WO2021204216A1 - 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

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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WO2021204216A1
WO2021204216A1 PCT/CN2021/086016 CN2021086016W WO2021204216A1 WO 2021204216 A1 WO2021204216 A1 WO 2021204216A1 CN 2021086016 W CN2021086016 W CN 2021086016W WO 2021204216 A1 WO2021204216 A1 WO 2021204216A1
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nucleotide sequence
nucleotide
seq
sirna
nucleotides
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PCT/CN2021/086016
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French (fr)
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梁子才
李海涛
张鸿雁
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北京瑞博开拓医药科技有限公司
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Priority to CN202180022722.5A priority Critical patent/CN115698288A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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 gene expression of a novel coronavirus 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.
  • Corona Virus Disease 2019 (Corona Virus Disease 2019, hereinafter referred to as COVID-19, formerly known as NCP, or New Coronavirus Pneumonia) refers to the disease caused by the novel coronavirus infection discovered in 2019, which is the severe acute respiratory syndrome coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, hereinafter referred to as new coronavirus or SARS-COV-2 virus, formerly referred to as 2019-nCov) is an acute respiratory infectious disease caused by infection.
  • severe acute respiratory syndrome coronavirus 2 severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, hereinafter referred to as new coronavirus or SARS-COV-2 virus, formerly referred to as 2019-nCov
  • 2019-nCov severe acute respiratory syndrome coronavirus 2
  • SARS-COV-2 virus, SARS, and MERS are all ⁇ -coronaviruses.
  • SARS-COV-2 virus is classified into ⁇ genus B subgroup. It is a single-stranded RNA virus. This single-stranded RNA virus has multiple mutants. For example, the full length of the gene is 29903bp (NC_045512.2) or the full length is The 29867bp (MT066156.1) or 29835bp (MT188341.1) single-stranded RNA virus has a common receptor ACE2 with the SARS virus (SARS-Cov) that appeared in 2002-2003.
  • SARS-COV-2 virus is mainly amplified and expressed in the lungs, which makes the lungs consolidate to varying degrees, with the exudation of monocytes and macrophages, the proliferation and shedding of type II alveolar epithelial cells, and focal hemorrhage and necrosis of lung tissue. Pulmonary interstitial fibrosis, etc.; part of the epithelium of the pulmonary bronchial mucosa falls off and mucus (plug) is formed in the cavity.
  • SARS-COV-2 virus is also amplified and expressed in spleen, heart, blood vessels, liver, gallbladder, kidney, and brain tissues. Cause the disease of these organs.
  • siRNAs and their modified sequences provided in the present disclosure can specifically inhibit the expression of SARS-COV-2 virus genes, and achieve protection against diseases or physiology caused by the expression of SARS-COV-2 virus.
  • the treatment or prevention of the condition thus completing the present invention.
  • the present disclosure provides a siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II.
  • the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, the The nucleotide sequence II is at least partially reverse complementary to the first nucleotide sequence, which is a nucleotide sequence in the target RNA, and the target RNA refers to SARS-COV- 2 Viral RNA.
  • the first nucleotide sequence is a nucleotide sequence in the conserved region sequence of the target RNA.
  • the present disclosure provides an siRNA capable of inhibiting the expression of SARS-COV-2 viral genes.
  • the siRNA contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently modified. Or unmodified nucleotides, wherein 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 At least partially reverse complementary to form a double-stranded region, and the nucleotide sequence I and the nucleotide sequence II are selected from the group of the following sequences i)-viii):
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are the same in length and have no more than 3 nucleotide differences
  • nucleotide sequence II is the same as SEQ ID NO: 2
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 1 is A
  • Z 2 is U
  • the nucleotide sequence I contains a nucleotide Z 3 whose position corresponds to Z 1
  • the nucleotide sequence II contains a nucleoside whose position corresponds to Z 2 Acid Z 4
  • the Z 4 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 5 is A
  • Z 6 is U
  • the nucleotide sequence I contains the nucleotide Z 7 whose position corresponds to Z 5
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 6 Acid Z 8
  • the Z 8 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 9 is A
  • Z 10 is U
  • the nucleotide sequence I contains the nucleotide Z11 whose position corresponds to Z 9
  • the nucleotide sequence II contains the nucleotide whose position corresponds to Z 10 Z 12
  • the Z 12 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 are the same in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 13 is A
  • Z 14 is U
  • the nucleotide sequence I contains the nucleotide Z 15 whose position corresponds to Z 13
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 14 Acid Z 16
  • the Z 16 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 242
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 17 is A
  • Z 18 is U
  • the nucleotide sequence I contains the nucleotide Z 19 whose position corresponds to Z 17
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 18 Acid Z 20
  • the Z 20 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 302
  • the nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 21 is A
  • Z 22 is U
  • the nucleotide sequence I contains the nucleotide Z 23 whose position corresponds to Z 21
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 22 Acid Z 24
  • the Z 24 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 362
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 25 is A
  • Z 26 is U
  • the nucleotide sequence I contains the nucleotide Z 27 whose position corresponds to Z 25
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 26 Acid Z 28
  • the Z 28 is the first nucleotide at the 5'end of the antisense strand.
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 422
  • nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences:
  • Z 29 is A
  • Z 30 is U
  • the nucleotide sequence I contains the nucleotide Z 31 whose position corresponds to Z 29
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 30 Acid Z 32
  • the Z 32 is the first nucleotide at the 5'end of the antisense strand.
  • the present disclosure provides a pharmaceutical composition containing the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides an siRNA conjugate containing the siRNA provided in the present disclosure and a conjugating group conjugated to the siRNA.
  • the present disclosure provides that the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are prepared for the treatment and/or prevention of related SARS-COV-2 virus gene expression. Use in medicine for diseases.
  • the present disclosure provides a method for treating and/or a disease caused by a novel coronavirus, the method comprising adding an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure Give to subjects suffering from diseases caused by the novel coronavirus.
  • the present disclosure provides a method for inhibiting SARS-COV-2 viral gene expression in a cell, the method comprising adding an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure Contact with the cell.
  • the present disclosure provides a kit containing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
  • Figure 1 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure and reference siRNA in HEK293A cells in vitro.
  • Figure 2 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure and reference siRNA in HEK293A cells in vitro.
  • Figure 3 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • Figure 4A is a bar graph showing the SARS-COV-2 viral RNA load of the siRNA of the present disclosure and the reference siRNA in Vero-infected cells for 24 hours.
  • Figure 4B is a bar graph showing the SARS-COV-2 viral RNA load of the siRNA of the present disclosure and the reference siRNA in Vero-infected cells for 48 hours.
  • Fig. 5A is a line graph showing the concentration-cell survival rate of the cell survival rate after the siRNA of the present disclosure and the reference siRNA are transfected into Vero cells by lipo2000.
  • Fig. 5B is a graph showing the concentration-cell survival rate of the cell survival rate after the free uptake of the siRNA of the present disclosure and the reference siRNA after being transfected into Vero cells.
  • Figure 6 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • Figure 7 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • Figure 8 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • Figure 9 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • Figure 10 shows the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • FIG. 11A is a bar graph showing the SARS-COV-2 viral RNA load in vivo of humanized ACE2 transgenic mice infected with the SARS-COV-2 virus.
  • FIG. 11B is a bar graph showing the SARS-COV-2 viral RNA load in vivo of humanized ACE2 transgenic mice infected with SARS-COV-2 virus.
  • Figure 12A is a bar graph showing the SARS-COV-2 viral RNA load in the lung tissue of humanized ACE2 transgenic mice infected with the SARS-COV-2 virus of the siRNA of the present disclosure.
