WO2023088455A1 - 一种核酸、含有该核酸的药物组合物与siRNA缀合物及制备方法和用途 - Google Patents

一种核酸、含有该核酸的药物组合物与siRNA缀合物及制备方法和用途 Download PDF

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WO2023088455A1
WO2023088455A1 PCT/CN2022/133121 CN2022133121W WO2023088455A1 WO 2023088455 A1 WO2023088455 A1 WO 2023088455A1 CN 2022133121 W CN2022133121 W CN 2022133121W WO 2023088455 A1 WO2023088455 A1 WO 2023088455A1
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nucleotide
sirna
nucleotide sequence
nucleotides
seq
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French (fr)
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梁子才
张鸿雁
高山
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to a nucleic acid capable of inhibiting the expression of complement component 3 (CC3) gene and a pharmaceutical composition containing the nucleic acid and siRNA conjugate.
  • the present disclosure also relates to preparation methods and uses of these nucleic acids, pharmaceutical compositions and siRNA conjugates.
  • MG Myasthenia gravis
  • AchR-Ab acetylcholine receptor antibody
  • AchR cellular immunity dependent
  • complement acetylcholine receptor
  • NMJ neuromuscular junction
  • Complement component 3 is one of the key targets for the treatment of myasthenia gravis.
  • AchR-Ab binds to AchR to form an antibody-receptor complex, thereby forming C3 convertase and activating CC3 through the complement system cascade, thereby binding to cells and further forming C5 convertase, and in cells
  • Membrane attack complex MAC is formed on the surface, causing cell lysis and massive destruction of AchR, which leads to barriers to transmission of acetylcholine in the post-synaptic neuromuscular junction (NMJ) and muscle weakness.
  • CC3 can be silenced at the gene level, immune-related diseases related to the complement system, such as myasthenia gravis, can be effectively treated.
  • Small interfering RNA small interfering RNA
  • siRNA can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), so as to achieve the purpose of treating diseases. If we can inhibit the expression of CC3 gene, block the production of complement component 3, and suppress the occurrence of abnormal immune response from the mRNA level, it will undoubtedly be the most ideal treatment method.
  • RNAi RNA interference
  • the inventors of the present disclosure unexpectedly found that the following siRNA and its modified sequence provided by the present disclosure can specifically inhibit the expression of CC3 gene, and the pharmaceutical composition or siRNA conjugate containing the siRNA can specifically target the liver, thereby It can inhibit the expression of CC3 gene in the liver and realize the purpose of treating or preventing myasthenia gravis.
  • the inventors also invented siRNA and pharmaceutical compositions with higher activity.
  • the present disclosure provides an siRNA capable of inhibiting CC3 gene expression
  • the siRNA contains a sense strand and an antisense strand
  • each nucleotide in the siRNA is independently a modified or unmodified nucleotide
  • 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 partly reverse complementary to form a double strand region
  • the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1, and there are no more than 3 nucleotide differences
  • the nucleotide sequence II is different from the nucleotide sequence shown in SEQ ID NO:
  • the nucleotide sequences shown in 2 are equal in length and differ by no more than 3 nucleotides:
  • Z1 is A
  • Z2 is U
  • nucleotide sequence I contains a nucleotide Z3 corresponding to Z1
  • nucleotide sequence II contains a nucleotide Z3 corresponding to Z2
  • Nucleotide Z4 said Z4 being the first nucleotide at the 5' end of said antisense strand.
  • the present disclosure provides a pharmaceutical composition comprising 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 conjugation group conjugated to the siRNA.
  • the present disclosure provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing myasthenia gravis.
  • the present disclosure provides a method for treating and/or preventing myasthenia gravis, the method comprising administering an effective amount of the disclosed siRNA and/or pharmaceutical composition and/or siRNA conjugate to the needy of subjects.
  • the present disclosure provides a method of inhibiting CC3 gene expression in a cell, the method comprising contacting the cell with an effective amount of the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure.
  • the present disclosure provides a kit comprising an siRNA and/or a pharmaceutical composition and/or a siRNA conjugate of the present disclosure.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure have higher CC3 mRNA inhibitory activity, and/or can treat, prevent or alleviate pathological conditions or disease symptoms caused by CC3 gene expression.
  • the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure exhibits excellent target mRNA inhibitory activity in in vitro cell experiments.
  • the siRNA conjugate provided by the present disclosure has an inhibition rate of target sequence expression in the psiCHECK system in vitro as high as 98.7%, showing an excellent effect of inhibiting CC3 gene expression.
  • high inhibitory activities of 86.37% and 81.5% can also be achieved.
  • the siRNA provided by the present disclosure exhibited higher CC3 mRNA inhibitory activity in monkey liver primary cells, and at a siRNA concentration of 50 nM, the siRNA conjugates of the present disclosure inhibited CC3 mRNA in monkey liver primary cells.
  • the rate can reach 97.4%.
  • the siRNA conjugates provided by the present disclosure show excellent inhibitory effect on CC3 mRNA in animal model in vivo experiments.
  • the siRNA conjugate provided by the present disclosure shows a high inhibitory effect on CC3 mRNA in transgenic mice, and at a dose of 3 mg/kg, the inhibitory rate on CC3 mRNA in mice is 49.5%.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure can inhibit the expression of CC3 gene, effectively treat and/or prevent the symptoms of myasthenia gravis, and have a good application prospect.
  • CC3 mRNA refers to the mRNA having the sequence shown in Genbank accession number NM_000064.4.
  • target gene used in the present disclosure refers to the gene that transcribes the above-mentioned CC3 mRNA
  • target mRNA refers to the above-mentioned CC3 mRNA.
  • the uppercase letters C, G, U, and A represent the base composition of nucleotides;
  • the lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is methoxy Modified nucleotides;
  • the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that between the two nucleotides adjacent to the left and right of the letter s It is a phosphorothioate subunit connection;
  • P1 indicates that the adjacent nucleotide on the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, and
  • the letter combination VP indicates the letter combination
  • the nucleotide adjacent to the right of VP is a vinyl phosphate-modified nucleotide
  • the letter combination Ps indicates that the nucleotide adjacent to the right of
  • fluorinated modified nucleotide refers to the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with fluorine
  • non-fluorinated modified nucleotide refers 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.
  • Nucleotide analog means a nucleic acid capable of replacing nucleotides, but is structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymus
  • BNA bridged nucleic acid
  • methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • the expression "complementary” or “reverse complementary” can be used interchangeably and has the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases on one strand are combined with the bases on the other strand.
  • the bases of each pair 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) is paired.
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch means in the art that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary form.
  • 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, which means that the base type of the nucleotide at the same position has changed between the former and the latter, For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is recognized as a difference between the two nucleotide sequences. There is a nucleotide difference 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 occurs at that position.
  • Abasic nucleotides refer to monomeric compounds formed after nucleic acid bases in nucleotides are replaced by other groups or hydrogen atoms, such other groups include but are not limited to substituted or unsubstituted aryl or heteroaryl groups.
  • nucleoside monomer refers to the The type and sequence of nucleotides in siRNA or siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also known as 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 this disclosure are all commercially available.
  • conjugate means that two or more chemical moieties each having a specific function are covalently linked to each other; accordingly, a “conjugate” is Refers to the compound formed by covalent linkage between the various chemical moieties.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugates should be understood as the general term for siRNA conjugates, the general term for siRNA conjugates shown in formula (305) and formula (307), or formula (305), formula (307), formula (308) according to the context siRNA conjugates indicated.
  • conjuggate 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 disclosure.
  • substituted or substituted groups such as substituted alkyl, substituted alkoxy, substituted amino, substituted aliphatic, substituted A heteroaliphatic, substituted acyl, substituted aryl, or substituted heteroaryl of .
  • a “substituted” or “substituted” group refers to a group formed by replacing a hydrogen atom in the group with one or more substituents.
  • substituted alkoxy refers to a group formed by replacing one or more hydrogen atoms in an alkoxy group with a substituent.
  • the substituent is selected from the group consisting of C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 - C 10 haloalkyl, -OC 1 -C 10 alkyl, -OC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-OH, -OC 1 -C 10 haloalkyl, -SC 1 -C 10 alkyl, -SC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-SH, -SC 1 -C 10 haloalkyl, halogen substituent, -OH, -SH, -NH 2 , - C 1 -C 10 alkyl-NH 2 , -N(C 1 -C 10 alkyl)(C 1 -C 10 alkyl), -NH(C 1 -C 10 alkyl), -N(C 1 - C 10 alkyl
  • the substituent is C 1 -C 3 alkyl, C 6 -C 8 aryl, -OC 1 -C 3 alkyl, -OC 1 -C 3 alkylphenyl, halogen, - One of OH, -NH 2 , cyano or nitro.
  • substituents are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically infeasible, and/or inherently unstable .
  • alkyl refers to straight and branched chains having the specified number of carbon atoms, typically 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residue having a specific number of carbons all branched and straight chain forms having that number of carbons are intended to be encompassed; thus, for example, "butyl” is meant to include n-butyl, sec-butyl , isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to the same residues as alkyl but with two points of attachment.
  • alkenyl means an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double Obtained by removing a molecule of hydrogen.
  • the group can be in the cis or trans configuration of the double bond.
  • Typical 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 base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1- group, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl and the like.
  • 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 residues as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond formed through 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, etc.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residues as alkynyl but with two points of attachment.
  • alkoxy refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy, etc.
  • Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through oxygen bridges.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbons of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e. contains cyclic rings according to the 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 the same residues as aryl but with two points of attachment.
  • cycloalkyl refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. Rings may 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 or iodo, and the term “halogen” includes fluoro, chloro, bromo or iodo.
  • haloalkyl refers to an alkyl group as defined above in which the indicated number of carbon atoms is substituted by one or more, up to the maximum permissible number, halogen atoms.
  • haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic ring group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise stated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. A heteroatom in a heterocyclyl group can be unoxidized or oxidized. One or more nitrogen atoms, if present, may be unquaternized or quaternized. A heterocyclyl group is partially saturated or fully saturated. A heterocyclyl group can be attached to the rest of the molecule through any ring atom.
  • heterocyclic groups include, but are not limited to: dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl base, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl ), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), thiamorpholinyl
  • hydroxy protecting groups can be used in the present disclosure.
  • protecting groups render chemical functional groups insensitive to specific reaction conditions and can be added to and removed from that functional group in a 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 ed, John Wiley & Sons, New York, 1991, cited in Each of the above-mentioned documents is incorporated herein in its entirety.
  • protecting groups are stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups useful herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanth-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups useful 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 a marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of rhesus monkeys), mice, pigs, horses, donkeys, cows, sheep, rats, rabbits, and any species poultry.
  • non-human primates e.g., rhesus or other types of rhesus monkeys
  • mice pigs, horses, donkeys, cows, sheep, rats, rabbits, and any species poultry.
  • Treatment refers to means of obtaining a beneficial or desired result, including but not limited to therapeutic benefit.
  • Treatment benefit means eradicating or ameliorating the underlying disorder being treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, whereby improvement is observed in the subject, although the subject may still be afflicted by the underlying disorder.
  • prevention refers to the means of obtaining a beneficial or desired result, including but not limited to prophylactic benefit.
  • siRNA, siRNA conjugates, or pharmaceutical compositions may be administered to subjects at risk of developing a particular disease, or to subjects reporting one or more physical symptoms of a disease, even if possible A diagnosis of the disease has not yet been made.
  • the present disclosure provides an siRNA capable of inhibiting CC3 gene expression.
  • the siRNA of the present disclosure 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, and will not be repeated here.
  • the siRNA of the present disclosure contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, so
  • the antisense strand contains a section of nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and SEQ ID
  • the nucleotide sequence shown in NO:1 is equal in length, and there are no more than 3 nucleotide differences, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2, and there are not many At 3 nucleotide differences:
  • Z1 is A
  • Z2 is U
  • nucleotide sequence I contains a nucleotide Z3 corresponding to Z1
  • nucleotide sequence II contains a nucleotide Z3 corresponding to Z2
  • Nucleotide Z4 said Z4 being the first nucleotide at the 5' end of said antisense strand.
  • corresponding position refers to being at the same position in the nucleotide sequence, counting from the same end of the nucleotide sequence.
  • the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide at a position corresponding 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: 1 There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:2.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 includes a difference at the Z4 position, and Z4 is selected from A, C or g. In some embodiments, the nucleotide difference is a difference at the Z4 position, Z4 being selected from A, C or G. In some embodiments, Z3 is a nucleotide complementary to Z4 .
  • the siRNAs with the above-mentioned nucleotide differences have higher target mRNA inhibitory ability, and these siRNAs containing 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 substantially reverse complementary refers to two core There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementarity refers to no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity It 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:
  • said Z4 is the first nucleotide at the 5' end of the antisense strand
  • Z3 is selected from A, U, G or C
  • Z4 is a nucleotide complementary to Z3 ; in some embodiments Among them, Z 3 is A and Z 4 is U.
  • the lengths 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 length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/ 25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand and the antisense strand of the siRNA is 19/21, 21/23 or
  • the sense strand further comprises nucleotide sequence III
  • the antisense strand further comprises nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each 1-4 nuclei Nucleotide
  • the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary
  • the nucleotide sequence III is connected to 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 expression in the target mRNA that is identified by SEQ ID A nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by NO:1 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, and the core The base of the nucleotide sequence IV is A; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, according to the 5' end In the direction to the 3' end, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The lengths of both nucleotide sequences III and IV are 3 nucleotides.
  • the base composition of the nucleotide sequence III is UCU, and the base composition of the nucleotide sequence IV is AGA; at this time, the length ratio of the sense strand and the antisense strand is 22/22; or, the lengths of nucleotide sequences III and IV are both 4 nucleotides, according to the direction from the 5' end to the 3' end, the core
  • the base composition of the nucleotide sequence III is CUCU, and the base composition of the nucleotide sequence IV is AGAG; at this time, the length ratio of the sense strand and the antisense strand is 23/23.
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and according to the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is Cu , the base composition of the nucleotide sequence IV is AG; 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 bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the antisense strand also contains a nucleotide sequence V, and the length of the nucleotide sequence V is 1 to 3 nucleotides, which is connected to the 3' end of the antisense strand to form an antisense The 3' overhang of the 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, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can 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 improve the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding position of the target mRNA.
