated modified nucleotide; a lowercase letter s indicates that between the two adjacent nucleotides to the left and right of the letter s It is a phosphorothioate group connection; P1 indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • P1 represents a specifically modified VP, Ps, or P, where the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is a nucleotide modified by vinyl phosphate.
  • Ps indicates that the adjacent nucleotide to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the adjacent nucleotide to the right of the letter P is a 5'-phosphate nucleotide .
  • the “fluoro-modified nucleotide” refers to a nucleotide formed by replacing the hydroxyl group at the 2'position of the ribose group of a nucleotide with fluorine
  • the “non-fluoro-modified nucleotide” refers to Nucleotides or nucleotide analogs formed by substituting a non-fluorine group for the hydroxyl group at the 2'position of the ribose group of a nucleotide.
  • Nucleotide analogue refers to a nucleic acid that can replace nucleotides, but has a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymus The group of pyrimidine deoxyribonucleotides. Such as heteronucleotides, bridged nucleotides (BNA for short) or acyclic nucleotides.
  • the "methoxy-modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the expressions "complementary” or “reverse complement” can be used interchangeably, and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand and the other strand The bases are paired in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) matches.
  • Each base pair includes a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, bases at corresponding positions are not paired 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 is no more than one base mismatch between two nucleotide sequences; “complete reverse complement” means that there is no base mismatch between two nucleotide sequences.
  • nucleotide difference between a nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed compared with the latter. For example, when one nucleotide base in the latter is A, when the corresponding nucleotide base at the same position in the former is U, C, G, or T, it is considered to be one of the two nucleotide sequences There is a nucleotide difference at this position. In some embodiments, when an abasic nucleotide or its equivalent is substituted for the nucleotide at the original position, it can also be considered that there is a nucleotide difference at that position.
  • the nucleoside monomer refers to the preparation method according to the The type and sequence of nucleotides in the siRNA or siRNA conjugate, the modified or unmodified nucleoside phosphoramidite monomers used in the solid-phase synthesis of phosphoramidites (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites).
  • Phosphoramidite solid-phase synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in the present disclosure are all commercially available.
  • siRNA conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; correspondingly, “conjugate” is Refers to the compound formed by covalent linkage between the various chemical parts.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugate should be understood as the general term of siRNA conjugate, the general term of siRNA conjugate represented by formula (305) and formula (307), or formula (305), formula (307), formula (308) SiRNA conjugate shown.
  • conjuggated molecule should be understood as a specific compound that can be conjugated to siRNA through a reaction, and ultimately form the siRNA conjugate of the present disclosure.
  • a dash that is not between two letters or between two symbols is used to indicate the point of attachment of substituents.
  • alkyl refers to straight and branched chains having a specified number of carbon atoms, the number is usually 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 Or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residues having a specific number of carbons it is intended to cover all branched and straight chain forms having that number of carbons; therefore, for example, "butyl” means including n-butyl, sec-butyl , Isobutyl and tert-butyl; "propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to residues that are the same as alkyl but have two points of attachment.
  • alkenyl refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon double bond, which is obtained from adjacent carbon atoms of the parent alkyl group. Obtained by removing one molecule of hydrogen. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl Group), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-ene-1- But-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon triple bond, the carbon-carbon triple bond is derived from adjacent carbon atoms of the parent alkyl group Obtained by removing two molecules of hydrogen.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like.
  • the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to residues that are the same as alkynyl but have two points of attachment.
  • alkoxy refers to an alkyl group with a specified number of carbon atoms connected through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, S-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentoxy and so on.
  • Alkoxy groups generally have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by oxygen bridges.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, that is, contains a ring according to Hückel theory , Delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include but are not limited to groups such as phenyl, fluorenyl and naphthyl.
  • Arylene is a subset of aryl and refers to residues that are the same as aryl but have two points of attachment.
  • cycloalkyl refers to a non-aromatic carbocyclic ring, usually having 3 to 7 ring carbon atoms. The ring can be saturated or have one or more carbon-carbon double bonds.
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.
  • halogen substituent or “halo” refers to fluoro, chloro, bromo and iodo, and the term “halogen” includes fluoro, chloro, bromo and iodo.
  • haloalkyl refers to an alkyl group as defined above in which the specified number of carbon atoms is replaced by one or more halogen atoms up to the maximum allowable number.
  • haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise stated in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include a fused ring or a bridged ring system. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group can be connected to the rest of the molecule through any ring atom.
  • heterocyclic groups include but are not limited to: dioxanyl, thienyl[1,3]dithianyl (thienyl[1,3]dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidine Group, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trisulfanyl (trithianyl) ), tetrahydropyranyl, thio
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, that is, contains a cyclic delocalization according to Hückel's theory (4n +2) ⁇ -electron system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized.
  • heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodiaxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl (benzo[b][1,4]dioxepinyl), benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl (benzodioxox)
  • hydroxyl protecting groups can be used in this disclosure.
  • the protective group makes the chemical functional group insensitive to specific reaction conditions, and can be added and removed from the functional group in the molecule without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d, John Wiley & Sons, New York, 1991, as cited In this way, the above-mentioned documents are incorporated into this article as a whole.
  • the protecting group is stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthene-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4"-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (for example, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any kind of poultry .
  • treat can be used interchangeably herein. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefits.
  • therapeutic benefit means eradicating or improving the underlying barrier to be treated.
  • therapeutic benefits are obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be afflicted by the underlying disorder.
  • prevent and “prevent” are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits.
  • the conjugate or composition can be administered to a subject who is at risk of suffering from a particular disease, or to a subject who reports one or more physiological symptoms of the disease, even though the diagnosis of the disease Not yet made.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and those skilled in the art know that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.
  • the siRNA may be the first siRNA.
  • the first siRNA contains a sense strand and an antisense strand, and each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 2 The length is equal, and no more than 3 nucleotide differences:
  • Z 1 is U
  • Z 2 is A
  • nucleotide sequence I contains the nucleotide Z 3 whose position corresponds to Z 1
  • nucleotide sequence II contains the nucleotide Z 4 whose position corresponds to Z 2
  • the Z 4 is the The first nucleotide at the 5'end of the antisense strand.
  • positional correspondence refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence.
  • the first nucleotide at the 3'end of the nucleotide sequence I is the nucleotide whose position corresponds to the first nucleotide at the 3'end of SEQ ID NO:1.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 2.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the difference at position Z 4 , and Z 4 is selected from U, C or G. In some embodiments, the nucleotide difference is a difference at the Z 4 position, and Z 4 is selected from U, C, or G. In some embodiments, Z 3 is a nucleotide that is complementary to Z 4 . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and siRNA conjugates containing these nucleotide differences are also within the protection scope of the present disclosure.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary.
  • the term “substantially reverse complementary” refers to the presence of no more than 3 base mismatches between two nucleotide sequences; “substantially reverse complementary” refers to two nucleotides There is no more than one base mismatch between the sequences; “complete reverse complementarity” means that there is no base mismatch between two nucleotide sequences.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:
  • Z 4 is the first nucleotide at the 5'end of the antisense strand
  • Z 3 is selected from A, U, G or C
  • Z 4 is a nucleotide complementary to Z 3 ; in some embodiments Where Z 3 is U, Z 4 is A;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores. Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence
  • the second nucleotide sequence refers to the sequence ID of the target mRNA
  • NO: 1 represents a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide
  • the base of the nucleotide sequence III is A
  • the base of the nucleotide sequence IV is U
  • the length ratio of the sense strand and the antisense strand is 20/20
  • the base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or,
  • the length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is GCA, and the base composition of nucleotide sequence IV is UGC; At this
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CA , The base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the base of the nucleotide sequence III, the base of the nucleotide sequence IV is also determined.
  • the siRNA may be a second siRNA.
  • the second siRNA contains a sense strand and an antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 61 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 62 The length is equal, and no more than 3 nucleotide differences:
  • Z 5 is A and Z 6 is U;
  • nucleotide sequence I contains the nucleotide Z 7 whose position corresponds to Z 5
  • nucleotide sequence II contains the nucleotide Z 8 whose position corresponds to Z 6
  • the Z 8 is the The first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 62.
  • the II and the nucleotide sequence SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 include differences in location Z 8, and Z 8 is selected from A, C or G.
  • the nucleotide difference is a difference at the Z 8 position, and Z 8 is selected from A, C, or G.
  • Z 7 is a nucleotide that is complementary to Z 8 .