  • FIG. 12B is a bar graph showing the SARS-COV-2 viral RNA load of the humanized ACE2 transgenic mice infected with the SARS-COV-2 virus in vivo (in vivo) tracheal tissue of the siRNA of the present disclosure.
  • Figure 13 is a bar graph showing the SARS-COV-2 viral RNA load in the lung tissue of humanized ACE2 transgenic mice infected with the SARS-COV-2 virus of the siRNA of the present disclosure.
  • Figure 14 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • Figure 15 is a bar graph showing the target gene inhibitory activity of different concentrations of siRNA of the present disclosure in HEK293A cells in vitro.
  • COVID-19 refers to the new type of coronavirus disease discovered in 2019, or known as the new type of coronavirus pneumonia.
  • SARS-COV-2 virus refers to a new type of coronavirus that causes COVID-19 and its variants.
  • the variant refers to a virus in which the SARS-COV-2 virus gene is mutated.
  • SARS-COV-2 virus gene refers to a gene of SARS-COV-2 virus and its variants, for example, the sequence shown in Genbank registration number NC_045512.2.
  • target gene used in the present disclosure refers to the gene of the aforementioned SARS-COV-2 virus and its mutants, especially the sequence shown in Genbank registration number NC_045512.2.
  • target RNA refers to the RNA expressed by the aforementioned SARS-COV-2 virus gene and its mutants, especially the RNA corresponding to the gene whose Genbank registration number is NC_045512.2.
  • capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy group Modified nucleotides; a lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; a lowercase letter s indicates that between the two adjacent nucleotides to the left and the right of the letter s It is a phosphorothioate group connection; P1 indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide or a nucleotide modified by a 5'-phosphate analogue, and the letter combination VP represents the letter combination VP The adjacent nucleotide on the right is a vinyl phosphate modified nucleotide, and the letter combination Ps indicates that the adjacent nucleotide on
  • fluorinated modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2'position of the ribose group of a nucleotide with fluorine
  • non-fluorinated modified nucleotide refers to Nucleotides or nucleotide analogs formed by substituting a non-fluorine group for the hydroxyl group at the 2'position of the ribose group of a nucleotide.
  • Nucleotide analogue refers to a nucleic acid that can replace nucleotides, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymus The group of pyrimidine deoxyribonucleotides. Such as heteronucleotides, bridged nucleotides (BNA) or acyclic nucleotides.
  • the "methoxy-modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the expressions "complementary” or “reverse complementary” can be used interchangeably, and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are connected to the other strands. The bases on the pair are paired in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) matches.
  • Each base pair includes a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary manner.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” “Means that there is no more than one base mismatch between two nucleotide sequences; “complete reverse complementation” means that there is no base mismatch between two nucleotide sequences.
  • nucleotide difference between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed compared with the latter. For example, when one nucleotide base in the latter is A, when the corresponding nucleotide base at the same position in the former is U, C, G, or T, it is regarded as one of the two nucleotide sequences. There is a nucleotide difference at this position. In some embodiments, when an abasic nucleotide or its equivalent is substituted for the nucleotide at the original position, it can also be considered that there is a nucleotide difference at that position.
  • the nucleoside monomer refers to the preparation method according to the The type and sequence of nucleotides in the siRNA or siRNA conjugates, the modified or unmodified nucleoside phosphoramidite monomers used in the solid-phase synthesis of phosphoramidites (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites).
  • Phosphoramidite solid phase synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in the present disclosure are all commercially available.
  • conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; correspondingly, “conjugate” is Refers to the compound formed by covalent linkage between the various chemical moieties.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugate of the present disclosure is sometimes referred to simply as "conjugate”.
  • An siRNA conjugate should be understood as a general term for multiple siRNA conjugates or an siRNA conjugate represented by a certain chemical formula according to the context.
  • a "conjugated molecule” should be understood as a specific compound that can be conjugated to siRNA through a reaction, and ultimately form the siRNA conjugate of the present disclosure.
  • optionally substituted alkyl includes “alkyl” and “substituted alkyl” as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically impractical, and/or inherently unstable. .
  • alkyl refers to straight and branched chains with a specified number of carbon atoms, the number is usually 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 Or 1 to 6 carbon atoms.
  • a C1-C6 alkyl group contains straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residue having a specific number of carbons it is intended to cover all branched and straight chain forms having that number of carbons; therefore, for example, "butyl” means 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 residues that are the same as alkyl but have two points of attachment.
  • alkenyl refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon double bond, which is obtained from adjacent carbon atoms of the parent alkyl group. Obtained by removing one molecule of hydrogen. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl Group), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-ene-1- But-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon triple bond, which is obtained from adjacent carbon atoms of the parent alkyl group. Obtained by removing two molecules of hydrogen.
  • Typical alkynyl groups include but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like.
  • 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 residues that are the same as alkynyl but have two points of attachment.
  • alkoxy refers to an alkyl group with a specified number of carbon atoms connected through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, S-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy and so on.
  • Alkoxy groups generally have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by oxygen bridges.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbons of 6 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, that is, contains a ring according to Hückel's theory , Delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl.
  • Arylene is a subset of aryl and refers to residues that are the same as aryl but have two points of attachment.
  • 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 specified number of carbon atoms is replaced by one or more halogen atoms up to the maximum allowable number.
  • haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise stated in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include a fused ring or a bridged ring system. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group can be connected to the rest of the molecule through any ring atom.
  • heterocyclic groups include, but are not limited to: dioxanyl, thienyl[1,3]dithianyl (thienyl[1,3]dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidine Group, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl (trithianyl) ), tetrahydropyranyl, thiomorph
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, that is, contains a cyclic delocalization according to Hückel's theory (4n +2) ⁇ -electron system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized.
  • heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodiaxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl (benzo[b][1,4]dioxepinyl), benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl, benzodioxin
  • hydroxyl protecting groups can be used in this disclosure.
  • the protecting group makes the chemical functionality insensitive to specific reaction conditions, and can be added to and removed from the functionality in the molecule without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d, John Wiley & Sons, New York, 1991, as cited in In this way, the above-mentioned documents are incorporated into this article in their entirety.
  • the protecting group is stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthene-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4"-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (for example, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, rabbits, and any species Of poultry.
  • treatment refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefits.
  • “Therapeutic benefit” means eradicating or improving the underlying barriers being treated.
  • the therapeutic benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be afflicted by the underlying disorder.
  • prevention refers to methods of obtaining beneficial or desired results, including but not limited to preventive benefits.
  • siRNA, siRNA conjugates or pharmaceutical compositions can be administered to subjects who are at risk of developing a particular disease, or to subjects who report one or more physiological symptoms of the disease, even if possible The diagnosis of the disease has not yet been made.
  • the present disclosure provides the first to eighth siRNA capable of inhibiting SARS-COV-2 viral gene expression.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and those skilled in the art know that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.
  • the nucleotide sequence II in the antisense strand of the siRNA described in the present disclosure is partially or completely reverse complementary to the first nucleotide sequence to achieve the specificity between the antisense strand of the siRNA and the first nucleotide sequence
  • the expression of SARS-COV-2 virus gene is silenced and the amplification of SARS-COV-2 virus is blocked. Therefore, as long as the nucleotide sequence II in the antisense strand of the siRNA can be specifically combined with the first nucleotide sequence, and the expression of the SARS-COV-2 virus gene can be silenced, the SARS-COV-2 virus can be blocked.
  • the first nucleotide sequence can be located at any position of the target RNA.
  • the first nucleotide sequence is located in the conserved region of the target RNA, in other words, the first nucleotide sequence is a nucleotide sequence in the conserved region sequence of the target RNA.
  • the conserved region sequence refers to a sequence that is at least partially reverse complementary to nucleotide sequence II when the SARS-COV-2 virus is mutated with a mutation rate of no more than 3 nucleotides.