  • the ratio of the lengths 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 target mRNA silencing activity.
  • the nucleotides at the corresponding positions of the target mRNA refer to the nucleotides or nucleotide sequences adjacent to the 5' end of a nucleotide sequence of the target mRNA.
  • the nucleotide sequence of the target mRNA is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, or substantially reverse to the nucleotide sequence composed of nucleotide sequence II and nucleotide sequence IV That stretch of nucleotide sequence that is complementary or fully reverse complementary.
  • 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 a nucleotide sequence as shown in SEQ ID NO:7
  • the antisense strand contains a nucleotide sequence as shown in SEQ ID NO:8:
  • said Z4 is the first nucleotide at the 5' end of the antisense strand
  • Z3 is selected from A, U, G or C
  • Z4 is a nucleotide complementary to Z3 .
  • the siRNAs described in the present disclosure are siCCa1 and siCCa2 listed in Table 1.
  • each nucleotide in the siRNA of the present disclosure is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleosides
  • the siRNA of the present disclosure contains at least one modified nucleotide.
  • modified nucleotide refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or having Nucleotides with modified bases.
  • the modified nucleotides will not cause obvious 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 disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate with a modification group
  • at least a part of the phosphate group and/or the 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 modification group and/or Or a ribose group with a modifying 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 by 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 stability in plasma and gene silencing efficiency in animal experiments.
  • the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, according to the direction from the 5' end to the 3' end, the nucleotide sequence I At least the 7th, 8th, and 9th nucleotides are fluorine-modified nucleotides; according to the direction from the 5' end to the 3' end, at least the 2nd, 6th, 14th, and 16th positions of the nucleotide sequence II The nucleotides are fluorinated modified nucleotides.
  • the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and the fluorinated modified nucleotides in the nucleotide sequence I are not more than 5, And, according to the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides in the nucleotide sequence I are fluorinated modified nucleotides; in the nucleotide sequence II There are no more than 7 fluorinated modified nucleotides, and at least the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides.
  • the nucleus at the 7th, 8th, and 9th or 5th, 7th, 8th, and 9th positions of the nucleotide sequence I The nucleotides are fluorinated modified nucleotides, and the nucleotides in 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 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides, and in the antisense strand Nucleotides at the remaining positions are non-fluorinated modified nucleotides.
  • fluorine-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, which has the structure shown in the following formula (7).
  • Non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine 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 substituted by a non-fluorine group A sort of.
  • Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorine group are well known to those skilled in the art. 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, 2'-deoxynucleotides.
  • the 2'-alkoxy-modified nucleotides are methoxy-modified nucleotides (2'-OMe), as shown in formula (8).
  • the 2'-substituted alkoxy modified nucleotide for example, may be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), such as formula (9 ) shown.
  • the 2'-amino modified nucleotide (2'-NH 2 ) is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is shown in formula (11):
  • Nucleotide analogs are capable of replacing nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymidine ribonucleotides group of ribonucleotides.
  • the nucleotide analogs can be isonucleotides, bridged nucleic acid (BNA for short) or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNAs may contain five-membered, six-membered, or seven-membered ring bridge structures with "fixed" C3'-endosugar constrictions. Typically the bridge is incorporated at the 2'-,4'-position of the ribose to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA etc., wherein, LNA is as shown in formula (12), ENA is as shown in formula (13), and cET BNA is as shown in formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (15), and GNA is shown in 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 isonucleotide can 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 non-fluorine groups as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA.
  • each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' ribose group - Nucleotides in which a hydroxyl group is substituted by a methoxy group.
  • 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, in the sense strand, the 7th, 8th, and 9th positions of the nucleotide sequence I Or the 5th, 7th, 8th, and 9th nucleotides are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand Among them, the 2nd, 6th, 14th, 16th or 2nd, 6th, 8th, 9th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides, and the antisense 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 5th, 7th, 8th and 9th positions of the nucleotide sequence I in the sense strand of the siRNA
  • the nucleotides are fluorine-modified nucleotides
  • the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides
  • the siRNA The 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the nucleotide sequence II in the antisense strand are fluorinated nucleotides
  • the nucleotides at the rest of the antisense strand of the 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 fluorine-modified nucleotides, the sense of the siRNA
  • the nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and, according to the direction from the 5' end to the 3' end, the 2nd, 6th, and 14th positions of the nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at position 16 and 16 are fluorine-modified nucleotides, and the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides;
  • the 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the sense strand of the siRNA
  • the nucleotides at the remaining positions are methoxy-modified nucleotides, and, according to the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th positions of the nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at the position are fluorine-modified nucleotides, and the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.
  • the siRNA provided by the present disclosure is any one of siCCa1-M1, siCCa1-M2, siCCa1-M3, siCCa2-M1, siCCa2-M2, siCCa2-M3 listed in Table 1.
  • the siRNA with the above modification is not only low in cost, but also makes it difficult for ribonuclease in the blood to cut nucleic acid, thereby increasing the stability of nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the above-mentioned modified siRNA has a higher activity of inhibiting target mRNA.
  • the phosphate group with the modification group is a phosphorothioate subunit formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, The phosphate group with the modification group is a phosphorothioate subunit having a structure as shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
  • the phosphorothioate subunit linkage is present at at least one of the following positions: the first and the second of either end of the sense strand or the antisense strand between the nucleotides; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above.
  • phosphorothioate subunit linkages are present at all of the above positions except the 5' end of the sense strand.
  • phosphorothioate subunit linkages are present at all of the above positions except the 3' end of the sense strand.
  • the phosphorothioate subunit linkage is present in at least one of the following positions:
  • the siRNA provided by the present disclosure is any one of siCCa1-M1S, siCCa1-M2S, siCCa1-M3S, siCCa2-M1S, siCCa2-M2S, siCCa2-M3S listed in Table 1.
  • the 5' terminal nucleotide of the antisense strand of the siRNA 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 may have the following 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 a 5'-phosphate modification shown in formula (2)
  • the 5'-phosphate analog modified nucleotide is a nucleotide containing a 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 by the present disclosure is siCCa1-M1P1, siCCa1-M2P1, siCCa1-M3P1, siCCa2-M1P1, siCCa2-M2P1, siCCa2-M3P1, siCCa1-M1SP1, siCCa1-M2SP1, Any one of siCCa1-M3SP1, siCCa2-M1SP1, siCCa2-M2SP1, and siCCa2-M3SP1.
  • the inventors of the present disclosure unexpectedly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosome stability, but also has higher target mRNA inhibitory activity.
  • the siRNA provided in the present disclosure can be obtained by conventional siRNA preparation methods in the art (such as solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services.
  • a modified nucleotide group can be introduced into the siRNA described in the present disclosure by using a correspondingly modified nucleoside monomer, a method for preparing a correspondingly modified nucleoside monomer and introducing a modified nucleotide group Methods of siRNA are also well known to those skilled in the art.
  • the present disclosure provides a pharmaceutical composition containing the above-mentioned siRNA as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (magnetic nanoparticles, such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon nanotubes ( carbon nanotubes), mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine acid (poly(L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (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(aminoethylethylene phosphate) (poly(2-
  • the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:( 1-500), in some embodiments, the above-mentioned weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds routinely 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 tris hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5 buffer.
  • the protective agent may be at least one of 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 makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmol/kg (mOsm/kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • 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 can be used for subcutaneous, intramuscular or intravenous injection administration, but can also be administered to the lungs by spraying, or administered to other organs (such as the liver) through the lungs by spraying.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposomal formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipid.
  • the organic amine, helper lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety) or its One or more of pharmaceutically acceptable salts or derivatives, helper lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) described in CN103380113A or a pharmaceutically acceptable salt thereof:
  • Each X 101 or X 102 is independently O, S, NA or CA, wherein A is hydrogen or a C 1 -C 20 hydrocarbon chain;
  • Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 or R 107 is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic Aphatic groups, cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic groups, substituted or unsubstituted, branched or straight-chain acyl groups, substituted or unsubstituted, branched or straight chain aryl, substituted or unsubstituted, branched or straight chain heteroaryl;
  • x is an integer of 1-10;
  • R 103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl, the alkyl A radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 can be any of the following formulas (204)-(213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201) Possible points of attachment of the nitrogen atom in , where each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in CN103380113A.
  • the organic amine is an organic amine shown in formula (214) and/or an organic amine shown in formula (215):
  • the helper lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxyl (polyethylene glycol)]-2000.
  • the molar ratio among the organic amine, the helper lipid and the pegylated lipid is (19.7-80):(19.7-80 ):(0.3-50), such as (50-70):(20-40):(3-20).
  • the particles of the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, more typically, the liposome
  • the average diameter of the particles is from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the liposome particles have an average diameter of about 30, 40, 50, 60, 70 nm , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the weight of siRNA and total lipid is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18.
  • the weight ratio of siRNA to total lipid of the present disclosure is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1 :11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, only the siRNA provided by the present disclosure can be used to replace the existing siRNA; in some In the embodiment, it can be prepared according to the following method:
  • the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid around room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more of, for example, can be ethanol.
  • the siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an aqueous siRNA solution.
  • concentration of the buffered saline solution is 0.05-0.5M, such as 0.1-0.2M
  • the pH of the buffered saline solution is adjusted to 4.0-5.5, such as 5.0-5.2
  • amount of the buffered saline solution is such that the concentration of siRNA does not exceed 0.6mg /mL, for example, can be 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and/or potassium acetate.
  • the lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation.
  • the volume ratio of lipid solution and siRNA aqueous solution is 1:(2-5), for example, it can be 1:4.
  • the incubated liposome preparation Concentrate or dilute the incubated liposome preparation, remove impurities, and sterilize to obtain the pharmaceutical composition provided by the present disclosure, whose physical and chemical parameters are pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200nm, polydispersity index not higher than 0.30, osmotic pressure 250-400mOsm/kg; for example, physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100nm, more The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or simultaneously with the removal of impurities.
  • the method for removing impurities can adopt various existing methods, for example, can use tangential flow system, hollow fiber column, ultrafiltration under 100K Da condition, ultrafiltration exchange solution is phosphate buffer saline (PBS) of pH7.4.
  • PBS phosphate buffer saline
  • Various existing methods can be used for the sterilization method, for example, filtration sterilization on a 0.22 ⁇ m filter can be used.
  • the present disclosure provides an siRNA conjugate comprising the above-mentioned siRNA and a conjugation group conjugated to the siRNA.
  • the conjugation group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the siRNA, the linker and the targeting group or the The delivery auxiliary groups are sequentially linked covalently or non-covalently, each targeting group is selected from ligands capable of binding to cell surface receptors, each delivery auxiliary group is selected from ligands capable of increasing the siRNA conjugation Biocompatibility of the drug in the delivery target organ or tissue.
  • conjugate means that two or more chemical moieties each having a specific function are covalently linked to each other; correspondingly, “conjugate” means A compound formed by covalent linkage between the individual chemical moieties.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugates should be understood as a general term of multiple siRNA conjugates or siRNA conjugates represented by a certain chemical formula according to the context.
  • a "conjugate 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 disclosure.
  • the conjugation group comprises at least one targeting group that is pharmaceutically acceptable, and in some embodiments further comprises a linker, and the siRNA, the linker and the targeting group
  • the groups are linked sequentially.
  • the siRNA molecule may be non-covalently or covalently conjugated to the conjugating group, for example may be covalently conjugated to the conjugating group.
  • the conjugation site between the siRNA and the conjugating group can be at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site between the siRNA and the conjugation group is at the 3' end of the sense strand of the siRNA.
  • the conjugate group can be attached to the phosphate group, the 2'-position hydroxyl group or the base of the nucleotide. In some embodiments, the conjugate group can also be connected to the hydroxyl group at the 3'-position, at this time, the nucleotides are connected by 2'-5' phosphodiester bonds.
  • the conjugation group is usually connected to the phosphate group of the nucleotide; when the conjugation group is connected to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base.
  • 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.
  • the types of these linkers, targeting groups and connection methods with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
  • the siRNA and the conjugated group may be linked by acid-labile or reducible chemical bonds, and these chemical bonds can be degraded in the acidic environment of the endosome of the cell, thereby making the siRNA a free state.
  • the conjugation group can be attached to the sense strand of siRNA, so as to minimize the impact of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting group can be a ligand routinely used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference This article.
  • the pharmaceutically acceptable targeting group can be selected from one or more of the 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, manna Sugar, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoprotein, asialosugar residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.
  • lipophilic molecules such as cholesterol, bile acids, Vitamins (
  • 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 the liver surface asialoglycoprotein receptor (ASGPR).
  • the types of these ligands are well known to those skilled in the art, and their role is generally to bind to specific receptors on the surface of target cells and mediate delivery of siRNA linked to ligands to target cells.
  • the pharmaceutically acceptable targeting moiety can be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells.
  • each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).
  • the ligand is a sugar or a 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 may be a monosaccharide, disaccharide or 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 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 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 can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D- Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannose, ⁇ -D-mannose, ⁇ -D-mannose, ⁇ -D-mannose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-pyranose Galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, glucosamine, sialic acid, galactosamine, N
  • the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent , Two prices, three prices, four prices. It should be understood that the monovalent, divalent, trivalent, and tetravalent mentioned here refer to the formation of siRNA conjugates between siRNA molecules and conjugation groups containing galactose or N-acetylgalactosamine molecules as targeting groups.
  • the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4.
  • the pharmaceutically acceptable targeting group is N-acetylgalactosamine.
  • the siRNA described in the present disclosure when the siRNA described in the present disclosure is conjugated to a conjugation group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugation group comprising N-acetylgalactosamine, 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.
  • linkers, targeting groups and connection methods with siRNA please refer to the disclosure content of WO2015006740A2, the entire content of which is incorporated herein by reference.
  • the linker in the siRNA conjugates of the present disclosure has a structure as shown in formula (301):
  • k is an integer of 1-3;
  • L A has a structure containing an amide bond as shown in formula (302)
  • L B has a structure containing N-acylpyrrolidine as shown in formula (303), containing carbonyl and oxygen atoms
  • LC is based on hydroxymethyl Linking groups for aminomethane, dimethylolaminomethane or trishydroxymethylaminomethane;
  • n 302 , q 302 and p 302 are each independently an integer of 2-6, optionally, n 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer of 4-16, which can be Optionally, n 303 is an integer of 8-12, Indicates the site where the group is covalently attached.