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64:
  • Z 8 is the first nucleotide at the 5'end of the antisense strand
  • Z 7 is selected from A, U, G, or C
  • Z 8 is a nucleotide complementary to Z 7 ; in some embodiments Where Z 7 is A, Z 8 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 61 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is AA, and the base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is GAA, and the base composition of nucleotide sequence IV is UUC; At
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is AA , The base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the base of the nucleotide sequence III, the base of the nucleotide sequence IV is also determined.
  • the third siRNA is the third siRNA
  • the siRNA may be a third siRNA.
  • the third siRNA contains a sense strand and an antisense strand, and each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 122 The length is equal, and no more than 3 nucleotide differences:
  • Z 13 is A
  • Z 14 is U
  • nucleotide sequence I includes the nucleotide Z 15 whose position corresponds to Z 13
  • nucleotide sequence II includes the nucleotide Z 16 whose position corresponds to Z 14
  • the Z 16 is the The first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II and SEQ ID NO: 122 has no more than one nucleotide difference between the nucleotide sequences shown.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 includes the difference at position Z 16 , and Z 16 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z 16 position, and Z 16 is selected from A, C, or G. In some embodiments, Z 15 is a nucleotide that is complementary to Z 16 . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124:
  • Z 16 is the first nucleotide at the 5'end of the antisense strand
  • Z 15 is selected from A, U, G, or C
  • Z 16 is a nucleotide complementary to Z 15 ; in some embodiments Where Z 15 is A, Z 16 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 121 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, The base of nucleotide sequence IV is A; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequences III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is UU, and the base composition of nucleotide sequence IV is AA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is UUU, and the base composition of nucleotide sequence IV is AAA; At this
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is AA , The base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the base of the nucleotide sequence III, the base of the nucleotide sequence IV is also determined.
  • the siRNA may be the fourth siRNA.
  • the fifth siRNA contains a sense strand and an antisense strand, each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 181 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 182 The length is equal, and no more than 3 nucleotide differences:
  • Z 17 is A
  • Z 18 is U
  • nucleotide sequence I includes the nucleotide Z 19 at a position corresponding to Z 17 and the nucleotide sequence II includes the nucleotide Z 20 at the position corresponding to Z 18 , and the Z 20 is the The first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181, and/or the nucleotide sequence II and SEQ ID NO: No more than 1 nucleotide difference between the nucleotide sequences shown in 182.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 includes the difference at position Z 20 , and Z 20 is selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position 20 Z, Z 20 is selected from A, C or G. In some embodiments, Z 19 is a nucleotide complementary to Z 20 . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 183
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 184:
  • Z 20 is the first nucleotide at the 5'end of the antisense strand
  • Z 19 is selected from A, U, G, or C
  • Z 20 is a nucleotide complementary to Z 19 ; in some embodiments Where Z 19 is A and Z 20 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 181 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is G, and the nuclear The base of nucleotide sequence IV is C; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GG, and the base composition of nucleotide sequence IV is CC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is UGG, and the base composition of nucleotide sequence IV is CCA;
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GG , The base composition of nucleotide sequence IV is CC; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the base of the nucleotide sequence III, the base of the nucleotide sequence IV is also determined.
  • the siRNA may be the fifth siRNA.
  • the fifth siRNA contains a sense strand and an antisense strand, each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 241 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 242 The length is equal, and no more than 3 nucleotide differences:
  • Z 21 is A and Z 22 is U;
  • the nucleotide sequence I contains the nucleotide Z 23 whose position corresponds to Z 21
  • the nucleotide sequence II contains the nucleotide Z 24 whose position corresponds to Z 22
  • the Z 24 is the reverse The first nucleotide at the 5'end of the sense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241, and/or the nucleotide sequence II and SEQ No more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:242.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 includes the difference at position Z 24 , and Z 24 is selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position 24 Z, Z 24 is selected from A, C or G. In some embodiments, Z 23 is a nucleotide complementary to Z 24 . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 304:
  • Z 16 is the first nucleotide at the 5'end of the antisense strand
  • Z 23 is selected from A, U, G, or C
  • Z 24 is a nucleotide complementary to Z 23 ; in some embodiments Where Z 23 is A, Z 24 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • This second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 241 in the target mRNA and has the same length as the nucleotide sequence IV .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, The base of nucleotide sequence IV is G; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GC, and the base composition of nucleotide sequence IV is GC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from 5'end to 3'end, the base composition of nucleotide sequence III is AGC, and the base composition of nucleotide sequence IV is GCU; At this time, the
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GC , The base composition of nucleotide sequence IV is GC; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the base of the nucleotide sequence III, the base of the nucleotide sequence IV is also determined.