  • the conserved region sequence refers to a sequence that is at least partially reverse complementary to nucleotide sequence II when the SARS-COV-2 virus is mutated with a mutation rate of no more than 1 nucleotide. In some embodiments, the conserved region sequence refers to a sequence that is at least partially reverse complementary to nucleotide sequence II when the SARS-COV-2 virus is mutated without mutation.
  • the nucleotide sequence II is substantially reverse complementary, substantially completely reverse complementary, or completely reverse complementary to the first stretch of nucleotide sequence.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • the lengths of the nucleotide sequence I and the nucleotide sequence II are the same or different, and are 15-40 nucleotides or 18-30 nucleotides respectively.
  • siRNA applicable to the present disclosure, and does not mean that the present invention is limited to the siRNA listed below.
  • the siRNA may be the first siRNA.
  • the first siRNA contains a sense strand and an antisense strand, each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 1 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 2 The length is equal, and no more than 3 nucleotide differences:
  • Z 1 is A
  • Z 2 is U
  • the nucleotide sequence I contains a nucleotide Z 3 whose position corresponds to Z 1
  • the nucleotide sequence II contains a nucleoside whose position corresponds to Z 2 Acid Z 4
  • the Z 4 is the first nucleotide at the 5'end of the antisense strand.
  • positional correspondence refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence.
  • the first nucleotide at the 3'end of the nucleotide sequence I is the nucleotide whose position corresponds to the first nucleotide at the 3'end of SEQ ID NO:1.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences 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 ID NO: There are no more than 2 nucleotide differences between the nucleotide sequences shown in 2.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and/or the nucleotide sequence II and SEQ ID NO:1 ID NO: There is no more than one nucleotide difference between the nucleotide sequences shown in 2.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the difference at position Z 4 , and Z 4 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z 4 position, and Z 4 is selected from A, C, or G. In some embodiments, Z 3 is a nucleotide that is complementary to Z 4. SiRNAs with the above-mentioned nucleotide differences have higher target RNA inhibitory ability, and these siRNAs with 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 complement refers to two nuclei There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complementation refers to the presence of no more than 1 base mismatch between 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:
  • Z 4 is the first nucleotide at the 5'end of the antisense strand
  • Z 3 is selected from A, U, G, or C
  • Z 4 is a nucleotide complementary to Z 3 ; in some embodiments Where Z 3 is A, Z 4 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the 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 siRNA sense strand and antisense strand is 19/21, 21/23, or
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • NO: 1 represents a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U. ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of the acid sequence III is UA, and the base composition of the nucleotide sequence IV is UA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is CUA, and the base composition of nucleotide sequence IV is UAG; at this time, the sense strand and the reverse The length ratio of the sense strand is 22/22; alternatively, 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 base composition of the nucleotide sequence III is GCUA, the base composition of nucleotide sequence IV is UAGC; 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UA , The base composition of nucleotide sequence IV is UA; 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 siRNA may be a second siRNA.
  • the second siRNA contains a sense strand and an antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 61 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 62 The length is equal, and no more than 3 nucleotide differences:
  • Z 5 is A
  • Z 6 is U
  • the nucleotide sequence I contains the nucleotide Z 7 whose position corresponds to Z 5
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 6 Acid Z 8
  • the Z 8 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ ID NO: There are no more than 2 nucleotide differences between the nucleotide sequences shown in 62.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 62.
  • the II and the nucleotide sequence SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 include differences in location Z 8, and Z 8 is selected from A, C or G.
  • the nucleotide difference is a difference at the Z 8 position, and Z 8 is selected from A, C, or G.
  • Z 7 is a nucleotide that is complementary to Z 8.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64:
  • Z 8 is the first nucleotide at the 5'end of the antisense strand
  • Z 7 is selected from A, U, G, or C
  • Z 8 is a nucleotide complementary to Z 7 ; in some embodiments Where Z 7 is A, Z 8 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 61 and has the same length as the nucleotide sequence IV in the target RNA .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U. ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of acid sequence III is CA
  • the base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is UCA
  • the base composition of nucleotide sequence IV is UGA
  • the sense strand and the reverse The length ratio of the sense strand is 22/22
  • the lengths of nucleotide sequences III and IV are both 4 nucleotides
  • the base composition of the nucleotide sequence III is GUCA
  • the base composition of nucleotide sequence IV is UGAC
  • 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CA , The base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the third siRNA is the third siRNA
  • the siRNA may be a third siRNA.
  • the third siRNA contains a sense strand and an antisense strand, and each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 121 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 122 The length is equal, and no more than 3 nucleotide differences:
  • Z 9 is A
  • Z 10 is U
  • the nucleotide sequence I contains the nucleotide Z 11 whose position corresponds to Z 9
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 10
  • Acid Z 12 the Z 12 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II and SEQ There are no more than 2 nucleotide differences between the nucleotide sequences shown in ID NO: 122.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II and SEQ ID NO: 122 has no more than one nucleotide difference between the nucleotide sequences shown.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 includes the difference at position Z 12 , and Z 12 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z 12 position, and Z 12 is selected from A, C, or G. In some embodiments, Z 11 is a nucleotide complementary to Z 12. SiRNAs with the above-mentioned nucleotide differences have higher target RNA inhibitory ability, and these siRNAs with nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124:
  • Z 12 is the first nucleotide at the 5'end of the antisense strand
  • Z 11 is selected from A, U, G or C
  • Z 12 is a nucleotide complementary to Z 11 ; in some embodiments Where Z 11 is U, Z 12 is A;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • This second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 121 and has the same length as the nucleotide sequence IV in the target RNA .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of the acid sequence III is UG, and the base composition of the nucleotide sequence IV is CA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is GUG, and the base composition of nucleotide sequence IV is CUC; at this time, the sense strand and the reverse The length ratio of the sense strand is 22/22; alternatively, 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 base composition of the nucleotide sequence III is UGUG, the base composition of nucleotide sequence IV is CACA; 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UG , The base composition of nucleotide sequence IV is CA; 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 siRNA may be the fourth siRNA.
  • the fourth siRNA contains a sense strand and an antisense strand, each nucleotide in the fourth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 181 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 182 The length is equal, and no more than 3 nucleotide differences:
  • Z 13 is A
  • Z 14 is U
  • the nucleotide sequence I contains the nucleotide Z 15 whose position corresponds to Z 13
  • the nucleotide sequence II contains the nucleoside whose position corresponds to Z 14 Acid Z 16
  • the Z 16 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181, and/or the nucleotide sequence II and SEQ There are no more than 2 nucleotide differences between the nucleotide sequences shown in ID NO: 182.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181, and/or the nucleotide sequence II and SEQ ID NO: No more than 1 nucleotide difference between the nucleotide sequences shown in 182.
  • the II and the nucleotide sequence SEQ ID NO: nucleotide differences between nucleotide sequence 182 includes difference 16 at a position Z, and the Z 16 is selected from A, C or G.
  • the nucleotide difference is a difference at the Z 16 position, and Z 16 is selected from A, C, or G.
  • Z 15 is a nucleotide that is complementary to Z 16.
  • SiRNAs with the above-mentioned nucleotide differences have higher target RNA inhibitory ability, and these siRNAs with 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.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 183
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 184:
  • Z 16 is the first nucleotide at the 5'end of the antisense strand
  • Z 15 is selected from A, U, G, or C
  • Z 16 is a nucleotide complementary to Z 15 ; in some embodiments Where Z 15 is A, Z 16 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 181 and has the same length as the nucleotide sequence IV in the target RNA .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of acid sequence III is UG, and the base composition of nucleotide sequence IV is CA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is UUG, and the base composition of nucleotide sequence IV is CAA; at this time, the sense strand and the reverse The length ratio of the sense strand is 22/22; alternatively, 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 base composition of the nucleotide sequence III is UUUG, the base composition of nucleotide sequence IV is CAAA; 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UG , The base composition of nucleotide sequence IV is CA; 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 siRNA may be the fifth siRNA.