  • each LA is connected to one of the targeting groups through an ether bond, and is connected through the oxygen atom of the hydroxyl group in the L C part and the L C part to form an ether bond;
  • L B is connected through the formula (303)
  • the carbonyl in the formula (303) forms a amide bond with the nitrogen atom of the amino group in the LC part, and forms a phosphate bond or a phosphorothioate bond with the siRNA through the oxygen atom in the formula (303).
  • the siRNA conjugate provided by the present disclosure has a structure as shown in formula (305):
  • Nu represents the siRNA provided by the present disclosure.
  • the linker in the siRNA conjugates of the present disclosure has the structure shown in formula (306):
  • n 306 is an integer of 0-3, and each p 306 is independently an integer of 1-6, Indicates the site where the group is covalently attached; the linking group is connected by an ether bond with the targeting group through the oxygen atom marked by *; the linking group is connected by at least one of the oxygen atoms marked by # One is connected to the siRNA by forming a phosphate bond or a phosphorothioate bond, and the rest are connected with an oxygen atom marked by # to form a hydroxyl group, or to form a C 1 -C 3 alkyl group to form a C 1 -C 3 alkane Oxygen;
  • siRNA conjugates of the present disclosure have a structure as shown in formula (307):
  • Nu represents the siRNA provided by the present disclosure.
  • the siRNA conjugate has a structure as shown in formula (308):
  • n1 is an integer selected from 1-3, and n3 is an integer selected from 0-4;
  • n1, m2 or m3 are independently an integer selected from 2-10;
  • R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each independently H, or are selected from the group consisting of the following groups: C 1 -C 10 alkyl, C 1 -C 10 haloalkane and C 1 -C 10 alkoxy;
  • R 3 is a group of structure shown in formula A59:
  • E 1 is OH, SH or BH 2
  • Nu is the siRNA of the present disclosure
  • L can be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:
  • Ra is selected from the group consisting of groups of formula A27-A45:
  • Rb is C 1 -C 10 alkyl
  • L is defined as a linear alkylene group for convenience, it may not be a linear group or be named differently, such as amine or alkenyl as a result of the substitutions and/or substitutions described above.
  • the length of L1 is the number of atoms in the chain connecting two attachment points.
  • a ring obtained by substituting a carbon atom of the linear alkylene group, such as a heterocyclylene or heteroarylene, is counted as one atom.
  • each M1 represents a targeting group, and its definition and optional range are the same as those of the above-mentioned targeting group.
  • each M1 is independently selected from one of the ligands that have affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
  • n1 can be an integer of 1-3, and n3 can be an integer of 0-4 , ensuring that the number of M 1 targeting groups in the siRNA conjugate is at least 2; in some embodiments, n1+n3 ⁇ 2, so that the number of M 1 targeting groups is at least 3, This makes it easier for the M1 targeting group to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the entry of the siRNA conjugate into cells through endocytosis.
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • the spatial positions between multiple M1 targeting groups can be adapted to the M1 targeting group and liver surface asialic acid
  • R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C
  • R 10 , R 11 , R 12 , R 13 , R 14 , or R 15 are each independently selected from H, methyl, and ethyl.
  • R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are all H.
  • R 3 is a group with the structure shown in formula A59, wherein E 1 is OH, SH or BH 2 , based on the availability of raw materials for preparation, in some embodiments, E 1 is OH or SH.
  • R2 is to realize the connection with the N atom on the nitrogen-containing skeleton and A59.
  • nitrogen-containing skeleton refers to a chain structure in which carbon atoms connected with R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected to N atoms.
  • R2 may be any linking group capable of linking the A59 group to the N atom on the nitrogen-containing backbone in an appropriate manner.
  • the siRNA conjugate shown in formula (308) is prepared by a solid-phase synthesis process, the R2 group needs to contain a connection site connected to the N atom on the nitrogen-containing skeleton at the same time and the linking site to P in R3 .
  • R can be B5, B6, B5' or B6':
  • the value range of q 2 may be an integer of 1-10, and in some embodiments, q 2 is an integer of 1-5.
  • L1 is to connect the M1 targeting group to N on the nitrogen-containing backbone, providing liver targeting function for the siRNA conjugate shown in formula (308).
  • L1 is selected from a linked combination of one or more of the groups of formulas A1-A26.
  • L1 is selected from a combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13.
  • L1 is selected from a connection combination of at least two of A1, A4, A8, A10 and A11.
  • L1 is selected from a connection combination of at least two of A1, A8, and A10.
  • L can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, the length of L is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atom.
  • each j1 is independently an integer of 2-10, and in some embodiments, j1 is an integer of 3-5.
  • each j2 is independently an integer of 2-10, and in some embodiments, each j2 is independently an integer of 3-5.
  • R' is C 1 -C 4 alkyl, and in some embodiments, R' is one of methyl, ethyl and isopropyl.
  • Ra is one of A27, A28, A29, A30 and A31, and in some embodiments, Ra is A27 or A28.
  • Rb is C 1 -C 5 alkyl, and in some embodiments, Rb is one of methyl, ethyl, isopropyl and butyl.
  • each of j1, j2, R', Ra, Rb in formulas A1-A26 is selected so that the M 1 targeting group is connected to the N atom on the nitrogen-containing skeleton, and the M 1 targeting The spatial position between the orientation groups is more suitable for the binding of the M 1 targeting group to the asialoglycoprotein receptor on the liver surface.
  • the siRNA conjugate has the formula (403), (404), (405), (406), (407), (408), (409), (410), (411 ), ( 412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):
  • the P atom in the formula A59 can be connected to any possible position in the siRNA sequence, for example, the P atom in the formula A59 can be connected to any nucleotide of the sense strand or the antisense strand of the siRNA; In some embodiments, the P atom in the formula A59 is connected to any nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in Formula A59 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in Formula A59 is attached to the end of the sense strand of the siRNA.
  • the terminus refers to the first 4 nucleotides counted from one end of the sense strand or the antisense strand.
  • the P atom in Formula A59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in Formula A59 is attached to the 3' end of the sense strand of the siRNA.
  • the siRNA conjugate represented by formula (308) enters the cell, upon unwinding, a separate siRNA antisense strand can be released to block CC3 mRNA translation Protein process that inhibits CC3 gene expression.
  • the P atom in formula A59 can be connected to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide 'Bases or bases of nucleotides. In some embodiments, the P atom in formula A59 can be linked to the 2' position, 3' position or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond.
  • the P atom in the formula A59 is connected to the oxygen atom formed after dehydrogenation of the 3' hydroxyl of the siRNA sense strand 3' terminal nucleotide (at this time, the P atom in the formula A59 can also be regarded as siRNA contained in the P atom in the phosphate group), or the P atom in the formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the sense strand of the siRNA, or the P atom in the formula A59 The P atom is attached to the nucleotide by replacing the hydrogen in the 5' hydroxyl of the 5' terminal nucleotide of the siRNA sense strand.
  • the inventors of the present disclosure unexpectedly found that the siRNAs of the present disclosure have significantly improved stability in plasma and higher CC3 mRNA silencing activity, and siRNA conjugates containing these siRNAs exhibited higher CC3 mRNA silencing activity. Accordingly, in some embodiments, the siRNA of the present disclosure may be one of the siRNAs shown in Table 1.
  • the uppercase letters C, G, U, and A indicate the base composition of nucleotides;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates The nucleotide adjacent to the left side of the letter f is a fluorinated nucleotide;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by phosphorothioate subunits;
  • P1 indicates the right side of the P1
  • One adjacent nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • P1 is VP, Ps or P representing a specific modification
  • the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is vinyl phosphate (5'-(E)- Vinylphosphonate, E-VP) modified nucleotides
  • the letter combination Ps indicates that the adjacent nucleotide on the right side of the letter combination Ps is a phosphorothioate modified nucleotide
  • the capital letter P indicates that the right side of the letter P is the same
  • the adjacent nucleotide is a 5'-phosphate nucleotide.
  • each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, and the non-bridging nucleotides in the phosphodiester bond or phosphorothioate bond are
  • the oxygen atom or sulfur atom has a negative charge, and it can exist in the form of hydroxyl or mercapto, and the hydrogen ions in the hydroxyl or mercapto can also be partially or completely replaced by cations.
  • the cation may be any cation, such as one of metal cations, ammonium ions NH 4 + , and organic ammonium cations.
  • the cation is selected from one or more of alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations.
  • the alkali metal ions may be K + and/or Na +
  • the cations formed by tertiary amines may be ammonium ions formed by triethylamine and/or ammonium ions formed by N,N-diisopropylethylamine.
  • the siRNA or siRNA conjugates described herein may exist at least in part in the form of a salt.
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond is at least partially combined with a sodium ion
  • the siRNA or siRNA conjugate described in the present disclosure is a sodium salt or a partial sodium salt form exists. Therefore, when referring to siRNA or siRNA conjugates described in the present disclosure, including but not limited to siRNA conjugates represented by any structural formula described in the present disclosure, it is intended to cover the sodium salt of the siRNA or siRNA conjugates or partial sodium salt form.
  • modified nucleotide groups can be introduced into the siRNAs described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are either commercially available or prepared using known methods.
  • siRNA conjugates can be synthesized by methods that have been described in detail in the prior art.
  • WO2015006740A2 describes the preparation methods of various siRNA conjugates in detail.
  • the siRNA conjugates of the present disclosure are obtained by means well known to those skilled in the art.
  • the preparation method of the structure represented by formula (305) is described in WO2014025805A1
  • the preparation method of the structure represented by formula (307) is described by Rajeev et al. in ChemBioChem 2015, 16, 903-908.
  • Chinese patent application CN110959011A also discloses in detail the method for preparing the siRNA conjugate represented by formula (308).
  • the contents of the above documents are incorporated herein in their entirety by reference.
  • siRNA conjugates of the present disclosure can also be used in combination with other pharmaceutically acceptable adjuvants, which can be one or more of various preparations or compounds routinely used in the art.
  • pharmaceutically acceptable adjuvants can be one or more of various preparations or compounds routinely used in the art.
  • description of the pharmaceutical composition please refer to the above-mentioned information about the present disclosure. Description of the pharmaceutical composition.
  • siRNA composition and siRNA conjugate of the present disclosure
  • the present disclosure provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing myasthenia gravis.
  • the present disclosure provides a method for preventing and/or treating myasthenia gravis, the method comprising administering an effective amount of siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to those in need of subjects.
  • the siRNA and/or pharmaceutical composition and/or siRNA conjugate disclosed herein can be used for preventing and/or treating myasthenia gravis, or for preparing a medicament for preventing and/or treating myasthenia gravis.
  • the diseases may share one or more risk factors, causes or consequences.
  • the term "administration/administration” refers to a method or approach that at least partially localizes the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure at a desired site to produce a desired effect
  • the siRNAs, pharmaceutical compositions and/or siRNA conjugates of the present disclosure are placed into a subject.
  • Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, local administration results in delivery of more of the siRNA conjugate to a specific site compared to the subject's systemic circulation; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to the subject's basic systemic circulation.
  • an administration is employed that delivers the drug to the liver.
  • Administration to a subject may be by any suitable route known in the art, including, but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual).
  • oral or parenteral routes such as intravenous, intramuscular, subcutaneous, transdermal Drugs, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual).
  • the administration frequency can be once or more every day, every week, every two weeks, every three weeks, every month, every two months, every quarter, every six months or every year.
  • the dosage of the siRNA, the pharmaceutical composition or the siRNA conjugate described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, body weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., by determining the LD50 (the lethal dose causing 50% of the population to die) and the ED50 (in dose response, the dose eliciting 50% of the maximum response intensity, In the qualitative response, it refers to the dose that causes 50% of the test subjects to have a positive response).
  • a range of dosage for use in humans can be derived based on the data obtained from cell culture assays and animal studies.
  • siRNA conjugates can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in some embodiments 0.05-20 mg/kg body weight kg body weight, 0.1-15 mg/kg body weight in other embodiments, 0.1-10 mg/kg body weight in other embodiments;
  • siRNA dosage May be 0.001-50 mg/kg body weight, in some embodiments 0.01-10 mg/kg body weight, in some embodiments 0.05-5 mg/kg body weight, in some embodiments 0.1-3 mg/kg body weight.
  • siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure into cells, the purpose of inhibiting the expression of CC3 gene in cells can also be achieved through the mechanism of RNA interference.
  • the amount of siRNA in the provided siRNA, pharmaceutical composition and/or siRNA conjugate is generally such an amount: it is enough to reduce the expression of target mRNA, and cause An extracellular concentration of 1 pM to 1 ⁇ M, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM at the surface of the target cell.
  • the amount necessary to achieve this local concentration will vary depending on various factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local or systemic), etc. .
  • the concentration at the site of delivery can be significantly higher than the concentration at the surface of the target cell or tissue.
  • the present disclosure provides a kit comprising an effective amount of at least one of an siRNA of the present disclosure, a pharmaceutical composition, and a siRNA conjugate.
  • kits described herein can provide the siRNA, the pharmaceutical composition, and/or the siRNA conjugate in one container.
  • a kit described herein may comprise a container providing a pharmaceutically acceptable excipient.
  • the kit may also contain other components, such as stabilizers or preservatives.
  • the kits described herein can comprise at least one additional therapeutic agent in a container other than the container providing the siRNA, pharmaceutical composition, and/or siRNA conjugate described herein.
  • the kit may comprise a mixing kit for mixing the siRNA, the pharmaceutical composition and/or the siRNA conjugate with a pharmaceutically acceptable carrier and/or excipient or other ingredients, if any. manual.
  • the siRNA and the pharmaceutically acceptable carrier and/or adjuvant as well as the siRNA, pharmaceutical composition and/or siRNA conjugate, and/or the pharmaceutically acceptable adjuvant can be in any form Provided, for example, in liquid form, dry form or lyophilized form.
  • the siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or siRNA conjugate and/or pharmaceutically acceptable adjuvant are substantially pure and/or sterile .
  • sterile water can be provided in kits of the present disclosure.