  • nucleotide sequence V nucleic acid sequence
  • nucleotide modification in siRNA and modification sequence is applicable to any one of the above-mentioned first siRNA to fifth siRNA. That is, if there is no specific indication, the following description of siRNA should be regarded as describing the first siRNA, the second siRNA, the third siRNA, the fourth siRNA and the fifth siRNA one by one. For example, if no specific siRNA is specified, "the siRNA also contains the nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA or the fifth siRNA It also contains the nucleotide sequence V".
  • the length of the sense strand and the antisense strand are different, and the antisense strand also contains a nucleotide sequence V.
  • the length of the nucleotide sequence V is 1 to 3 nucleotides.
  • the 3'end of the antisense strand constitutes the 3'overhang of the antisense strand.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26.
  • the length of the nucleotide sequence V is 2 nucleotides. Therefore, the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure may be 19/21, 21/23, or 23. /25.
  • Each nucleotide in the nucleotide sequence V can be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand with the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23. At this time, the siRNA of the present disclosure has better mRNA silencing activity.
  • the nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to a nucleotide sequence of the target mRNA at the 5'end.
  • the nucleotide sequence of the target mRNA is the same as the nucleotide sequence Sequence II is substantially reverse complementary or completely reverse complementary, or a nucleotide sequence that is substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of nucleotide sequence II and nucleotide sequence IV.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 6.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8:
  • Z 4 is the first nucleotide at the 5'end of the antisense strand
  • Z 3 is selected from A, U, G or C
  • Z 4 is a nucleotide complementary to Z 3 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68:
  • Z 8 is the first nucleotide at the 5'end of the antisense strand
  • Z 7 is selected from A, U, G, or C
  • Z 8 is a nucleotide complementary to Z 7 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 125
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128:
  • Z 16 is the first nucleotide at the 5'end of the antisense strand
  • Z 15 is selected from A, U, G, or C
  • Z 16 is a nucleotide complementary to Z 15 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 185
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 186.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 187
  • the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 188:
  • Z 20 is the first nucleotide at the 5'end of the antisense strand
  • Z 19 is selected from A, U, G or C
  • Z 20 is a nucleotide complementary to Z 19 .
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 245, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 246.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 247
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 248:
  • Z 24 is the first nucleotide at the 5'end of the antisense strand
  • Z 23 is selected from A, U, G or C
  • Z 24 is a nucleotide complementary to Z 23 .
  • the siRNA described in the present disclosure is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf2:
  • Sense chain 5'-GGAACUCAAUAAAGUGCUU-3' (SEQ ID NO: 9)
  • Antisense strand 5'-AAGCACUUUAUUGAGUUCCUG-3' (SEQ ID NO: 10)
  • Antisense strand 5'-AAGCACUUUAUUGAGUUCCUGCG-3' (SEQ ID NO: 12)
  • Antisense strand 5'-UUCAAAGCACUUUAUUGAGUU-3' (SEQ ID NO: 70)
  • Antisense strand 5'-UUCAAAGCACUUUAUUGAGUUCC-3' (SEQ ID NO: 72).
  • Antisense strand 5'-UUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO: 130)
  • Antisense strand 5'-UUUCACUUUCUUGGGCUCCAAAC-3' (SEQ ID NO: 132)
  • Antisense strand 5'-UCUUUCACUUUCUUGGGCUCC-3' (SEQ ID NO: 190)
  • Antisense strand 5'-UCUUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO: 192)
  • Antisense strand 5'-UGGUCUUUCACUUUCUUGGGC-3' (SEQ ID NO: 250)
  • Antisense strand 5'-UGGUCUUUCACUUUCUUGGGCUC-3' (SEQ ID NO: 252)
  • the siRNA has a nucleotide sequence (ie, a nucleic acid base sequence) shown in siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1 or siFXIIf2.
  • a nucleotide sequence ie, a nucleic acid base sequence
  • the nucleotides in the siRNA of the present disclosure are each independently a modified or unmodified nucleotide.
  • the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides.
  • the siRNA of the present disclosure contains at least one modified nucleotide.
  • modified nucleotides refers to nucleotides or nucleotide analogs formed by replacing the 2'hydroxyl group of the ribose group of nucleotides with other groups, or nucleosides
  • the base on the acid is the nucleotide of the modified base.