  • the fifth siRNA contains a sense strand and an antisense strand, and each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 241 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 242 The length is equal, and no more than 3 nucleotide differences:
  • Z 17 is A and Z 18 is U; the nucleotide sequence I contains the nucleotide Z 18 whose position corresponds to Z 17 and the nucleotide sequence II contains the nucleoside whose position corresponds to Z 14 Acid Z 19 , the Z 20 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241, and/or the nucleotide sequence II and SEQ There are no more than 2 nucleotide differences between the nucleotide sequences shown in ID NO:242.
  • the nucleotide sequence I has the nucleotide sequence shown in SEQ ID NO: 713, and the nucleotide sequence II is substantially reverse complementary or completely complementary to the nucleotide sequence I. Reverse complementary nucleotide sequence.
  • nucleotide sequence I there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241, and/or the nucleotide sequence II and SEQ ID NO: 242 shows no more than one nucleotide difference between the nucleotide sequences.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 includes the difference at position Z 20 , and Z 20 is selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position 20 Z, Z 20 is selected from A, C or G. In some embodiments, Z 19 is a nucleotide that is complementary to Z 20. SiRNAs with the above-mentioned nucleotide differences have higher target RNA inhibitory ability, and these siRNAs with nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 243
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 244:
  • Z 20 is the first nucleotide at the 5'end of the antisense strand
  • Z 19 is selected from A, U, G, or C
  • Z 20 is a nucleotide complementary to Z 19 ; in some embodiments Where Z 19 is A, Z 20 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 241 and has the same length as the nucleotide sequence IV in the target RNA .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U. ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of the acid sequence III is UA, and the base composition of the nucleotide sequence IV is UA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is CUA, and the base composition of nucleotide sequence IV is UAG; at this time, the sense strand and the reverse The length ratio of the sense strand is 22/22; alternatively, 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 base composition of the nucleotide sequence III is ACUA, the base composition of nucleotide sequence IV is UAGU; 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UA , The base composition of nucleotide sequence IV is UA; 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 fifth siRNA described in the present disclosure that can inhibit the expression of SARS-COV-2 virus gene completely matches the corresponding fragment of the viral RNA sequence in the Beijing Xinfadi G614D mutant strain.
  • the fifth siRNA provided by the present disclosure has base substitutions or mutations at the characteristic sites of the sense strand or antisense strand. The same or similar inhibitory activity of base substitution or unmutated siRNA.
  • the fifth siRNA provided in the present disclosure shows the same or similar inhibitory activity against the mutant SARS-CoV-2 viral RNA as against the unmutated SARS-CoV-2 viral RNA.
  • the siRNA may be a sixth siRNA.
  • the sixth siRNA contains a sense strand and an antisense strand, and each nucleotide in the sixth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 301 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 302 The length is equal, and no more than 3 nucleotide differences:
  • Z 21 is A and Z 22 is U; the nucleotide sequence I contains the nucleotide Z 22 whose position corresponds to Z 21 , and the nucleotide sequence II contains the nucleoside whose position corresponds to Z 22 Acid Z 23 , the Z 24 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301, and/or the nucleotide sequence II and SEQ There are no more than 2 nucleotide differences between the nucleotide sequences shown in ID NO:302.
  • 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: 301, and/or the nucleotide sequence II and SEQ ID NO: 302 shows no more than one nucleotide difference between the nucleotide sequences.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 302 includes the difference at position Z 24 , and Z 24 is selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position 24 Z, Z 24 is selected from A, C or G. In some embodiments, Z 23 is a nucleotide complementary to Z 24. SiRNAs with the above nucleotide differences have higher target RNA inhibition ability, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 304:
  • Z 24 is the first nucleotide at the 5'end of the antisense strand
  • Z 23 is selected from A, U, G, or C
  • Z 24 is a nucleotide complementary to Z 23 ; in some embodiments Where Z 23 is A, Z 24 is U;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 301 and has the same length as the nucleotide sequence IV in the target RNA .
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of the acid sequence III is UG, and the base composition of the nucleotide sequence IV is CA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is AUG, and the base composition of nucleotide sequence IV is CAU; at this time, the sense strand and the reverse The length ratio of the sense strand is 22/22; alternatively, 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 base composition of the nucleotide sequence III is AAUG, the base composition of nucleotide sequence IV is CAUU; 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UG , The base composition of nucleotide sequence IV is CA; 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 siRNA may be a seventh siRNA.
  • the seventh siRNA contains a sense strand and an antisense strand, each nucleotide in the seventh siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I It is the same length as the nucleotide sequence shown in SEQ ID NO: 361, and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same length as the nucleotide sequence shown in SEQ ID NO: 362 , And no more than 3 nucleotide differences:
  • Z 25 is A
  • Z 26 is U
  • the nucleotide sequence I contains the nucleotide Z27 whose position corresponds to Z 25
  • the nucleotide sequence II contains the nucleotide whose position corresponds to Z 26 .
  • Z 28 the Z 28 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361, and/or the nucleotide sequence II and SEQ There are no more than 2 nucleotide differences between the nucleotide sequences shown in ID NO: 362.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361, and/or the nucleotide sequence II and SEQ ID NO: 362 shows no more than one nucleotide difference between the nucleotide sequences.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 362 includes the difference at position Z 28 , and Z 28 is selected from A, C or G.
  • the nucleotide difference as a difference at a position of Z 28, Z 28 is selected from A, C or G.
  • Z 27 is a nucleotide that is complementary to Z 4. SiRNAs with the above nucleotide differences have higher target RNA inhibition ability, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 363
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 364:
  • Z 28 is the first nucleotide at the 5'end of the antisense strand
  • Z 27 is selected from A, U, G or C
  • Z 28 is a nucleotide complementary to Z 27 ; in some embodiments Among them, Z 27 is A and Z 28 is U.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II.
  • the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence.
  • the nucleotide sequence represented by NO: 361 has a nucleotide sequence that is adjacent to the 5'end and has the same length as the nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U. ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of acid sequence III is CA
  • the base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is UCA
  • the base composition of nucleotide sequence IV is UGA
  • the sense strand and the reverse The length ratio of the sense strand is 22/22
  • the lengths of nucleotide sequences III and IV are both 4 nucleotides
  • the base composition of the nucleotide sequence III is CUCA
  • the base composition of nucleotide sequence IV is UGAG
  • 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CA , The base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and the nucleotide sequence IV are completely reverse complementary, therefore, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are also determined.
  • the siRNA may be an eighth siRNA.
  • the eighth siRNA contains a sense strand and an antisense strand, each nucleotide in the eighth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 421 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 422 The length is equal, and no more than 3 nucleotide differences:
  • Z 29 is A and Z 30 is U; the nucleotide sequence I contains the nucleotide Z 31 whose position corresponds to Z 29 , and the nucleotide sequence II contains the nucleoside whose position corresponds to Z 30 Acid Z 32 , the Z 8 is the first nucleotide at the 5'end of the antisense strand.
  • the sense strand only includes nucleotide sequence I
  • the antisense strand only includes nucleotide sequence II.
  • nucleotide sequence I there are no more than 2 nucleotide differences between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421, and/or the nucleotide sequence II and SEQ There are no more than 2 nucleotide differences between the nucleotide sequences shown in ID NO:422.
  • the nucleotide sequence I has the nucleotide sequence shown in SEQ ID NO: 787, 861 or 935, and the nucleotide sequence II is substantially the opposite of the nucleotide sequence I.