  • the reagents and culture media used in the following examples are all commercially available, and the operations such as nucleic acid electrophoresis and real-time PCR used are all referred to in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). method to proceed.
  • siRNA conjugate 1 was synthesized.
  • the siRNA conjugate is the siRNA conjugate formed after conjugating the L-9 conjugate molecule in Preparation Example 1 of CN110959011A and the siRNA numbered siCCa1-M1S in Table 2.
  • siRNA conjugate 1 in Table 2 was prepared, the only difference being that the sense strand and antisense strand of the siRNA contained in siRNA conjugate 1 were as shown in Table 2 As shown; according to the nucleic acid sequence of the siRNA conjugate numbered siCCa1-M1S in the following Table 2, the nucleoside monomers were connected one by one to synthesize the sense strand and the antisense strand of the siRNA.
  • siRNA conjugate 1 After diluting siRNA conjugate 1 to a concentration of 0.2 mg/mL (calculated as siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25°C)), the An instrument (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier) was used for molecular weight detection.
  • Sense strand theoretical value, 7530.70, measured value, 7531.20; antisense strand: theoretical value, 7062.67, measured value, 7061.84, the measured value is consistent with the theoretical value, indicating that the synthesized siRNA conjugate 1 is a double-stranded nucleic acid sequence designed for the target .
  • siRNA conjugate 1 has a structure shown in formula (403), wherein Nu is the siRNA group formed by the siRNA corresponding to siCCa1-M1S in Table 2, and the conjugating group in the siRNA conjugate 1 is connected to the siRNA group The ribose 3' position of the 3' terminal nucleotide of the sense strand, and the siRNA conjugate is in the sodium salt form.
  • the uppercase letters C, G, U, and A indicate the base composition of nucleotides;
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f indicates The adjacent nucleotide on the left side of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by phosphorothioate subunits.
  • HepG2 cells were seeded in a 24-well plate at 7.5 ⁇ 10 4 cells/well. After 16 hours, when the cell growth density reached 70-80%, the complete H-DMEM medium in the culture wells was exhausted, and 500 ⁇ L Opti-MEM was added to each well for culture. Base (GIBCO company) continued to cultivate for 1.5h.
  • siRNA Conjugate 1 of the present disclosure was prepared into 10 ⁇ M, 1 ⁇ M and 0.1 ⁇ M siRNA conjugate working solutions with DEPC water, respectively.
  • each 1A solution contains 3 ⁇ L of the above-mentioned siRNA conjugate working solution at concentrations of 10 ⁇ M, 1 ⁇ M and 0.1 ⁇ M and 47 ⁇ L of Opti-MEM medium.
  • each 1B solution contains 1 ⁇ L Lipofectamine TM 2000 and 49 ⁇ L Opti-MEM medium.
  • a part of the 1B solution was mixed with the obtained 1A solution of each siRNA, and incubated at room temperature for 20 min to obtain a transfection complex 1X of each siRNA conjugate.
  • transfection complex 1X Mix a portion of 1B solution with 50 ⁇ L of Opti-MEM medium, and incubate at room temperature for 20 min to obtain transfection complex 1X’.
  • each transfection complex 1X In the culture wells, add each transfection complex 1X, mix evenly, and add 100 ⁇ L/well to obtain transfections with final concentrations of each siRNA conjugate of about 50 nM, 5 nM and 0.5 nM (calculated as siRNA). Transfection complex, each transfection complex 1X transfected 2 culture wells to obtain a transfection mixture containing siRNA conjugate, which was recorded as the test group.
  • the transfection complex 1X' was added at a rate of 100 ⁇ L/well to obtain a transfection mixture without siRNA, which was recorded as the blank control group.
  • H-DMEM complete medium containing 20% FBS was added to each well.
  • the 24-well plate was placed in a CO 2 incubator to continue culturing for 24 h.
  • RNAVzol purchased from Viglass Biotechnology (Beijing) Co., Ltd., Cat. No. N002
  • Viglass Biotechnology Beijing Co., Ltd., Cat. No. N002
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ L of the above cDNA-containing solution as a template, use SYBR qPCR SuperMix Plus Kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) provides 20 ⁇ L of reagents to configure the qPCR reaction system.
  • the PCR primer sequences used to amplify the target gene CC3 and the internal reference gene GAPDH are shown in Table 3 As indicated, the final concentration of each primer was 0.25 ⁇ M.
  • Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 30s, annealing at 60°C for 30s, and annealing at 72°C. After extending for 30 s at °C, repeating the above-mentioned denaturation, annealing, and extension process for a total of 40 times, a product W containing amplified target gene CC3 and internal reference gene GAPDH was obtained.
  • the product W was then incubated at 95°C for 15s, 60°C for 1min, and 95°C for 15s, and the melting curves of the target gene CC3 and the internal reference gene GAPDH in the product W were respectively measured by a real-time fluorescent quantitative PCR instrument, and the target gene CC3 and the internal reference gene GAPDH were obtained. Ct value.
  • the comparative Ct ( ⁇ Ct) method was used to perform relative quantitative calculation of the target gene CC3 in each test group, and the calculation method was as follows:
  • ⁇ Ct (test group) Ct (target gene of test group) – Ct (internal reference gene of test group)
  • ⁇ Ct (control group) Ct (control group target gene) – Ct (control group internal reference gene)
  • ⁇ Ct (test group) ⁇ Ct (test group) - ⁇ Ct (average of the control group)
  • ⁇ Ct (control group) ⁇ Ct (control group) - ⁇ Ct (control group average)
  • ⁇ Ct average of control group
  • ⁇ Ct control group
  • the expression level of CC3 mRNA in the test group was normalized, and the expression level of CC3 mRNA in the blank control group was defined as 100%.
  • Test group CC3 mRNA relative expression level 2 - ⁇ Ct (test group) ⁇ 100%
  • Test group CC3 mRNA inhibition rate (1- test group CC3 mRNA relative expression level) ⁇ 100%
  • the inhibition rate of CC3 mRNA by siRNA conjugate 1 is summarized in Table 4.
  • the mRNA inhibition rate is the arithmetic mean of the test group CC3 mRNA inhibition rates measured in two culture wells.
  • the siRNA conjugate 1 provided by the present disclosure has a higher inhibitory activity in the HepG2 cell line.
  • the CC3 mRNA inhibition rate is as high as 98.7%, and at 5nM and 0.5nM siRNA Concentration, also can reach 86.37% and 81.5% high inhibitory activity.
  • CM Culture medium The Primary Cell Solution
  • Thawing medium The Primary Cell Solution
  • FBS fetal bovine serum
  • CM seeding medium The Primary Cell Solution company
  • CM seeding medium The Primary Cell Solution company
  • CM Culture medium The Primary Cell Solution
  • siRNA Conjugate 1 was prepared into 20 ⁇ M (calculated as siRNA) working solution of siRNA Conjugate 1 with DEPC water.
  • each 2A solution contains 3 ⁇ L of the above-mentioned siRNA conjugate 1 working solution and 97 ⁇ L of cell maintenance medium in sequence.
  • each 2B solution contains 2 ⁇ L Lipofectamine TM 2000 and 98 ⁇ L cell maintenance medium.
  • a part of 2B solution was mixed with the obtained 2A solution of siRNA conjugate 1, respectively, and incubated at room temperature for 20 min to obtain transfection complex 2X of siRNA conjugate 1.
  • each siRNA transfection complex 2X was transfected into 2 culture wells to obtain a transfection mixture containing siRNA conjugate 1, which was recorded as the test group.
  • the blank transfection complex 2X' was added respectively, and the addition amount was 200 ⁇ L/well to obtain the transfection mixture without siRNA, which was recorded as the blank control group.
  • RNA in the cells in each well was extracted using the UNIQ-10 Column Total RNA Extraction Kit (purchased from Sangon, Cat. No. B511361-0100) according to the method described in the instructions to obtain solutions containing total RNA respectively.
  • RNA For each well of cells, a solution containing 1 ⁇ g of total RNA was taken respectively, and the reagents provided by the reverse transcription kit GoldenstarTM RT6 cDNA Synthesis Kit (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd., catalog number TSK301M) were used, wherein GoldenstarTM Oligo ( dT)17 was used as a primer, and 20 ⁇ L of a reverse transcription reaction system was prepared according to the reverse transcription operation steps in the kit instruction manual, and the total RNA of cells in each well was reverse transcribed.
  • the reverse transcription kit GoldenstarTM RT6 cDNA Synthesis Kit
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 50°C for 50 minutes, then incubate at 85°C for 5 minutes, and finally incubate at 4°C for 30 seconds. Add 80 ⁇ l of DEPC water to the system to obtain 100 ⁇ l of cDNA-containing solution.
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ L of the above cDNA-containing solution as a template, use SYBR qPCR SuperMix Plus Kit (purchased from Nearshore Protein Technology Co., Ltd., Cat. No. E096-01B) provides 20 ⁇ l of reagents to configure the qPCR reaction system.
  • the PCR primer sequences used to amplify the target gene CC3 and the internal reference gene GAPDH are shown in Table 5 As indicated, the final concentration of each primer was 0.25 ⁇ M. Place each qPCR reaction system containing the target gene CC3 on the ABI StepOnePlus Real-Time PCR instrument, and use a three-step method for amplification.
  • the amplification program is pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 30s, and at 55°C.
  • Anneal for 30s extend at 72°C for 30s, repeat the above-mentioned denaturation, annealing, and extension process 40 times, and obtain the product W containing the amplified target gene CC3; for the qPCR system containing the internal reference gene GAPDH, use the same method as above Perform qPCR amplification, the only difference is that the annealing temperature in the amplification program is 60°C, and the product W' containing the amplified internal reference gene GAPDH is obtained.
  • the products W and W' were then incubated at 95°C for 15s, 60°C for 1min, and 95°C for 15s in sequence, and the melting curves of the target gene CC3 and the internal reference gene GAPDH in the products W and W' were respectively measured by real-time fluorescent quantitative PCR instrument to obtain the target Ct values of gene CC3 and reference gene GAPDH.
  • the siRNA conjugate 1 provided by the present disclosure showed high CC3 mRNA inhibitory activity in monkey primary hepatocytes, and the CC3 mRNA inhibitory rate was as high as 97.4% at an siRNA concentration of 50 nM.
  • siRNA conjugate inhibits CC3 mRNA in mice (in vivo)
  • mice purchased from Shanghai N forcing Model Biotechnology Co., Ltd.
  • mice in the experimental group mice in the experimental group and mice in the blank control group, respectively. Rats were injected subcutaneously.
  • Each experimental group of mice was given siRNA conjugate 1 at a dose of 3 mg/kg mouse body weight (calculated as siRNA), and siRNA conjugate 1 contained 0.6 mg/mL (calculated as siRNA ) in the form of 1 ⁇ PBS solution of siRNA conjugates, with an administration volume of 5 mL/kg; each mouse in the blank control group was given 1 ⁇ PBS, with an administration volume of 5 mL/kg.
  • RNA later (Sigma Aldrich); 1mL Trizol (Sigma Company) was added to each liver tissue. ), crushed 3 times in a Tissuelyset II automatic tissue homogenizer, each time for 30s, to obtain liver tissue homogenate, add 0.2mL chloroform to it, and let it stand for 3min. Centrifuge at 12,000 rpm for 10 min at 4°C, and take 0.4 mL of the supernatant. Add 0.5mL isopropanol to the supernatant and let it stand at room temperature for 10min.
  • RNA in the liver tissue of each mouse take 10.5 ⁇ L of total RNA aqueous solution containing 1 ⁇ g of total RNA, use the reverse transcription kit Reverse Transcription System (purchased from Promega, Cat. No. A3500), and reverse The recording operation steps were prepared as 20 ⁇ L of reverse transcription reaction system, and the total RNA was reverse transcribed.
  • the reverse transcription conditions are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 42°C for 30 minutes, then incubate at 95°C for 5 minutes, and finally incubate at 4°C for 5 minutes. Add 80 ⁇ L of DEPC water to the recording reaction system to obtain a solution containing cDNA.
  • each reverse transcription reaction system 5 ⁇ L of the above-mentioned cDNA-containing solution was used as a template, and 20 ⁇ L of a qPCR reaction system was prepared using the reagents provided by the SYBR select Master Mix kit (Applied biosystem company), which was used to amplify the target gene CC3
  • the PCR primer sequences of the internal reference gene GAPDH are shown in Table 7 below, and the final concentration of each primer is 0.25 ⁇ M.
  • Each qPCR reaction system was placed on the ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method.
  • the amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30s, annealing at 60°C for 30s, and extension at 72°C for 30s. After repeating the above-mentioned denaturation, annealing, and extension processes for a total of 40 times, a product W containing amplified target gene CC3 and internal reference gene GAPDH was obtained. The product W was then incubated at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s. The real-time fluorescent quantitative PCR instrument collected the melting curves of the target gene CC3 and the internal reference gene GAPDH in the product W respectively, and obtained the Ct of the target gene CC3 and the internal reference gene GAPDH. value.
  • the comparative Ct ( ⁇ Ct) method was used to carry out relative quantitative calculation of the expression level of the target gene CC3 mRNA in each test group, and the calculation method was as described in Experimental Example 1 above.
  • siRNA conjugate 1 after giving siRNA conjugate 1 to mice at a dose of 3 mg/kg mouse body weight (in terms of siRNA), the inhibitory rate of siRNA conjugate 1 to CC3 mRNA in mice was 49.5%, showing that Better CC3 mRNA inhibitory effect.