  • the modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression. For example, 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 in the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate ester with a modified group
  • at least a 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 modified group and/ Or a ribose group with a modified group.
  • 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 in the present disclosure is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • the inventors of the present disclosure surprisingly found that the siRNA described in the present disclosure achieved a high balance of plasma stability and gene silencing efficiency in animal experiments.
  • the fluoro-modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and, in the direction from the 5'end to the 3'end, the nucleotide sequence I
  • the nucleotides at positions 7, 8, and 9 are fluorinated modified nucleotides; according to the direction from the 5'end to the 3'end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II Glycolic acid is a fluorinated modified nucleotide.
  • the fluoro-modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and there are no more than 5 fluoro-modified nucleotides in the nucleotide sequence I,
  • the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides;
  • the fluorine in the nucleotide sequence II There are no more than 7 modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides.
  • the nucleus at positions 7, 8, 9 or 5, 7, 8, and 9 of the nucleotide sequence I The nucleotides are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand.
  • the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the antisense strand
  • the nucleotides at the remaining positions are non-fluorinated modified nucleotides.
  • fluoromodified nucleotides refer to nucleotides in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refer to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of a nucleotide with a non-fluorine group.
  • each non-fluorinated modified nucleotide is independently selected from among nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group.
  • nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group is independently selected from among nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group.
  • nucleotides formed by replacing the hydroxyl group at the 2'position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these 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'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotides are methoxy modified nucleotides (2'-OMe), as shown in formula (8).
  • the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (9 ) Shown.
  • the 2'-amino modified nucleotide (2'-NH 2 ) is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is represented by formula (11):
  • Nucleotide analogs refer to nucleotides that can replace nucleotides in nucleic acids, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxynucleotides The group of ribonucleotides.
  • the nucleotide analogs can be isonucleotides, bridged nucleotides (BNA), or acyclic nucleotides.
  • BNA refers to nucleotides that are constrained or inaccessible.
  • BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosaccharide condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.
  • the BNA may be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):
  • Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of nucleotides.
  • acyclic nucleotides can be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is represented by formula (15) and GNA is represented by formula (16):
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotide refers to a compound formed by changing the position of the base in the nucleotide on the ribose ring.
  • the heteronucleotide may be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18):
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of heteronucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluorinated modified nucleotide is a methoxy modified nucleotide.
  • the methoxy modified nucleotide refers to the 2'of the ribose group. -Nucleotides formed by the substitution of hydroxy groups with methoxy groups.
  • the siRNA of the present disclosure is an siRNA with the following modifications: in the direction from the 5'end to the 3'end, in the sense strand, positions 7, 8, and 9 of the nucleotide sequence I Or the nucleotides at positions 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand Wherein, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense The nucleotides at the remaining positions in the chain are methoxy modified nucleotides.
  • the siRNA of the present disclosure is an siRNA with the following modifications: according to the direction from the 5'end to the 3'end, the siRNA is at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand
  • the nucleotides are fluoro-modified nucleotides
  • the nucleotides in the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides
  • the siRNA in the direction from the 5'end to the 3'end
  • the nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand are fluorinated modified nucleotides
  • the nucleotides at the remaining positions in the antisense strand of siRNA are methoxy Modified nucleotides;
  • the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the sense of siRNA
  • the nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of siRNA are methoxy-modified nucleotides;
  • the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are -fluoro-modified nucleotides, and the sense strand of the siRNA
  • the nucleotides at the remaining positions of the siRNA are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, 14th, and fourth nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotide at position 16 is a fluoro-modified nucleotide
  • the nucleotide at the remaining position of the antisense strand of the siRNA is a methoxy-modified nucleotide.
  • the siRNA provided in the present disclosure is siFXIIa1-M1, siFXIIa1-M2, siFXIIa1-M3, siFXIIa2-M1, siFXIIa2-M2, siFXIIa2-M3, siFXIIb1-M1, siFXIIb1-M2, siFXIIb1-M3, siFXIIb2- M1, siFXIIb2-M2, siFXIIb2-M3, siFXIId1-M1, siFXIId1-M2, siFXIId1-M3, siFXIId2-M1, siFXIId2-M2, siFXIId2-M3, siFXIIe1-M1, siFXIIe1-M2, siFXIIe1-M3, siFXIIe2-M1 Any one of siFXIIe2-M2, siFXIIe2-M3, siFXIIf1-M1, siFXII
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide.