  • a nucleotide sequence that is complementary or completely reverse complementary is complementary or completely reverse complementary.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421, and/or the nucleotide sequence II and SEQ There is no more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:422.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 422 includes the difference at position Z 32 , and Z 32 is selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position Z 32, Z 32 is selected from A, C or G. In some embodiments, Z 31 is a nucleotide complementary to Z 32. SiRNAs with the above nucleotide differences have higher target RNA inhibition ability, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.
  • nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 423
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 424:
  • Z 32 is the first nucleotide at the 5'end of the antisense strand
  • Z 31 is selected from A, U, G, or C
  • Z 32 is a nucleotide complementary to Z 31 ; in some embodiments Among them, Z 31 is A and Z 32 is U.
  • the sense strand further contains a nucleotide sequence III
  • the antisense strand further contains a nucleotide sequence IV.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores.
  • Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence,
  • the second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 421 and has the same length as the nucleotide sequence IV in the target RNA .
  • 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 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 length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleoside
  • the base composition of the acid sequence III is GU, and the base composition of the nucleotide sequence IV is AC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV Both are 3 nucleotides.
  • the base composition of nucleotide sequence III is GGU, and the base composition of nucleotide sequence IV is ACC; at this time, the sense strand and the reverse The length ratio of the sense strand is 22/22; alternatively, 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 base composition of the nucleotide sequence III is GGGU, the base composition of nucleotide sequence IV is AAAC; 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, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GU , The base composition of nucleotide sequence IV is AC; 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 eighth siRNA described in the present disclosure that can inhibit the expression of the SARS-COV-2 virus gene completely matches the corresponding fragment of the viral RNA sequence in the Beijing Xinfadi G614D mutant strain. And, in some embodiments, after base substitution or mutation at the characteristic site of the sense strand or antisense strand of the eighth siRNA provided in the present disclosure, the siRNA, the composition containing the siRNA, or the siRNA conjugate shows The inhibitory activity is the same as or similar to that of non-base substitution or non-mutated siRNA. In some embodiments, the eighth siRNA provided in the present disclosure shows the same or similar inhibitory activity against the mutant SARS-CoV-2 viral RNA as against the unmutated SARS-CoV-2 viral RNA.
  • nucleotide sequence V nucleic acid sequence
  • nucleotide modification in siRNA and modification sequence are applicable to any one of the above-mentioned first siRNA to eighth siRNA. That is, if there is no specific indication, the following description of siRNA should be regarded as the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA, the sixth siRNA, and the seventh siRNA. siRNA and the eighth siRNA are described one by one.
  • the siRNA also contains a nucleotide sequence V
  • the siRNA also contains a nucleotide sequence V
  • the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA , The sixth siRNA, the seventh siRNA or the eighth siRNA also contains the nucleotide sequence V.
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure may 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 siRNA sense strand and antisense strand is 19/21, 21/
  • the antisense strand further contains a nucleotide sequence V.
  • 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 strand. The 3'overhang of the chain.
  • the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26.
  • the length of the nucleotide sequence V is 2 nucleotides. Therefore, the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure may be 19/21, 21/23, or 23. /25.
  • Each nucleotide in the nucleotide sequence V can be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand with the target RNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target RNA. Therefore, in some embodiments, the ratio of the length of the sense strand and the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23. At this time, the siRNA of the present disclosure has better target RNA silencing activity.
  • the nucleotide at the corresponding position of the target RNA refers to the nucleotide or nucleotide sequence adjacent to a nucleotide sequence of the target RNA at the 5'end.
  • the nucleotide sequence of the target RNA is substantially reverse complementary or completely reverse complementary to the nucleotide sequence II, or substantially reverse to the nucleotide sequence composed of the nucleotide sequence II and the nucleotide sequence IV The nucleotide sequence that is complementary or completely 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 the nucleotide sequence shown in SEQ ID NO: 7
  • the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 8:
  • Z 4 is the first nucleotide at the 5'end of the antisense strand, Z 4 is selected from A, U, G or C, and Z 3 is a nucleotide complementary to Z 4.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68:
  • Z 8 is the first nucleotide at the 5'end of the antisense strand, Z 8 is selected from A, U, G or C, and Z 7 is a nucleotide complementary to Z 8.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 125
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128:
  • Z 12 is the first nucleotide at the 5'end of the antisense strand
  • Z 12 is selected from A, U, G, or C
  • Z 11 is a nucleotide complementary to Z 12.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 185
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 186.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 187
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 188:
  • Z 16 is the first nucleotide at the 5'end of the antisense strand, Z 16 is selected from A, U, G or C, and Z 15 is a nucleotide complementary to Z 16.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 245, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 246.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 247
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 248:
  • Z 20 is the first nucleotide at the 5'end of the antisense strand
  • Z 20 is selected from A, U, G, or C
  • Z 19 is a nucleotide complementary to Z 20.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 305
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 306.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 307
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 308:
  • Z 24 is the first nucleotide at the 5'end of the antisense strand, Z 24 is selected from A, U, G, or C, and Z 23 is a nucleotide complementary to Z 24.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 365
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 366.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 367
  • the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 368:
  • Z 28 is the first nucleotide at the 5'end of the antisense strand
  • Z 28 is selected from A, U, G, or C
  • Z 27 is a nucleotide complementary to Z 28.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 425
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 426.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 427
  • the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 428:
  • Z 32 is the first nucleotide at the 5'end of the antisense strand
  • Z 32 is selected from A, U, G, or C
  • Z 31 is a nucleotide complementary to Z 32.
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have a difference of 2 nucleotides.
  • the nucleotide sequence I has the nucleotide sequence I has the nucleotide sequence shown in SEQ ID NO: 713, 787, 861 or 935, and the nucleotide sequence II is the same as The nucleotide sequence I is a completely reverse complementary nucleotide sequence.
  • the siRNA described in the present disclosure is siCOVIDa1, siCOVIDa2, siCOVIDb1, siCOVIDb2, siCOVIDc1, siCOVIDc2, siCOVIDd1, siCOVIDd2, siCOVIDe1, siCOVIDe2, siCOVIDe1U, siCOVIDe2U, siCOVIDf1, siCOVIDf2, siCOVIDg1 listed in Table 1a-Table 1h , SiCOVIDg2, siCOVIDh1 and siCOVIDh2, siCOVIDh1U, siCOVIDh2U, siCOVIDh1R, siCOVIDh2R, siCOVIDh1G and siCOVIDh2G.
  • the nucleotides in the siRNA of the present disclosure are each independently a modified or unmodified nucleotide.
  • each nucleotide in the siRNA of the present disclosure is an unmodified nucleotide.
  • some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups will not cause the siRNA conjugate of the present disclosure to inhibit SARS-COV-2 The function of viral gene expression is significantly weakened or lost.
  • the siRNA of the present disclosure contains at least one modified nucleotide.
  • modified nucleotides refers to nucleotides or nucleotide analogs formed by replacing the 2'hydroxyl group of the ribose group of nucleotides with other groups, or nucleosides
  • the base on the acid is the nucleotide of the modified base.
  • the modified nucleotides will not cause the siRNA to significantly weaken or lose the function of inhibiting gene expression.
  • the 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 in the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate with a modified group base.
  • at least a part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modified group and/or has Modification of the ribose group of the group.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided in the present disclosure is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
  • the inventors of the present disclosure surprisingly found that the siRNA described in the present disclosure achieved a high balance of plasma stability and gene silencing efficiency in animal experiments.
  • the fluoro-modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and, in the direction from the 5'end to the 3'end, the nucleotide sequence I At least the nucleotides at positions 7, 8, and 9 are fluorinated modified nucleotides; in the direction from the 5'end to the 3'end, at least the 2, 6, 14, and 16 positions of the nucleotide sequence II The nucleotides are fluoro-modified nucleotides.