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Abstract

提供了一种抑制补体成分3(CC3)基因表达的siRNA，含有该siRNA的药物组合物和siRNA 缀合物。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，所述正义链含有核苷酸序列I，所述核苷酸序列Ⅰ与SEQ ID NO: 1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述反义链含有核苷酸序列Ⅱ，所述核苷酸序列Ⅱ与SEQID NO: 2所示的核苷酸序列长度相等，且不多于3个核苷酸差异。还提供了siRNA、药物组合物和siRNA 缀合物，可以有效治疗和/或预防补体成分3介导的相关疾病，例如重症肌无力症(MG)的方法。

Description

一种核酸、含有该核酸的药物组合物与siRNA缀合物及制备方法和用途 技术领域
本公开涉及一种能够抑制补体成分3(CC3)基因表达的核酸和含核酸的药物组合物与siRNA缀合物。本公开还涉及这些核酸、药物组合物与siRNA缀合物的制备方法和用途。
背景技术
重症肌无力(MG)是一种主要由乙酰胆碱受体抗体(AchR-Ab)介导、细胞免疫依赖、补体参与，累及神经肌肉接头(NMJ)突触后膜上乙酰胆碱受体(AchR)的获得性自身免疫学疾病。
补体成分3(CC3)是治疗重症肌无力的关键靶点之一。在重症肌无力患者中，AchR-Ab与AchR结合形成抗体-受体复合物，从而经由补体系统级联作用形成C3转化酶并活化CC3，从而与细胞结合并进一步形成C5转化酶，并在细胞表面组建攻膜复合物(MAC)，引起细胞溶解，并使AchR大量破坏，导致神经肌肉接头(NMJ)突触后膜传递乙酰胆碱障碍而产生肌无力。研究表明，通过特异性抑制CC3的作用，进而阻止攻膜复合物的形成，从而阻断攻膜复合物对神经肌肉接头的破坏以及后续促炎因子的产生，发挥免疫抑制作用，治疗重症肌无力。
鉴于此，若能从基因水平沉默CC3基因表达，可以有效治疗与补体系统有关的免疫相关疾病，例如重症肌无力。
小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以序列特异性的方式抑制或阻断任何感兴趣的目的基因的表达，从而达到治疗疾病的目的。若能从mRNA层面，抑制CC3基因表达，阻断补体成分3的生成，抑制异常免疫反应的发生，无疑将是最为理想的治疗手段。
发明内容
本公开的发明人意外发现，具有本公开提供的如下siRNA及其修饰序列能够特异性地抑制CC3基因的表达，含有该siRNA的药物组合物或siRNA缀合物能够特异性地靶向肝脏，从而可以抑制肝脏中CC3基因的 表达，实现治疗或预防重症肌无力的目的。此外，发明人还发明了具有较高活性的siRNA和药物组合物。
在一方面，本公开提供了一种能够抑制CC3基因表达的siRNA，该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，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，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-UGGCCCUACUGCAGCUAAZ 1-3'(SEQ ID NO:1)；
5'-Z 2UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:2)，
其中，Z 1为A，Z 2为U，并且，所述核苷酸序列I中包含位置对应于Z 1的核苷酸Z 3，所述核苷酸序列II中包含位置对应于Z 2的核苷酸Z 4，所述Z 4是所述反义链5'末端的第一个核苷酸。
在另一方面，本公开提供了一种药物组合物，所述药物组合物含有本公开的siRNA和药学上可接受的载体。
在又一方面，本公开提供了一种siRNA缀合物，所述siRNA缀合物含有本公开提供的siRNA以及缀合连接至该siRNA的缀合基团。
在又一方面，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防重症肌无力的药物中的用途。
在又一方面，本公开提供了一种治疗和/或预防重症肌无力的方法，所述方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予有需要的受试者。
在又一方面，本公开提供了一种抑制细胞中CC3基因表达的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物与所述细胞接触。
在又一方面，本公开提供了一种试剂盒，所述试剂盒含有本公开的siRNA和/或药物组合物和/或siRNA缀合物。
以引用的方式并入
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本文，其程度与每一单独的出版物、专利以及专利申请均专门并且单独地以引用的方式并入本文的程度相同。
有益效果
本公开提供的siRNA、药物组合物和siRNA缀合物具有较高的CC3mRNA抑制活性，和/或能治疗、预防或缓解CC3基因表达引起的病理状况或疾病症状。
在一方面，本公开提供的siRNA、药物组合物或siRNA缀合物在体外细胞实验中显示出优异的靶mRNA抑制活性。例如，在50nM浓度下，本公开提供的siRNA缀合物在体外psiCHECK系统中的目标序列表达量抑制率高达98.7％，显示出优异的抑制CC3基因表达的效果。在5nM和0.5nM的siRNA浓度下，也能达到86.37％和81.5％的高抑制活性。又例如，本公开提供的siRNA在猴肝原代细胞中显示出较高的CC3 mRNA抑制活性，在50nM的siRNA浓度下，本公开的siRNA缀合物在猴肝原代细胞中对CC3 mRNA抑制率可达97.4％。
在另一方面，本公开提供的siRNA缀合物在动物模型体内实验中显示出优异的CC3 mRNA抑制效果。例如，本公开提供的siRNA缀合物在转基因小鼠体内显示出较高的CC3 mRNA抑制效果，在3mg/kg的剂量下，对小鼠体内CC3 mRNA的抑制率为49.5％。
由此说明，本公开提供的siRNA、药物组合物以及siRNA缀合物能够抑制CC3基因的表达，有效治疗和/或预防重症肌无力症状，具有良好的应用前景。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
在本公开中，CC3 mRNA是指具有Genbank注册号为NM_000064.4所示序列的mRNA。进一步地，若无其它说明，本公开中所使用的术语“靶基因”是指转录上述CC3 mRNA的基因，术语“靶mRNA”是指上述CC3 mRNA。
定义
在上文及下文中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；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亚基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。
在上文及下文中，所述“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。“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。所述“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。
在本文的上下文中，表述“互补”或“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，一条链的碱基与另一条链上的碱基各自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。无碱基核苷酸是指核苷酸中的核酸碱基被其它基团或氢原子代替后形成的单体化合物，该其它基团包括不限于取代或未取代的芳香基或杂芳基。
在上文及下文中，特别是在描述本公开的siRNA、含siRNA的组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNA phosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。
在本公开的上下文中，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siRNA缀合物的总称、式(305)和式(307)所示的siRNA缀合物总称，或式(305)、式(307)、式(308)所示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开 的siRNA缀合物的特定化合物。
在上文或下文中，“经取代的”或“被取代的”基团，如经取代的烷基、经取代的烷氧基、经取代的氨基、经取代的脂族基团、经取代的杂脂族基团、经取代的酰基、经取代的芳基或经取代的杂芳基。其中，如无其他说明，“经取代的”或“被取代的”基团是指该基团中的氢原子被一个或多个取代基所替代而形成的基团。例如，“经取代的烷氧基”是指烷氧基中的一个或多个氢原子被取代基所替代而形成的基团。本领域技术人员能够理解，可用于本公开应用的化合物中可以包含各种取代基，只要是该取代基的引入不会影响本公开的功能，能够实现本公开的目的，就可用于本公开。在一些实施方式中，所述取代基选自于由以下基团所组成的组：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤代烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)。在一些实施方式中，所述取代基是C 1-C 3烷基、C 6-C 8芳基、-OC 1-C 3烷基、-OC 1-C 3烷基苯基、卤素、-OH、-NH 2、氰基或硝基中的一种。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或本身不稳定的任何取代或取代模式。
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1至20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C 1-C 6烷基包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲 丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两分子氢而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个连接点的残基。
如本文所使用的，“烷氧基”是指通过氧桥连接的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥连接的碳原子。
如本文所使用的，“芳基”是指通过从环碳原子中除去氢原子而衍生自芳香族单环或多环烃环系统形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的至少一个环是完全不饱和的，即，包含根据Hückel理论的环状、离域的(4n+2)π-电子体系。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的子集，指与芳基相同、但具有两个连接点的残基。
如本文所使用的，“环烷基”是指非芳香碳环，通常具有3至7个环 碳原子。环可以是饱和的，或具有一个或多个碳-碳双键。环烷基的实例包括环丙基、环丁基、环戊基、环戊烯基、环己基和环己烯基，以及桥联和笼状环基团，如降冰片烷(norbornane)。
如本文所使用的，“卤素取代基”或“卤代”指氟代、氯代、溴代或碘代，术语“卤素”包括氟、氯、溴或碘。
如本文所使用的，“卤代烷基”是指指定数量的碳原子被一个或多个、直至最大允许数量的卤素原子取代的如上述所定义的烷基。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。
“杂环基”是指稳定的3-至18-元非芳香族环基，包含2-12个碳原子和1-6个杂原子，所述杂原子选自氮、氧和硫。除非说明书中另有说明，杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环基中的杂原子可以是未氧化的或者是被氧化的。一个或多个氮原子(如果存在的话)可以是未被季铵化的或者是被季铵化的。杂环基是部分饱和或完全饱和的。杂环基可以通过任何环原子连接至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1,3]二硫酰基(thienyl[1,3]dithianyl)、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异噁唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂吡咯烷基、噁唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三硫酰基(trithianyl)、四氢吡喃基、硫代吗啉基(thiomorpholinyl)、硫杂吗啉基(thiamorpholinyl)、1-氧代硫吗啉基(1-oxo-thiomorpholinyl)和1,1-二氧代硫吗啉基(1,1-dioxo-thiomorpholinyl)。
在本公开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添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。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，本文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-苯基氧杂蒽-9-基(Pixyl)和9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方式中，本文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)。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开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、兔和任何种类的家禽。
如本文所使用的“治疗”指的是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。
如本文所使用的“预防”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作出。
siRNA
在一方面，本公开提供了一种能够抑制CC3基因表达的siRNA。
本公开的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，在此不再赘述。
本公开的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，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且 不多于3个核苷酸差异：
5'-UGGCCCUACUGCAGCUAAZ 1-3'(SEQ ID NO:1)；
5'-Z 2UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:2)，
其中，Z 1为A，Z 2为U，并且，所述核苷酸序列I中包含位置对应于Z 1的核苷酸Z 3，所述核苷酸序列II中包含位置对应于Z 2的核苷酸Z 4，所述Z 4是所述反义链5'末端的第一个核苷酸。
在上文与下文中，“位置对应”是指从核苷酸序列相同端起算，处于核苷酸序列中相同的位置。例如，核苷酸序列I的3'端第1个核苷酸是位置对应于SEQ ID NO:1的3'端第1个核苷酸的核苷酸。
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间的核苷酸差异包括Z 4位置处的差异，且Z 4选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 4位置处的差异，Z 4选自A、C或G。在一些实施方式中，Z 3是与Z 4互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶mRNA抑制能力，而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，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碱基错配。
在一些实施方式中，核苷酸序列I是SEQ ID NO:3所示的核苷酸序列，核苷酸序列II是SEQ ID NO:4所示的核苷酸序列：
5'-UGGCCCUACUGCAGCUAAZ 3-3'(SEQ ID NO:3)；
5'-Z 4UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:4)，
其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸；在一些实施方式中，Z 3为A，Z 4为U。
所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。这样，本公开提供的siRNA正义链和反义链的长度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。在一些实施方式中，所述siRNA正义链和反义链的长度比为19/21、21/23或23/25。
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长度各自为1-4个核苷酸；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者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端。在一些实施方式中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:1表示的核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。
在一些实施方式中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UCU，核苷酸序列IV的碱基组成为AGA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CUCU，核苷酸序列IV的碱基组成为AGAG；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度 为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU，核苷酸序列IV的碱基组成为AG；此时，正义链和反义链的长度比为21/21。
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。
在一些实施方式中，所述反义链还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3'末端，构成反义链的3'突出端。由此，本公开提供的siRNA正义链和反义链的长度比可以是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或23/26。在一些实施方式中，所述核苷酸序列V的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/23或23/25。
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约合成成本，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高siRNA反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方式中，本公开的siRNA的正义链和反义链的长度之比为19/21或21/23，此时，本公开的siRNA具有更好的靶mRNA沉默活性。
靶mRNA的相应位置的核苷酸是指与靶mRNA的一段核苷酸序列在5'末端相邻的核苷酸或核苷酸序列。该段靶mRNA的核苷酸序列是与核苷酸序列II实质上反向互补或完全反向互补，或者与核苷酸序列II和核苷酸序列IV构成的核苷酸序列实质上反向互补或完全反向互补的那段核苷酸序列。
在一些实施方式中，所述siRNA的正义链含有如SEQ ID NO:5所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:6所示的核苷酸序列：
5'-UGGCCCUACUGCAGCUAAZ 3-3'(SEQ ID NO:5)；
5'-Z 4UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:6)；
或者，所述siRNA的正义链含有如SEQ ID NO:7所示的核苷酸序 列，所述反义链含有如SEQ ID NO:8所示的核苷酸序列：
5'-CUUGGCCCUACUGCAGCUAAZ 3-3'(SEQ ID NO:7)；
5'-Z 4UUAGCUGCAGUAGGGCCAAGAG-3'(SEQ ID NO:8)；
其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸。