  • the modified siRNA not only has low cost, but also makes it difficult for ribonuclease in blood to cleave nucleic acid, thereby increasing the stability of nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. At the same time, the above modifications did not significantly reduce the inhibitory performance of siRNA.
  • the phosphate group with a modification group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the The phosphate group with a modified group is a phosphorothioate group having a structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of the siRNA and maintain the high specificity and affinity of base pairing.
  • the phosphorothioate group linkage exists in at least one of the following positions: the first and second cores of either end of the sense strand or the antisense strand Between nucleotides; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.
  • phosphorothioate linkages are present at all the above positions except the 5'end of the sense chain.
  • the phosphorothioate group linkages are present at all the aforementioned positions except the 3'end of the sense chain.
  • the phosphorothioate group linkage is present in at least one of the following positions:
  • the siRNA provided by the present disclosure is siFXIIa1-M1S, siFXIIa1-M2S, siFXIIa1-M3S, siFXIIa2-M1S, siFXIIa2-M2S, siFXIIa2-M3S, siFXIIb1-M1S, siFXIIb1-M2S, siFXIIb1-M3S, siFXIIb1-M3S, M1S, siFXIIb2-M2S, siFXIIb2-M3S, siFXIId1-M1S, siFXIId1-M2S, siFXIId1-M3S, siFXIId2-M1S, siFXIId2-M2S, siFXIId2-M3S, siFXIIe1-M1S, siFXIIe1-e1-M1S, siFXIIe
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lowercase letter s means that the two nucleotides on the left and right of the letter are connected by phosphorothioate groups.
  • the 5'terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides or 5'-phosphate analog modified nucleotides are well known to those skilled in the art, for example, 5'-phosphate nucleotides can have the following formula (2) structure:
  • R is selected from H, OH, methoxy, fluorine;
  • Base represents a nucleic acid base, selected from A, U, C, G or T.
  • the 5'-phosphate nucleotide is a nucleotide containing 5'-phosphate modification represented by formula (2)
  • the 5'-phosphate analog modified nucleotide is a nucleotide containing vinyl phosphate ( 5'-(E)-vinylphosphonate, E-VP) modified nucleotides, as shown in formula (3), or phosphorothioate modified nucleotides, as shown in formula (5).
  • the siRNA provided in the present disclosure is siFXIIa1-M1P1, siFXIIa1-M2P1, siFXIIa1-M3P1, siFXIIa2-M1P1, siFXIIa2-M2P1, siFXIIa2-M3P1, siFXIIa1-M1SP1, siFXIIa1-M2SP1, siFXIIa1-M3SP1, siFXIIa1-M3SP1 M1SP1, siFXIIa2-M2SP1, siFXIIIa2-M3SP1, siFXIIIa1U-M1P1, siFXIIIa1U-M2P1, siFXIIIa1U-M3P1, siFXIIIa2U-M1P1, siFXIIIa2U-M2P1, siFXIIIa2U-M3P1, siFXIIa2U-M3P1, siFXI
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand
  • Antisense strand

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Abstract

L'invention concerne un ARNsi destiné à inhiber l'expression du gène du facteur XII (FXII), et une composition pharmaceutique et un conjugué contenant l'ARNsi. Les nucléotides de l'ARNsi sont chacun indépendamment des nucléotides modifiés ou non modifiés. L'ARNsi contient un brin sens et un brin antisens. Le brin sens contient une séquence nucléotidique I ayant une longueur égale à celle de la séquence nucléotidique représentée dans SEQ ID NO: 1 et n'ayant pas plus de trois nucléotides différents de ceux de la séquence nucléotidique représentée dans SEQ ID NO: 1. Le brin antisens contient une séquence nucléotidique II ayant une longueur égale à celle de la séquence nucléotidique représentée dans SEQ ID NO: 2 et n'ayant pas plus de trois nucléotides différents de ceux de la séquence nucléotidique représentée dans SEQ ID NO: 2. L'ARNsi et la composition pharmaceutique, et le conjugué correspondant peuvent traiter et/ou prévenir l'angio-oedème héréditaire (AOH) et/ou la thrombose.
PCT/CN2020/072813 2019-01-18 2020-01-17 Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation Ceased WO2020147847A1 (fr)

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EP4262822A4 (fr) * 2020-12-18 2024-10-02 Ionis Pharmaceuticals, Inc. Composés et méthodes pour moduler le facteur xii

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