  • the fluoro-modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and there are no more than 5 fluoro-modified nucleotides in the nucleotide sequence I, In addition, according to the direction from the 5'end to the 3'end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides; in the nucleotide sequence II There are no more than 7 fluoro-modified nucleotides, and at least the 2, 6, 14, 16 nucleotides of the nucleotide sequence II are fluoro-modified nucleotides.
  • the core at positions 7, 8, 9 or 5, 7, 8, and 9 of the nucleotide sequence I Glycolic acid is a fluorinated modified nucleotide, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand , The nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense strand The nucleotides at the remaining positions are non-fluorinated modified nucleotides.
  • fluoromodified nucleotides refer to nucleotides in which the hydroxy group at the 2'position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refer to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group.
  • each non-fluorinated modified nucleotide is independently selected from among nucleotides or nucleotide analogues formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorinated group. A sort of.
  • nucleotides formed by replacing the hydroxyl group at the 2'position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotides are methoxy modified nucleotides (2'-OMe), as shown in formula (8).
  • the 2'-substituted alkoxy-modified nucleotides can be, for example, 2'-O-methoxyethyl modified nucleotides (2'-MOE), as shown in formula (9 ) Shown.
  • the 2'-amino modified nucleotide (2'-NH2) is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is represented by formula (11):
  • Nucleotide analogs refer to nucleotides that can replace nucleotides in nucleic acids, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxynucleotides The group of ribonucleotides.
  • the nucleotide analogs can be isonucleotides, bridged nucleotides (BNA for short), or acyclic nucleotides.
  • BNA refers to nucleotides that are constrained or inaccessible.
  • BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosaccharide condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):
  • Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of nucleotides.
  • acyclic nucleotides may be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotide refers to a compound formed by changing the position of the base in the nucleotide on the ribose ring.
  • the heteronucleotide may be a compound formed by moving a base from the 1'-position of the ribose ring to the 2'-position or the 3'-position, 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 the non-fluorine group as described above.
  • the nucleotide analog is selected from one of heteronucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluorinated modified nucleotide is a methoxy modified nucleotide.
  • the methoxy modified nucleotide refers to the 2'of the ribose group. -Nucleotides formed by the substitution of a hydroxy group with a methoxy group.
  • the siRNA of the present disclosure is an siRNA with the following modifications: in the direction from the 5'end to the 3'end, in the sense strand, the 7th, 8th, and 9th positions of the nucleotide sequence I Or the nucleotides at positions 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand Wherein, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense The nucleotides at the remaining positions in the chain are methoxy modified nucleotides.
  • the siRNA of the present disclosure is an siRNA with the following modifications: in 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 fluoro-modified nucleotides
  • the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides
  • the siRNA in the direction from the 5'end to the 3'end, the siRNA
  • the nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand are fluorinated modified nucleotides
  • the nucleotides at the remaining positions of the antisense strand of siRNA are methoxy groups. Modified nucleotides;
  • the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the sense of the siRNA
  • the nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at position 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides;
  • the nucleotides at positions 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are -fluoro-modified nucleotides, and the sense strand of the siRNA
  • the nucleotides at the remaining positions of the siRNA are methoxy-modified nucleotides, and according to the direction from the 5'end to the 3'end, the second, sixth, 14th, and second nucleotide sequence II in the antisense strand of the siRNA
  • the nucleotides at position 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides.
  • the siRNA provided in the present disclosure is siCOVIDa1-M1, siCOVIDa1-M2, siCOVIDa1-M3, siCOVIDa2-M1, siCOVIDa2-M2, siCOVIDa2-M3, siCOVIDb1-M1, siCOVIDb1 listed in Table 1a-Table 1h -M2, siCOVIDb1-M3, siCOVIDb2-M1, siCOVIDb2-M2, siCOVIDb2-M3, siCOVIDc1-M1, siCOVIDc1-M2, siCOVIDc1-M3, siCOVIDc2-M1, siCOVIDc2-M2, siCOVIDc2-M3, siCOVIDd1-M1, siCOVIDd1-M2 , SiCOVIDd1-M3, siCOVIDd2-M1, siCOVIDd2-M2, siCOVIDc2-M3, siCOVIDd1-M1, siCOVIDd1-M2 , SiCOVIDd1-M3,
  • the modified siRNA not only has low cost, but also makes it difficult for ribonuclease in blood to cleave nucleic acid, thereby increasing the stability of nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the above-mentioned modified siRNA has a higher activity of inhibiting target RNA.
  • the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group with a modification group.
  • the phosphate ester group with a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the The phosphate group with a modified group is a phosphorothioate group with a structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and affinity of base pairing.
  • the phosphorothioate group linkage exists in at least one of the following positions: the first and second cores of either end of the sense strand or the antisense strand Between the nucleotides; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.
  • the phosphorothioate group linkages are present at all the above positions except the 5'end of the sense chain.
  • the phosphorothioate group linkages are present at all of the above-mentioned positions except the 3'end of the sense chain.
  • the phosphorothioate group linkage is present in at least one of the following positions:
  • the siRNA provided by the present disclosure is siCOVIDa1-M1S, siCOVIDa1-M2S, siCOVIDa1-M3S, siCOVIDa2-M1S, siCOVIDa2-M2S, siCOVIDa2-M3S, siCOVIDa1-M1S1, siCOVIDa1 listed in Table 1a-Table 1h -M2S1, siCOVIDa1-M3S1, siCOVIDa2-M1S1, siCOVIDa2-M2S1, siCOVIDa2-M3S1, siCOVIDb1-M1S, siCOVIDb1-M2S, siCOVIDb1-M3S, siCOVIDb2-M1S, siCOVIDb2-M2S, siCOVIDb2-M1S, siCOVIDb2-M2S, siCOVIDb2-M3S, siCOVIDb1S, siCOVIDb2-M2S, siCOVIDb2-M3
  • the 5'terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides or 5'-phosphate analog modified nucleotides are well known to those skilled in the art, for example, 5'-phosphate nucleotides may have the following structure:
  • R is selected from H, OH, methoxy, and 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 represented by 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 in the present disclosure is siCOVIDa1-M1P1, siCOVIDa1-M2P1, siCOVIDa1-M3P1, siCOVIDa2-M1P1, siCOVIDa2-M2P1, siCOVIDa2-M3P1, siCOVIDa1-M1SP1, siCOVIDa1 listed in Table 1a-Table 1h.
  • the inventors of the present disclosure unexpectedly discovered that the above-mentioned siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosome stability, but also has higher target RNA inhibitory activity.
  • the siRNA provided in the present disclosure can be obtained by conventional siRNA preparation methods in the art (for example, solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis already has commercial customized services.
  • the modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications, a method for preparing nucleoside monomers with corresponding modifications, and the introduction of modified nucleotide groups The method of siRNA is also well known to those skilled in the art.
  • the present disclosure provides a pharmaceutical composition, which contains the above-mentioned siRNA as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier conventionally 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, polyethylenimine (PEI), 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(2-aminoethyl ethylene phosphate) (PPEEA)
  • the content of the siRNA and the pharmaceutically acceptable carrier may be a conventional content, and there is no special requirement.
  • the weight ratio of the siRNA to the pharmaceutically acceptable carrier may be 1:(1-500), and in some embodiments, the above-mentioned weight ratio is 1:(1-50).
  • the pharmaceutical composition may also include other pharmaceutically acceptable auxiliary materials, which may be one or more of various formulations or compounds conventionally used in the art.