在一些实施方式中，本公开所述siRNA为表1中列出的siCCa1和siCCa2。
如前所述，本公开的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，本公开的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，本公开的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公开的siRNA抑制CC3基因表达的功能明显削弱或丧失。
在一些实施方式中，本公开的siRNA至少含有1个修饰的核苷酸。在本公开的上下文中，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，本公开提供的siRNA的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，本公开提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。
本公开的发明人惊奇地发现，本公开所述的siRNA在动物实验中获 得了血浆中稳定性和基因沉默效率的高度平衡。
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，并且，按照5'末端到3'末端的方向，所述核苷酸序列I的至少第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列II的至少第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，所述核苷酸序列I中氟代修饰的核苷酸不多于5个，并且，按照5'末端到3'末端的方向，所述核苷酸序列I的至少第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列II中氟代修饰的核苷酸不多于7个，并且，所述核苷酸序列II的至少第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5'末端到3'末端的方向，在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。
在本公开的上下文中，“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，其具有以下式(7)所示的结构。“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'-经取代的烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-脱氧核苷酸中的一种。
在一些实施方式中，2'-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2'-OMe)，如式(8)所示。在一些实施方式中，2'-经取代的烷氧基修饰的核苷酸，例如可以是2'-O-甲氧基乙基修饰的核苷酸(2'-MOE)，如式(9)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH 2)如式(10)所示。在一些实施方式中，2'-脱氧核苷酸(DNA)如式(11)所示：
Figure PCTCN2022133121-appb-000001
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。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3'-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2'-、4'-位处以提供一个2',4'-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：
Figure PCTCN2022133121-appb-000002
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
Figure PCTCN2022133121-appb-000003
上述式(15)和式(16)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。在一些实施方式中，异核苷酸可以是碱基从核糖环的1'-位移动至2'-位或3'-位而形成的化合物，如式(17)或(18)所示。
Figure PCTCN2022133121-appb-000004
上述式(17)-式(18)化合物中，Base表示核酸碱基，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、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均为甲氧基修饰的核苷酸，在上文和下文中，所述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。
在上文及下文中，“氟代修饰的核苷酸”、“2'-氟修饰的核苷酸”、“核糖基团的2'-羟基被氟取代的核苷酸”和“具有2'-氟代核糖基的核苷酸”意义相同，均指核苷酸的2'-羟基被氟取代，而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”、“2'-甲氧基修饰的核苷酸”、“核糖基团的2'-羟基被甲氧基取代的核苷酸”和“具有2'-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基团的2'-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按 照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，所述siRNA的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、8、9、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5'末端到3'末端的方向，所述siRNA的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5'末端到3'末端的方向，所述siRNA的正义链中核苷酸序列I的第7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸。
在一些实施方式中，本公开提供的siRNA为表1中列出的siCCa1-M1、siCCa1-M2、siCCa1-M3、siCCa2-M1、siCCa2-M2、siCCa2-M3中的任意一种。
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。同时，上述修饰的siRNA具有较高的抑制靶mRNA的活 性。
在一些实施方式中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方式中，具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亚基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯亚基：
Figure PCTCN2022133121-appb-000005
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯亚基连接存在于由以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯亚基连接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯亚基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯亚基连接存在于以下位置中的至少一处：
所述正义链的5'末端第1个核苷酸和第2个核苷酸之间；
所述正义链的5'末端第2个核苷酸和第3个核苷酸之间；
所述正义链的3'末端第1个核苷酸和第2个核苷酸之间；
所述正义链的3'末端第2个核苷酸和第3个核苷酸之间；
所述反义链的5'末端第1个核苷酸和第2个核苷酸之间；
所述反义链的5'末端第2个核苷酸和第3个核苷酸之间；
所述反义链的3'末端第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'末端第2个核苷酸和第3个核苷酸之间。
在一些实施方式中，本公开提供的siRNA为表1中列出的siCCa1-M1S、siCCa1-M2S、siCCa1-M3S、siCCa2-M1S、siCCa2-M2S、siCCa2-M3S 中的任意一种。
在一些实施方式中，所述siRNA反义链的5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。
常用的所述5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5'-磷酸核苷酸可具有如下结构：
Figure PCTCN2022133121-appb-000006
再如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开了如下4种5'-磷酸类似物修饰的核苷酸：
Figure PCTCN2022133121-appb-000007
其中，R选自H、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。
在一些实施方式中，5'-磷酸核苷酸为式(2)所示的含有5'-磷酸修饰的核苷酸，5'-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。
在一些实施方式中，本公开提供的siRNA为表1中列出的siCCa1-M1P1、siCCa1-M2P1、siCCa1-M3P1、siCCa2-M1P1、siCCa2-M2P1、siCCa2-M3P1、siCCa1-M1SP1、siCCa1-M2SP1、siCCa1-M3SP1、siCCa2-M1SP1、siCCa2-M2SP1、siCCa2-M3SP1中的任意一种。
本公开的发明人意外发现，本公开提供的siRNA不仅具有显著增强 的血浆和溶酶体稳定性，还具有较高的靶mRNA抑制活性。
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
药物组合物
本公开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，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe 3O 4或Fe 2O 3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。
在一些实施方式中，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，在一些实施方式中，siRNA与药学上可接受的载体的重量比可以为1:(1-500)，在一些实施方式中，上述重量比为1:(1-50)。
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。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
Figure PCTCN2022133121-appb-000008
其中：
每个X 101或X 102各自独立地是O、S、N-A或C-A，其中A是氢或C 1-C 20烃链；
每个Y 101或Z 101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO 2；
每个R 101、R 102、R 103、R 104、R 105、R 106或R 107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R 102是氢；
并且，如果n或m中的至少一个是2，那么R 103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
Figure PCTCN2022133121-appb-000009
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R 103是多胺。在其它实施方式中，R 103是缩酮。 在一些实施方式中，在式(201)中的R 101和R 102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R 103可以是下述式(204)-式(213)中的任一个：
Figure PCTCN2022133121-appb-000010
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数， 每个“HCC”代表烃链，且每个*显示R 103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
Figure PCTCN2022133121-appb-000011
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；
所述聚乙二醇化脂质为1,2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80):(19.7-80):(0.3-50)，例如可以为(50-70):(20-40):(3-20)。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1:1至约1:50、从约1:1至约1:30、从约1:3至约1:20、从约1:4至约1:18、从约1:5至约1:17、从约1:5至约1:15、从约1:5至约1:12、从约1:6至约1:12或从约1:6至约1:10的范围内，例如，本公开的siRNA与全部脂质的重量比为约1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的siRNA替代现有siRNA即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1：(2-5)，例如可以为1:4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
siRNA缀合物
本公开提供了一种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。在一些实施方式中，所述缀合基团包含接头和药学上可接受的靶向基团和/或递送辅助基团，并且，所述siRNA、所述接头和所述靶向基团或者所述递送辅助基团依次共价或非共价连接，每个所述靶向基团选自能够和细胞表面受体结合的配体，每个递送辅助基团选自能够增加所述siRNA缀合物在递送目标器官或组织中的生物相容性的基团。
在本公开的上下文中，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”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表示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的特定化合物。
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，在一些实施方式中还进一步包含接头(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'端或5'端，也可在反义链的5'端，还可以在siRNA的内部序列中。在一些实施方式中，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.
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，通过引用的方式将其整体内容并入本文。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，以引用的方式将其全部公开内容并入本文。
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，以引用的方式将其全部公开内容并入本文。
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，当本公开所述的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，当本公开所述的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，通过引用的方式将其整体内容并入本文。
在一些实施方式中，本公开的siRNA缀合物中的接头具有如式(301)所示的结构：
Figure PCTCN2022133121-appb-000012
其中，k为1-3的整数；
L A具有如式(302)所示的包含酰胺键的结构，L B具有如式(303)所示的包含N-酰基吡咯烷的结构，含有羰基和氧原子，L C为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团；
Figure PCTCN2022133121-appb-000013
其中，n 302、q 302和p 302各自独立地为2-6的整数，可选地，n 302、q 302和p 302各自独立地为2或3；n 303为4-16的整数，可选地，n 303为8-12的整数，
Figure PCTCN2022133121-appb-000014
表示基团共价连接的位点。
所述接头中，每个L A分别与一个所述靶向基团通过醚键连接，并通过L C部分中羟基的氧原子与L C部分形成醚键而连接；L B通过式(303)中的羰基与L C部分中氨基的氮原子形成酰胺键而连接，并通过式(303)中的氧原子与所述siRNA通过氧原子形成磷酸酯键或硫代磷酸酯键相连接。
在一些实施方式中，本公开提供的siRNA缀合物具有如式(305)所示的结构：
Figure PCTCN2022133121-appb-000015
其中，Nu表示本公开提供的siRNA。
在一些实施方式中，本公开的siRNA缀合物中的接头具有式(306)所示的结构：
Figure PCTCN2022133121-appb-000016
其中，n 306为0-3的整数，每个p 306独立地为1-6的整数，
Figure PCTCN2022133121-appb-000017
表示基团共价连接的位点；所述连接基团通过由*标出的氧原子与所述靶向基团形成醚键连接；所述连接基团由#标出的氧原子中的至少一个与所述siRNA形成磷酸酯键或硫代磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C 1-C 3烷基连接形成C 1-C 3烷氧基；
在一些实施方式中，本公开的siRNA缀合物具有如式(307)所示的结构：
Figure PCTCN2022133121-appb-000018
其中，Nu表示本公开提供的siRNA。
在一些实施方式中，所述siRNA缀合物具有如式(308)所示的结构：
Figure PCTCN2022133121-appb-000019
其中：
n1为选自1-3的整数，n3为选自0-4的整数；
m1、m2或m3独立地为选自2-10的整数；
R 10、R 11、R 12、R 13、R 14或R 15各自独立地为H，或选自于由以下基团所组成的组：C 1-C 10烷基、C 1-C 10卤代烷基以及C 1-C 10烷氧基；
R 3为式A59所示结构的基团：
Figure PCTCN2022133121-appb-000020
其中，E 1为OH、SH或BH 2，Nu为本公开的siRNA；
R 2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子可 选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，R 2可选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)；
每个L 1是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子可选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O) 2、C 2-C 10亚烯基、C 2-C 10亚炔基、C 6-C 10亚芳基、C 3-C 18亚杂环基和C 5-C 10亚杂芳基；并且其中，L 1可选地具有由以下基团所组成的组中的任何一个或多个的取代基：C 1-C 10烷基、C 6-C 10芳基、C 5-C 10杂芳基、C 1-C 10卤代烷基、-OC 1-C 10烷基、-OC 1-C 10烷基苯基、-C 1-C 10烷基-OH、-OC 1-C 10卤代烷基、-SC 1-C 10烷基、-SC 1-C 10烷基苯基、-C 1-C 10烷基-SH、-SC 1-C 10卤代烷基、卤素取代基、-OH、-SH、-NH 2、-C 1-C 10烷基-NH 2、-N(C 1-C 10烷基)(C 1-C 10烷基)、-NH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、氰基、硝基、-CO 2H、-C(O)O(C 1-C 10烷基)、-CON(C 1-C 10烷基)(C 1-C 10烷基)、-CONH(C 1-C 10烷基)、-N(C 1-C 10烷基)(C 1-C 10烷基苯基)、-NH(C 1-C 10烷基苯基)、-CONH 2，-NHC(O)(C 1-C 10烷基)、-NHC(O)(苯基)、-N(C 1-C 10烷基)C(O)(C 1-C 10烷基)、-N(C 1-C 10烷基)C(O)(苯基)、-C(O)C 1-C 10烷基、-C(O)C 1-C 10烷基苯基、-C(O)C 1-C 10卤烷基、-OC(O)C 1-C 10烷基、-SO 2(C 1-C 10烷基)、-SO 2(苯基)、-SO 2(C 1-C 10卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 10烷基)、- SO 2NH(苯基)、-NHSO 2(C 1-C 10烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 10卤代烷基)。
在一些实施方式中，L 1可选自于由A1-A26基团或其任意组合所组成的组，其中A1-A26的结构和定义如下所示：
Figure PCTCN2022133121-appb-000021
Figure PCTCN2022133121-appb-000022
其中，每个j1独立地为1-20的整数；每个j2独立地为1-20的整数；R'为C 1-C 10烷基；
Ra选自式A27-A45基团所组成的组：
Figure PCTCN2022133121-appb-000023
Figure PCTCN2022133121-appb-000024
Rb为C 1-C 10烷基；
Figure PCTCN2022133121-appb-000025
表示基团共价连接的位点。
技术人员会理解的是，尽管为了方便起见，L 1被定义为线性亚烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或取代而产生的胺或烯基。为了本公开内容的目的，L 1的长度是连接两个连接点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。
M 1表示靶向基团，其定义和可选择的范围与上述靶向基团相同。在一些实施方式中，每个M 1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。
当M 1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体时，在一些实施方式中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述siRNA缀合物中M 1靶向基团的个数至少为2；在一些实施方式中，n1+n3≥2，这样可以使得M 1靶向基团的个数至少为3，从而使得M 1靶向基团与肝表面去唾液酸糖蛋白受体更容易结合，进而促进所述siRNA缀合物通过内吞作用进入细胞。实验表明，当M 1靶向基团的个数大于3个时，M 1靶向基团与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方式中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。
在一些实施方式中，m1、m2或m3独立地选自2-10的整数时，可 以使多个M 1靶向基团之间的空间位置适合M 1靶向基团与肝表面去唾液酸糖蛋白受体的结合，为了使本公开提供的siRNA缀合物更为简单，更容易合成和/或降低成本，在一些实施方式中，m1、m2或m3各自独立地为2-5的整数，在一些实施方式中，m1＝m2＝m3。
本领域技术人员可以理解，当R 10、R 11、R 12、R 13、R 14或R 15各自独立地选自H、C 1-C 10烷基、C 1-C 10卤代烷基、以及C 1-C 10烷氧基中的一种时，不会改变本公开的siRNA缀合物的性质，均可以实现本公开的目的。在一些实施方式中，R 10、R 11、R 12、R 13、R 14或R 15各自独立地选自H、甲基和乙基。在一些实施方式中，R 10、R 11、R 12、R 13、R 14和R 15均为H。
R 3为式A59所示结构的基团，其中，E 1为OH、SH或BH 2，基于制备原料易获取性的考虑，在一些实施方式中，E 1为OH或SH。
R 2的选择是为了实现与含氮骨架上的N原子与A59的连接。在本公开的上下文中，“含氮骨架”是指连接有R 10、R 11、R 12、R 13、R 14和R 15的碳原子与N原子互相连接的链状结构。因此，R 2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N原子的连接基团。在一些实施方式中，在通过固相合成的工艺制备式(308)所示的siRNA缀合物的情况下，R 2基团中需要同时含有与含氮骨架上的N原子连接的连接位点和与R 3中的P相连接的连接位点。在一些实施方式中，R 2中所述与含氮骨架上的N原子连接的位点与N形成酰胺键，所述与R 3上的P原子连接的位点与P原子形成磷酸酯键；在一些实施方式中，R 2可以是B5、B6、B5'或B6'：
Figure PCTCN2022133121-appb-000026
Figure PCTCN2022133121-appb-000027
其中，
Figure PCTCN2022133121-appb-000028
表示基团共价连接的位点。
q 2的取值范围可以是1-10的整数，在一些实施方式中，q 2为1-5的整数。
L 1的作用是将M 1靶向基团与含氮骨架上的N连接，为式(308)所示的siRNA缀合物提供肝靶向功能。在一些实施方式中，L 1选自式A1-A26基团中的一种或多种的连接组合。在一些实施方式中，L 1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合。在一些实施方式中，L 1选自A1、A4、A8、A10和A11中至少2个的连接组合。在一些实施方式中，L 1选自A1、A8、A10中至少2个的连接组合。
在一些实施方式中，L 1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方式中，L 1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。
在一些实施方式中，每个j1独立地为2-10的整数，在一些实施方式中，j1为3-5的整数。在一些实施方式中，每个j2独立地为2-10的整数，在一些实施方式中，每个j2独立地为3-5的整数。R'为C 1-C 4烷基，在一些实施方式中，R'为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种，在一些实施方式中，Ra为A27或A28。Rb为C 1-C 5烷基，在一些实施方式中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方式中，在式A1-A26中各自对j1、j2、R'、Ra、Rb进行选择，以实现M 1靶向基团与含氮骨架上的N原子连接，并使M 1靶向基团之间的空间位置更适合M 1靶向基团与肝表面去唾液酸糖蛋白受体结合。
在一些实施方式中，该siRNA缀合物具有式(403)、(404)、(405)、 (406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：
Figure PCTCN2022133121-appb-000029
Figure PCTCN2022133121-appb-000030
Figure PCTCN2022133121-appb-000031
Figure PCTCN2022133121-appb-000032
Figure PCTCN2022133121-appb-000033
Figure PCTCN2022133121-appb-000034
Figure PCTCN2022133121-appb-000035
在一些实施方式中，式A59中的P原子可以连接到siRNA序列中任何可能的位置，例如，式A59中的P原子可以连接到siRNA正义链或反义链的任何一个核苷酸上；在一些实施方式中，式A59中的P原子连接到siRNA正义链的任何一个核苷酸上。在一些实施方式中，式A59中的P原子连接到siRNA正义链或反义链的端部；在一些实施方式中，式A59中的P原子连接到siRNA正义链的端部。所述端部指所述正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方式中，式A59中的P原子连接到siRNA正义链或反义链的末端；在一些实施方式中，式A59中的P原子连接到siRNA正义链的3'末端。在连接至siRNA的正义链的上述位置的情况下，式(308)所示的siRNA缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以阻断CC3 mRNA翻译蛋白 质的过程，抑制CC3基因表达。
在一些实施方式中，式A59中的P原子可以连接到siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5'位、核苷酸的2'位、核苷酸的3'位或核苷酸的碱基上。在一些实施方式中，式A59中的P原子可通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2'位、3'位或5'位。在一些实施方式中，式A59中的P原子连接在siRNA正义链3'末端核苷酸的3'羟基脱氢后形成的氧原子上(此时，式A59中的P原子也可以看作是siRNA中含有的磷酸基团中的P原子)，或者式A59中的P原子通过取代siRNA正义链中的一个核苷酸的2'-羟基中的氢与核苷酸连接，或者式A59中的P原子通过取代siRNA正义链5'末端核苷酸的5'羟基中的氢与核苷酸连接。
本公开的发明人意外发现，本公开的siRNA具有显著提高的血浆中稳定性和较高的CC3 mRNA沉默活性，含有这些siRNA的siRNA缀合物表现出更高的CC3 mRNA沉默活性。因此，在一些实施方式中，本公开的siRNA可以为表1中示出的siRNA中的一种。
表1本公开的siRNA序列
Figure PCTCN2022133121-appb-000036
Figure PCTCN2022133121-appb-000037
Figure PCTCN2022133121-appb-000038
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯亚基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。
本公开所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子，铵离子NH 4 +，有机铵阳离子中的一种。出于提高溶解性考虑，在一些实施方式中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是K +和/或Na +，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N,N-二异丙基乙胺形成的铵离子。因此，本公开所述siRNA或siRNA缀合物可以至少部分以盐的形式存在。在一些实施方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或siRNA缀合物以钠盐或部分钠盐的形式存在。因此，在提及本公开所述的siRNA或siRNA缀合物，包括但不限于本公开所述的任何结构式表示的siRNA缀合物时，均旨在涵盖该siRNA或siRNA缀合物的钠盐或部分钠盐形式。
本领域技术人员清楚知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中。制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本 领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。
本公开siRNA缀合物的制备
上述siRNA缀合物可以通过现有技术中已经详细描述的方法进行合成。例如，WO2015006740A2中详细描述了多种siRNA缀合物的制备方法。通过本领域技术人员熟知的方式，获得本公开的siRNA缀合物。如WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem 2015,16,903-908中描述了式(307)所示结构的制备方法。中国专利申请CN110959011A也详细公开了制备式(308)所示的siRNA缀合物的方法。以引用的方式将上述文献内容整体并入本文。
本公开的siRNA缀合物也可以与药学上可接受的其它辅料联用，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，详情可参见上文关于本公开的药物组合物的描述。
本公开的siRNA、药物组合物及siRNA缀合物的应用
在一些实施方式中，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防重症肌无力的药物中的用途。
在一些实施方式中，本公开提供了一种预防和/或治疗重症肌无力的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予有需要的受试者。
通过将本公开的siRNA活性成分给予有需要的受试者，可以通过RNA干扰的机制达到预防和/或治疗重症肌无力的目的。因此，本公开的siRNA和/或药物组合物和/或siRNA缀合物可用于预防和/或治疗重症肌无力，或用于制备用于预防和/或治疗重症肌无力的药物。所述疾病可共同具有一个或多个风险因素、成因或结果。
本文所使用的术语“给药/给予”是指通过使得至少部分地将本公开的siRNA、药物组合物和/或siRNA缀合物定位于期望的位点以产生期望效果的方法或途径，将本公开的siRNA、药物组合物和/或siRNA缀合物 放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者体循环相比将更多siRNA缀合物递送至特定位点；而全身给药导致将本公开的siRNA、药物组合物和/或siRNA缀合物递送至受试者的基本体循环。考虑到本公开旨在提供预防和/或治疗重症肌无力的手段，在一些实施方式中采用能够将药物递送至肝脏的给药方式。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月、每两个月、每季度、每半年或每年1次或多次。
本公开所述的siRNA、药物组合物或siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD 50(使50％的群体死亡的致死剂量)和ED 50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述的siRNA、药物组合物、和/或siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以siRNA的量计：(i)对于siRNA缀合物，其siRNA用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在一些实施方式中为0.05-20mg/kg体重，另一些实施方式中为0.1-15mg/kg体重，另一些实施方式中为0.1-10mg/kg体重；(ii)对于siRNA与药学上可接受的载体形成的药物组合物，其siRNA用量可以为0.001-50mg/kg体重，在一些实施方式中为0.01-10mg/kg体重，在一些实施方式中为0.05-5mg/kg体重，在一些实施方式中为0.1-3mg/kg体重。
另外，通过将本公开的siRNA和/或药物组合物和/或siRNA缀合物导入细胞，还可以通过RNA干扰的机制达到抑制细胞中CC3基因表达这一目的。
采用本公开提供的方法抑制CC3基因在细胞中表达，所提供的siRNA、药物组合物和/或siRNA缀合物中的siRNA用量一般是这样的量：其足以减少靶mRNA的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或0.05nM至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、递送途径(局部还是全身)等。在递送部位处的浓度可以显著高于在靶细胞或组织的表面处的浓度。
试剂盒
本公开提供了一种试剂盒，所述试剂盒包含有效量的本公开的siRNA、药物组合物和siRNA缀合物的至少一种。
在一些实施方式中，本文所述的试剂盒可在一个容器中提供siRNA、药物组合物和/或siRNA缀合物。在一些实施方式中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本文所述的试剂盒可在不同于提供本文所述siRNA、药物组合物和/或siRNA缀合物的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将siRNA、药物组合物和/或siRNA缀合物与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本公开的试剂盒中，所述siRNA和药学上可接受的载体和/或辅料以及所述siRNA、药物组合物和/或siRNA缀合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或siRNA缀合物和/或药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品， 所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor Laboratory Press(1989))所记载的方法进行。
制备例1 siRNA缀合物1的制备
本制备例合成了siRNA缀合物1。该siRNA缀合物为CN110959011A制备例1中的L-9缀合分子与表2中编号为siCCa1-M1S的siRNA缀合后形成的siRNA缀合物。
按照CN110959011A制备例1所述的制备方法，制备获得了以下表2中的siRNA缀合物1，区别仅在于，siRNA缀合物1中含有的siRNA的正义链和反义链分别如表2中所示；按照以下表2中编号为siCCa1-M1S的siRNA缀合物的核酸序列，分别逐一连接核苷单体，合成siRNA的正义链和反义链。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将siRNA缀合物1稀释至浓度为0.2mg/mL(以siRNA计)后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。正义链：理论值，7530.70，实测,7531.20；反义链：理论值，7062.67，实测值，7061.84，实测值与理论值一致，说明所合成的siRNA缀合物1是目标设计的双链核酸序列。siRNA缀合物1具有式(403)所示的结构，其中Nu是表2中siCCa1-M1S对应的siRNA形成的siRNA基团，该siRNA缀合物1中的缀合基团连接至siRNA基团正义链的3'末端核苷酸的核糖3'位，并且所述siRNA缀合物处于钠盐形式。
表2 siRNA缀合物
Figure PCTCN2022133121-appb-000039
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯亚基连接。
实验例1不同浓度的siRNA缀合物在HepG2细胞中对CC3 mRNA表达量的抑制效率检测
将HepG2细胞以7.5×10 4细胞/孔接种于24孔板中，16h后细胞生长密度达到70-80％时，吸尽培养孔中H-DMEM完全培养基，每孔加入500μL Opti-MEM培养基(GIBCO公司)继续培养1.5h。
用DEPC化水将本公开的siRNA缀合物1分别配制成10μM、1μM和0.1μM的siRNA缀合物工作液。
配制1A溶液，每份1A溶液依次含有上述10μM、1μM和0.1μM浓度的siRNA缀合物工作液3μL和Opti-MEM培养基47μL。
配制1B溶液，每份1B溶液含有1μL Lipofectamine TM 2000和49μL Opti-MEM培养基。
分别将一份1B溶液与得到的每个siRNA的1A溶液混合，分别室温下孵育20min，得到每个siRNA缀合物的转染复合物1X。
将一份1B溶液与Opti-MEM培养基50μL混合，室温下孵育20min，得到转染复合物1X’。
在培养孔中，分别加入每一个转染复合物1X，均匀混合，加入量为100μL/孔，得到每个siRNA缀合物终浓度分别约为50nM、5nM和0.5nM(以siRNA计)的转染复合物，每个转染复合物1X分别转染2个培养孔，得到含siRNA缀合物的转染混合物，记为测试组。
在另外2个培养孔中，分别加入转染复合物1X’，加入量为100μL/孔，得到不含siRNA的转染混合物，记为空白对照组。
将含siRNA缀合物的转染混合物在培养孔中转染4h后，每孔补加1mL含20％FBS的H-DMEM完全培养基。将24孔板置于CO 2培养箱继续培养24h。
随后，使用RNAVzol(购自威格拉斯生物技术(北京)有限公司，货号N002)根据说明书记载的方法提取各孔细胞中的总RNA。
对于每孔细胞，分别取1μg总RNA，使用反转录试剂盒GoldenstarTM  RT6 cDNA Synthesis Kit(购自北京擎科新业生物技术有限公司，货号TSK301M)提供的试剂，其中选取GoldenstarTM Oligo(dT)17作为引物，按试剂盒说明书中反转录操作步骤配置反转录反应体系20μL，对各孔细胞的总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于50℃孵育50min，然后85℃孵育5min，最后4℃孵育30s，反应结束后，向反转录反应体系中加入DEPC水80μl，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用