  • the other pharmaceutically acceptable auxiliary materials 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 of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5 Buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole per kilogram (mOsm/kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the pharmaceutical composition may be a liquid preparation, such as an injection, or a lyophilized powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation can be, but is not limited to, administered by subcutaneous, intramuscular or intravenous injection, and can also be, but not limited to, administered to the lungs by spraying, or administered to other organs and tissues (such as liver) by spraying through the lungs, Or the pharmaceutical composition can be delivered by means of oropharyngeal inhalation, or nasal administration.
  • the pharmaceutical composition is for spray administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter may also be referred to as an organic amine), auxiliary lipids and/or PEGylation Lipids.
  • the organic amine, auxiliary lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (incorporated in its entirety by reference) or the amine-containing transfection compounds described in Chinese patent application CN103380113A
  • One or more of pharmaceutically acceptable salts or derivatives, auxiliary lipids and pegylated lipids are examples of pharmaceutically acceptable salts or derivatives, auxiliary lipids and pegylated lipids.
  • the organic amine may be a compound represented by formula (201) described in CN103380113A or a pharmaceutically acceptable salt thereof:
  • Each of X101 and X102 is independently O, S, N-A or C-A, where A is hydrogen or a C1-C20 hydrocarbon chain;
  • R101, R102, R103, R104, R105, R106 and R107 is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, 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 Chain or straight chain aryl, substituted or unsubstituted, branched or straight chain heteroaryl;
  • x is an integer of 1-10;
  • R103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
  • g, e, and f are each independently an integer from 1 to 6
  • HCC represents a hydrocarbon chain
  • each *N represents a nitrogen atom in formula (201).
  • R103 is a polyamine. In other embodiments, R103 is a ketal. In some embodiments, each of R101 and R102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group, the alkyl group or Alkenyl groups have 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.
  • R103 can be any of the following formulas (204)-(213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R103 is the same as in formula (201)
  • the possible connection points of the nitrogen atom, where each H at any * position can be replaced to achieve the connection with 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 represented by formula (214) and/or an organic amine represented by formula (215):
  • the auxiliary lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is a lipid covalently conjugated to one or more polyethylene glycol moieties.
  • the pegylated lipid is a lipid to which one or more polyethylene glycol moieties are covalently conjugated.
  • the PEGylated lipid used in the transfection complexes of the present disclosure is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene Diol)-2000].
  • the molar ratio between the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80): (19.7-80 ): (0.3-50), for example (50-70): (20-40): (3-20).
  • cationic lipids are also included in the pharmaceutical composition.
  • the cationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • the molar ratio between the organic amine, the auxiliary lipid, the pegylated lipid and the cationic lipid is (19.7-80 ): (19.7-80): (0.3-50): (50-150), for example (50-70): (20-40): (3-20): (80-120).
  • the pharmaceutical composition particles 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, and more generally, the liposome
  • the average diameter of the particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70 , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the siRNA in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent, is combined with all lipids (such as organic amines, auxiliary lipids, PEGylated lipids and/or cationic lipids).
  • lipids such as organic amines, auxiliary lipids, PEGylated lipids and/or cationic lipids.
  • the weight ratio 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, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or In the range from about 1:6 to about 1:10, for example, the weight ratio of the siRNA of the present disclosure to all lipids 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 formulation when used.
  • the pharmaceutical composition formed by the siRNA provided in the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, but the siRNA provided in the present disclosure can be used to replace the existing siRNA; in some In the embodiment, it can be prepared as follows:
  • the organic amine, auxiliary lipid, PEGylated lipid and cationic lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the lipid solution is 2 25 mg/mL, for example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400 One or more of, for example, ethanol.
  • the siRNA provided in the present disclosure is dissolved in a buffered salt solution to obtain an aqueous siRNA solution.
  • concentration of the buffer salt solution is 0.05-0.5M, such as 0.1-0.2M, adjust the pH of the buffer salt solution to 4.0-5.5, such as 5.0-5.2, and the amount of the buffer salt solution is such that the concentration of siRNA does not exceed 0.6 mg /mL, for example, 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it may be 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 the lipid solution to the siRNA aqueous solution is 1:(2-5), for example, it may be 1:4.
  • the incubated liposome preparation is concentrated or diluted to remove impurities and sterilize to obtain the pharmaceutical composition provided by the present disclosure.
  • Its physical and chemical parameters are pH 6.5-8, encapsulation rate not less than 80%, and particle size 40-200nm, the polydispersity index is not higher than 0.30, the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, the encapsulation rate is not less than 90%, and the particle size is 60-100nm.
  • the dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • the concentration or dilution can be carried out before, after or at the same time as the impurities are removed.
  • Various existing methods can be used to remove impurities, such as a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is phosphate buffered saline (PBS) with pH 7.4.
  • PBS phosphate buffered saline
  • the method of sterilization can adopt various existing methods, for example, it can be filtered and sterilized on a 0.22 ⁇ m filter.
  • the present disclosure provides an siRNA conjugate containing the above-mentioned siRNA and a conjugating group conjugated to the siRNA.
  • the conjugating group includes at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence.
  • the targeting groups are 1-6.
  • 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 of the siRNA and the conjugating group can be at the 3'end or 5'end of the siRNA sense strand, or at the 5'end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugating group is at the 3'end of the sense strand of the siRNA.
  • the conjugating group may be attached to the phosphate group, the 2'-position hydroxyl group or the base of the nucleotide. In some embodiments, the conjugating group can also be connected to the 3'-position hydroxyl group, in which case the nucleotides are connected by 2'-5' phosphodiester bond.
  • the conjugating group is usually attached to the phosphate group of the nucleotide; when the conjugating group is attached to the internal sequence of the siRNA, the conjugating group is Usually attached to the ribose ring or base.
  • the literature Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silence in vivo in hepatocytes. ACS Chemical biology, 2015, 10 (5)
  • the siRNA and the conjugating group may be connected by acid-labile or reducible chemical bonds. Under the acidic environment of cell endosomes, these chemical bonds can be degraded, so that the siRNA becomes a free state.
  • the conjugating group can be attached to the sense strand of the siRNA to minimize the effect of conjugation on the activity of the siRNA.
  • each of the ligands is independently selected from a ligand capable of binding to cell surface receptors.
  • the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration.
  • the pharmaceutically acceptable targeting group may be selected from one or more of the following targeting molecules or ligands formed by derivatives thereof; ligands targeting receptors on the surface of liver cells Body, such as lipophilic molecules, such as cholesterol, bile acids, vitamins (such as vitamin E), lipid molecules of different chain length; polymers, such as polyethylene glycol; sugars, such as lactose, polylactose, mannose, semi Lactose, N-acetylgalactosamine (GalNAc); receptor ligands expressed by liver parenchymal cells, such as asialoglycoprotein, asialoglycan residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.) ), glucagon, neurotransmitter (such as adrenaline), growth factor, transferrin and other aptamers; antibodies; quantum dots.
  • At least one targeting group is a ligand capable of binding to the receptor of the novel coronavirus on the cell surface.
  • the types of these ligands are well known to those skilled in the art, and their role is to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligand to target cells.
  • at least one targeting group is a ligand that targets a receptor on the surface of lung cells, and in some embodiments, at least one targeting group is a ligand capable of targeting ACE2 or integrin ⁇ v ⁇ 6.
  • At least one targeting group is a polypeptide, such as a membrane-permeable peptide; or a small molecule ligand, such as a targeting group listed in Table 6 of WO2019010274A1, or various targeting groups described in WO2019089765A1 ( As described in paragraph [0094], the ligand numbered as Structure 1-Structure 37), the entire disclosure of which is incorporated herein by reference.
  • the inventors of the present disclosure unexpectedly discovered that the siRNA conjugate of the present disclosure exhibits higher SARS-COV-2 virus RNA silencing activity.
  • the siRNA of the present disclosure may be one of the siRNAs shown in Tables 1a-1h. Using these siRNAs, the siRNA conjugates of the present disclosure exhibit higher SARS-COV-2 viral RNA silencing activity.