Figure PCTCN2022133121-appb-000040
SYBR qPCR SuperMix Plus试剂盒(购自近岸蛋白质科技有限公司，货号E096-01B)提供的试剂配置qPCR反应体系20μL，其中，用于扩增目标基因CC3和内参基因GAPDH的PCR引物序列如表3所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为在95℃预变性10min，然后在95℃变性30s，在60℃退火30s，在72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因CC3和内参基因GAPDH的产物W。产物W随即依次经过95℃15s，60℃1min，95℃15s的孵育，通过实时荧光定量PCR仪分别测定获得产物W中目标基因CC3和内参基因GAPDH的溶解曲线，得到目标基因CC3和内参基因GAPDH的Ct值。
表3：引物信息
Figure PCTCN2022133121-appb-000041
采用比较Ct(ΔΔCt)法，对各测试组中目标基因CC3进行相对定量计算，计算方法如下：
ΔCt(测试组)＝Ct(测试组目标基因)–Ct(测试组内参基因)
ΔCt(对照组)＝Ct(对照组目标基因)–Ct(对照组内参基因)
ΔΔCt(测试组)＝ΔCt(测试组)-ΔCt(对照组平均)
ΔΔCt(对照组)＝ΔCt(对照组)-ΔCt(对照组平均)
其中，ΔCt(对照组平均)是对照组两个培养孔各自的ΔCt(对照组)的算术平均值。从而，测试组和对照组的每一培养孔均对应一个ΔΔCt值。
以对照组为基准，对测试组CC3 mRNA的表达水平进行归一化，定义空白对照组CC3 mRNA表达水平为100％，
测试组CC3 mRNA相对表达水平＝2 -ΔΔCt(测试组)×100％
测试组CC3 mRNA抑制率＝(1-测试组CC3 mRNA相对表达水平)×100％
siRNA缀合物1对CC3 mRNA的抑制率总结于表4中。对于同一测试组siRNA缀合物，mRNA抑制率是两个培养孔测定的测试组CC3 mRNA抑制率的算术平均值。
表4：不同浓度的siRNA缀合物在HepG2细胞中对CC3 mRNA的抑制率
Figure PCTCN2022133121-appb-000042
由表4的结果可见，本公开提供的siRNA缀合物1在HepG2细胞系中有较高的抑制活性，在50nM的siRNA浓度下，CC3 mRNA抑制率高达98.7％，在5nM和0.5nM的siRNA浓度下，也能达到86.37％和81.5％的高抑制活性。
实验例2 siRNA缀合物在猴原代肝细胞中对CC3 mRNA表达量的抑制效率检测
在I型胶原蛋白包被的12孔培养板中，每孔中加入1000μL的细胞维持培养基(CM Culture medium，The Primary Cell Solution)，于37℃在含5％CO 2/95％空气的培养箱中培养30min。将冻存的猴原代肝细胞(The Primary Cell Solution)在含有10％胎牛血清(FBS，RMBIO公司)的解冻培养基(Thawing medium，The Primary Cell Solution公司)中，在37℃下解冻2min，在700rpm速度下离心5min，弃去上清液，以接种培养基 (CM seeding medium，The Primary Cell Solution公司)溶解并使用细胞计数仪计数，随后加入接种培养基至活细胞量为4×10 5细胞/孔，于37℃在含5％CO 2/95％空气的培养箱中培养过夜。
弃去培养基，每孔添加1000μL的细胞维持培养基(CM Culture medium，The Primary Cell Solution)，得到猴原代肝细胞悬液。
用DEPC水将siRNA缀合物1配制成20μM(以siRNA计)的siRNA缀合物工作液。
配制2A溶液，使用siRNA缀合物1配制2A溶液，每份2A溶液依次含有上述siRNA缀合物1工作液3μL和细胞维持培养基97μL。
配制2B溶液，每份2B溶液含有2μL Lipofectamine TM 2000和细胞维持培养基98μL。
分别将一份2B溶液与得到的siRNA缀合物1的2A溶液混合，分别室温下孵育20min，得到siRNA缀合物1的转染复合物2X。
将一份2B溶液与细胞维持培养基100μL混合，在室温下孵育20min，得到空白转染复合物2X’。
在培养孔中，分别加入siRNA缀合物1的转染复合物2X，均匀混合，加入量为200μL/孔，得到每个siRNA缀合物终浓度(以siRNA计)分别约为50nM的转染复合物，每个siRNA的转染复合物2X分别转染2个培养孔，得到含siRNA缀合物1的转染混合物，记为测试组。
在另外2个培养孔中，分别加入空白转染复合物2X’，加入量为200μL/孔，得到不含siRNA的转染混合物，记为空白对照组。
将载有含siRNA缀合物1的转染混合物和含参比siRNA缀合物的转染混合物的12孔板置于含5％CO 2/95％空气的培养箱在37℃下继续培养24h。
随后，使用UNIQ-10柱式总RNA抽提试剂盒(购自生工公司，货号B511361-0100)根据说明书记载的方法提取各孔细胞中的总RNA，分别得到含总RNA的溶液。
对于每孔细胞，分别取含1μg总RNA的溶液，使用反转录试剂盒GoldenstarTM RT6 cDNA Synthesis Kit(购自北京擎科新业生物技术有限公司，货号TSK301M)提供的试剂，其中选取GoldenstarTM Oligo(dT)17作为引物，按试剂盒说明书中反转录操作步骤配制反转录反应体系20μL，对各孔细胞的总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于50℃孵育50min，然后85℃孵育5min，最后4℃孵育30s，反应结束后，向反转录反应体系中加入DEPC水80μl，得到含cDNA的溶液100μL。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用
Figure PCTCN2022133121-appb-000043
SYBR qPCR SuperMix Plus试剂盒(购自近岸蛋白质科技有限公司，货号E096-01B)提供的试剂配置qPCR反应体系20μl，其中，用于扩增目标基因CC3和内参基因GAPDH的PCR引物序列如表5所示，每条引物的终浓度为0.25μM。将包含目标基因CC3的各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为在95℃预变性10min，然后在95℃变性30s，在55℃退火30s，在72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因CC3的产物W；对于包含内参基因GAPDH的qPCR体系，使用与上述相同的方法进行qPCR扩增，区别仅在于，扩增程序中退火的温度为60℃，得到得到含有扩增了内参基因GAPDH的产物W’。产物W和W’随即依次经过95℃15s，60℃1min，95℃15s的孵育，通过实时荧光定量PCR仪分别测定获得产物W和W’中目标基因CC3和内参基因GAPDH的溶解曲线，得到目标基因CC3和内参基因GAPDH的Ct值。
表5：引物信息
Figure PCTCN2022133121-appb-000044
对于上述各测试组和对照组，分别进行2次上述定量PCR检测。
采用比较Ct(ΔΔCt)法，对各测试组中目标基因CC3进行相对定量 计算，计算方法如实验例1中所述。
得到转染了本公开的siRNA缀合物1后，猴原代肝细胞中CC3 mRNA的抑制率如下表6所示。
表6猴原代肝细胞中CC3 mRNA的抑制率
Figure PCTCN2022133121-appb-000045
由表6的结果可见，本公开提供的siRNA缀合物1在猴原代肝细胞中显示出很高的CC3 mRNA抑制活性，在50nM的siRNA浓度下，CC3 mRNA抑制率高达97.4％。
实验例3siRNA缀合物在小鼠体内(in vivo)对CC3 mRNA的抑制
将等级为SPF、5周龄的雌性hC3转基因小鼠(购买自上海南方模式生物科技股份有限公司)随机分为两组，每组5只小鼠，分别为实验组小鼠和空白对照组小鼠，以皮下注射的方式，每只实验组小鼠以3mg/kg小鼠体重的剂量(以siRNA计)给予siRNA缀合物1，siRNA缀合物1以含0.6mg/mL(以siRNA计)的siRNA缀合物的1×PBS溶液形式提供，给药体积均为5mL/kg；每只空白对照组小鼠给予1×PBS，给药体积均为5mL/kg。
以给药时间点作为第1天计算，在第22天处死动物，分别收集每只小鼠的肝脏组织，用RNA later(Sigma Aldrich公司)保存；向每份肝组织中加入1mL Trizol(Sigma公司)，在Tissuelyset II型全自动组织匀浆仪中破碎3次，每次30s，获得肝组织匀浆，向其中加入0.2mL氯仿，静置3min。在4℃下以12000rpm离心10min，取0.4mL上清。向上清中加入0.5mL异丙醇，室温下静置10min。4℃下以12000rpm离心10min，弃去上清。向沉淀中加入1mL乙醇洗涤沉淀，4℃下以12000rpm离心5min，弃去上清。沉淀中加入70μL DEPC化水，得到提取的总RNA溶液。
对于每一小鼠的肝脏组织总RNA，分别取包含1μg总RNA的总RNA水溶液10.5μL，使用反转录试剂盒Reverse Transcription System(购自 Promega公司，货号A3500)，按试剂盒说明书中反转录操作步骤配制为反转录反应体系20μL，对总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于42℃孵育30min，然后于95℃孵育5min，最后于4℃孵育5min，反应结束后，向反转录反应体系中加入DEPC水80μL，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μL做模板，使用SYBR select Master Mix试剂盒(Applied biosystem公司)提供的试剂配制qPCR反应体系20μL，其中，用于扩增目标基因CC3和内参基因GAPDH的PCR引物序列如下表7所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因CC3和内参基因GAPDH的产物W。产物W随即依次经过95℃1min，55℃30s，95℃30s的孵育，实时荧光定量PCR仪分别收集产物W中目标基因CC3和内参基因GAPDH的溶解曲线，得到目标基因CC3和内参基因GAPDH的Ct值。
表7引物序列信息
Figure PCTCN2022133121-appb-000046
采用比较Ct(ΔΔCt)法，对各测试组中目标基因CC3 mRNA的表达水平进行相对定量计算，计算方法如上述实验例1所述。
本实验例中，对小鼠以3mg/kg小鼠体重的剂量(以siRNA计)给予siRNA缀合物1后，siRNA缀合物1对小鼠体内CC3 mRNA的抑制率为49.5％，显示出较好的CC3 mRNA抑制效果。
以上详细描述了本公开的一些实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (31)

  1. 一种siRNA，所述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，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，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-UGGCCCUACUGCAGCUAAZ 1-3'(SEQ ID NO:1)；
    5'-Z 2UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:2)，
    其中，Z 1为A，Z 2为U，所述核苷酸序列I中包含位置对应于Z 1的核苷酸Z 3，所述核苷酸序列II中包含位置对应于Z 2的核苷酸Z 4，所述Z 4是所述反义链5'末端的第一个核苷酸。
  2. 如权利要求1所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。
  3. 如权利要求1或2所述的siRNA，其中，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间的核苷酸差异包括Z 4位置处的差异，且Z 4选自A、C或G。
  4. 如权利要求1-3中任一项所述的siRNA，其中Z 3是与Z 4互补的核苷酸。
  5. 如权利要求1-4中任一项所述的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。
  6. 如权利要求1-5中任一项所述的siRNA，其中，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸；并且所述核苷酸序列I是SEQ ID NO:3所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:4所示的核苷酸序列：
    5'-UGGCCCUACUGCAGCUAAZ 3-3'(SEQ ID NO:3)；
    5'-Z 4UUAGCUGCAGUAGGGCCA-3'(SEQ ID NO:4)，
    其中，Z 3选自A、U、G或C，Z 4是与Z 3互补的核苷酸。
  7. 如权利要求6所述的siRNA，其中Z 3为A，Z 4为U。
  8. 如权利要求1-7中任一项所述的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1-4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。
  9. 如权利要求8所述的siRNA，其中，所述核苷酸序列III和IV的长度均为1个核苷酸，所述核苷酸序列III的碱基为U；
    或者，所述核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CU；
    或者，所述核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UCU；
    或者，所述核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CUCU。
  10. 如权利要求1-9中任一项所述的siRNA，其中，所述反义链还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3'末端，构成反义链的3'突出端。
  11. 如权利要求10所述的siRNA，其中，所述核苷酸序列V的长度为2个核苷酸。
  12. 如权利要求10或11所述的siRNA，其中，所述核苷酸序列V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与靶mRNA相应位置的核苷酸互补。
  13. 如权利要求1-12中任一项所述的siRNA，其中，所述siRNA的正义链含有如SEQ ID NO:5所示的核苷酸序列，所述反义链含有如SEQ ID NO:6所示的核苷酸序列：
    5'-UGGCCCUACUGCAGCUAAZ 3-3'(SEQ ID NO:5)；
    5'-Z 4UUAGCUGCAGUAGGGCCAAG-3'(SEQ ID NO:6)；
    或者，所述siRNA的正义链含有如SEQ ID NO:7所示的核苷酸序列，所述反义链含有如SEQ ID NO:8所示的核苷酸序列：
    5'-CUUGGCCCUACUGCAGCUAA Z 3-3'(SEQ ID NO:7)；
    5'-Z 4UUAGCUGCAGUAGGGCCAAGAG-3'(SEQ ID NO:8)；
    其中，所述Z 4是反义链5'末端的第一个核苷酸，Z 3选自A、U、G或C，并且Z 4是与Z 3互补的核苷酸。
  14. 如权利要求1-13中任一项所述的siRNA，其中，所述siRNA为siCCa1或siCCa2：
    siCCa1
    正义链：5'-UGGCCCUACUGCAGCUAAA-3'(SEQ ID NO:9)
    反义链：5'-UUUAGCUGCAGUAGGGCCAAG-3'(SEQ ID NO:10)
    siCCa2
    正义链：5'-CUUGGCCCUACUGCAGCUAAA-3'(SEQ ID NO:11)
    反义链：5'-UUUAGCUGCAGUAGGGCCAAGAG-3'(SEQ ID NO:12)。
  15. 如权利要求1-14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。
  16. 如权利要求1-15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氟代修饰的核苷酸或非氟代修饰的核苷酸。
  17. 如权利要求16所述的siRNA，其中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，并且，按照5'末端到3'末端的方向，所述核苷酸序列I的至少第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列II的至少第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
  18. 如权利要求16或17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。
  19. 如权利要求18所述的siRNA，其中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，所述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。
  20. 如权利要求1-19中任一项所述的siRNA，其中，所述siRNA为siCCa1-M1、siCCa1-M2、siCCa1-M3、siCCa2-M1、siCCa2-M2、siCCa2-M3中的任意一种。
  21. 如权利要求15所述的siRNA，其中，所述具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亚基，所述硫代磷酸酯亚基连接存在于由以下位置组成的组中的至少一处：
    所述正义链的5'末端第1个核苷酸和第2个核苷酸之间；
    所述正义链的5'末端第2个核苷酸和第3个核苷酸之间；
    所述正义链的3'末端第1个核苷酸和第2个核苷酸之间；
    所述正义链的3'末端第2个核苷酸和第3个核苷酸之间；
    所述反义链的5'末端第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'末端第2个核苷酸和第3个核苷酸之间；
    所述反义链的3'末端第1个核苷酸和第2个核苷酸之间；以及
    所述反义链的3'末端第2个核苷酸和第3个核苷酸之间。
  22. 如权利要求1-21中任一项所述的siRNA，其中，所述siRNA为siCCa1-M1S、siCCa1-M2S、siCCa1-M3S、siCCa2-M1S、siCCa2-M2S、siCCa2-M3S中的任意一种。
  23. 如权利要求1-22中任一项所述的siRNA，其中，所述反义链的5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。
  24. 如权利要求23所述的siRNA，其中，所述siRNA为siCCa1-M1P1、siCCa1-M2P1、siCCa1-M3P1、siCCa2-M1P1、siCCa2-M2P1、siCCa2-M3P1、siCCa1-M1SP1、siCCa1-M2SP1、siCCa1-M3SP1、siCCa2-M1SP1、siCCa2-M2SP1、siCCa2-M3SP1中的任意一种。
  25. 一种药物组合物，其特征在于，该药物组合物含有权利要求1-24中任意一项所述的siRNA和药学上可接受的载体。
  26. 一种siRNA缀合物，所述siRNA缀合物含有权利要求1-24中任意一项所述的siRNA以及缀合连接至该siRNA的缀合基团。
  27. 如权利要求26所述的siRNA缀合物，其中，所述siRNA缀合物具有式(403)所示的结构，其中Nu是siCCa1-M1S，所述siRNA缀合物中的缀合基团连接至siRNA基团正义链的3'末端核苷酸的核糖3'位，并且所述siRNA缀合物处于钠盐形式。
  28. 权利要求1-24中任意一项所述的siRNA、权利要求25所述的药物组合物和/或权利要求26或27所述的siRNA缀合物在制备用于治疗和/或预防重症肌无力的药物中的用途。
  29. 一种治疗和/或预防重症肌无力的方法，其中，所述方法包括将有效量的权利要求1-24中任意一项所述的siRNA、权利要求25所述的药物组合物和/或权利要求26或27所述的siRNA缀合物给予患有重症肌无力的受试者。
  30. 一种抑制肝细胞中CC3基因表达的方法，该方法包括将有效量的权利要求1-24中任意一项所述的siRNA、权利要求25所述的药物组合物和/或权利要求26或27所述的siRNA缀合物与所述肝细胞接触。
  31. 一种试剂盒，其中，该试剂盒含有权利要求1-24任意一项所述的siRNA、权利要求25所述的药物组合物和/或权利要求26或27所述的siRNA缀合物。
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