  • capital letters C, G, U, A represent the base composition of nucleotides
  • capital letter R represents a degenerate base, specifically a random nucleotide base A or G
  • Y is a base complementary to R
  • the lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide
  • the lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleus Glycolic acid
  • lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by phosphorothioate groups
  • P1 indicates that the adjacent nucleotide on the right side of P1 is 5'-phosphate nucleotide or 5'- Phosphate analog modified nucleotides.
  • P1 represents a specifically modified VP, Ps, or P, where the letter combination VP indicates that the adjacent nucleotide to the right 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, and the capital letter P indicates the right side of the letter P The adjacent nucleotide is a 5'-phosphate nucleotide.
  • each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, and the phosphodiester bond or phosphorothioate bond is not bridged
  • the oxygen atom or sulfur atom has a negative charge, it can exist in the form of a hydroxyl group or a mercapto group, and the hydrogen ion in the hydroxyl group or mercapto group can also be partially or completely replaced by a cation.
  • the cation may be any cation, such as one of metal cation, ammonium ion NH4+, and organic ammonium cation.
  • the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations.
  • the alkali metal ion may be K+ and/or Na+
  • the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate described in the present disclosure may exist at least partially 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 sodium ions
  • the siRNA or siRNA conjugate described in the present disclosure is a sodium salt or a partial sodium salt. The form exists.
  • modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications.
  • the method of preparing nucleoside monomers with corresponding modifications and the method of introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.
  • siRNA of the present disclosure and pharmaceutical compositions and conjugates containing 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 the treatment and/or prevention of diseases caused by the novel coronavirus.
  • the disease caused by the novel coronavirus is COVID-19.
  • the present disclosure provides a method for preventing and/or a disease caused by a novel coronavirus, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure Subjects in need.
  • the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure can be used to prevent and/or treat diseases caused by the novel coronavirus, or be used to prevent and/or treat diseases caused by the novel coronavirus. medicine.
  • administration/administration refers to a method or approach that allows the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to be at least partially positioned at a desired site to produce a desired effect.
  • the siRNA, pharmaceutical composition, and/or siRNA conjugate of the present disclosure are placed in a subject.
  • the routes of administration suitable for the method of the present disclosure include local administration and systemic administration. Generally speaking, local administration results in the delivery of more siRNA conjugates to specific sites than in the subject's systemic circulation; while systemic administration results in the delivery of the siRNA, pharmaceutical compositions and/or siRNA conjugates of the present disclosure Basic systemic circulation to the subject.
  • an administration method capable of delivering drugs to the lungs is adopted.
  • the subject can be administered to the subject by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, and transdermal administration. Medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including oral administration and sublingual administration).
  • the frequency of administration can be one or more times per 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, pharmaceutical composition or 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, weight and sex of the subject. Toxicity and efficacy can be measured in cell culture or experimental animals through standard pharmaceutical procedures, such as measuring LD50 (lethal dose to 50% of the population) and ED50 (in quantitative response, it refers to the dose that can cause 50% of the maximum response intensity. The reaction middle refers to the dose that can cause a positive reaction in 50% of the test subjects). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies. In some embodiments, the dosage of the preparation made of the siRNA, pharmaceutical composition or siRNA conjugate during the administration process is adjusted according to different modes of administration.
  • 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, in other embodiments 0.1-15 mg/kg body weight, in other embodiments 0.1-10 mg/kg body weight;
  • siRNA dosage can be 0.001-50mg/kg body weight, in some embodiments 0.01-10mg/kg body weight, in some embodiments 0.05-5mg/kg body weight, in some embodiments 0.1-3mg/kg weight.
  • the present disclosure provides a method for inhibiting SARS-COV-2 virus gene expression in lung cells, the method comprising conjugating an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA of the present disclosure In contact with the lung cells, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are introduced into the lung cells, and the expression of SARS-COV-2 virus gene in the lung cells is inhibited through the mechanism of RNA interference. the goal of.
  • the amount of siRNA in the provided modified siRNA, pharmaceutical composition and/or siRNA conjugate is generally such an amount that it is sufficient to reduce the target
  • the expression of the gene results in 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 required to achieve this local concentration will vary with various factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, the delivery route (local or systemic), etc. .
  • the concentration at the delivery site 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 the modified siRNA, pharmaceutical composition and siRNA conjugate of the present disclosure.
  • kits described herein can provide modified siRNA in one container.
  • the kits described herein may include a container that provides pharmaceutically acceptable excipients.
  • the kit may also contain other ingredients, such as stabilizers or preservatives.
  • the kits described herein may contain at least one other therapeutic agent in a container other than the container that provides the modified siRNA described herein.
  • the kit may include instructions for mixing the modified siRNA with pharmaceutically acceptable carriers and/or excipients or other ingredients (if any).
  • the modified siRNA and a pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, pharmaceutical composition and/or siRNA conjugate and/or conjugate, and/ Or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dried form or lyophilized form.
  • the modified siRNA and pharmaceutically acceptable carriers and/or adjuvants and the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable adjuvants are substantially pure and/or Sterile.
  • sterile water can be provided in the kit of the present disclosure.
  • the siRNA, the composition containing the siRNA, and the siRNA conjugate provided by the present disclosure have good stability, high gene suppression activity, low cytotoxicity, and/or can significantly treat or alleviate the disease caused by the new coronavirus Symptoms of the disease.
  • the siRNA, the composition containing the siRNA (hereinafter sometimes referred to as the siRNA composition) or the siRNA conjugate provided in the present disclosure show excellent target gene inhibitory activity in in vitro cell experiments.
  • the siRNA, siRNA composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition rate of target gene expression.
  • the siRNA provided in the present disclosure shows a target RNA expression inhibition rate of 96.23% to 99.00% in HEK293A cells.
  • the siRNA provided in the present disclosure shows a target RNA expression inhibition rate of 91.53% to 96.99% in HEK293A cells. In some embodiments, at a siRNA concentration of 1 nM, the siRNA provided in the present disclosure shows a target RNA expression inhibition rate of 92.54%-97.75% in HEK293A cells. In some embodiments, at a final siRNA transfection concentration of 50 nM, the siRNA provided in the present disclosure in the Vero cell line infected with the SARS-COV-2 virus showed at least 37.5%, even as high as 48h after transfection. 97.9% SARS-COV-2 virus RNA inhibition rate.
  • the siRNA, the composition containing the siRNA, or the siRNA conjugate provided in the present disclosure may have higher stability and/or higher activity in vivo.
  • the siRNA, siRNA composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo % Inhibition rate of target gene expression.
  • the siRNA, siRNA composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo % SARS-COV-2 virus gene expression inhibition rate.
  • the siRNA, siRNA composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo % Inhibition rate of SARS-COV-2 virus gene expression in the lungs. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo % Of the SARS-COV-2 virus gene expression inhibition rate in the lungs of animal models.
  • the siRNA, siRNA composition, or siRNA conjugate provided by the present disclosure in some embodiments exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80% in vivo , 90% or 95% of the SARS-COV-2 virus gene expression inhibition rate in tracheal tissues in animal models. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided in the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo Inhibition rate of SARS-COV-2 virus gene expression in the lungs of% of human subjects. In some embodiments, the siRNA provided in the present disclosure shows a relative inhibition rate of 85.71% to 94.20% of viral RNA load in lung tissues of mice.
  • the siRNA provided in the present disclosure shows a relative inhibition rate of 98.19%-99.71% of viral RNA load in tracheal tissues of mice. In some embodiments, the siRNA provided in the present disclosure shows a relative inhibition rate of 49.5% of viral RNA load in lung tissues of mice. In some embodiments, the siRNA provided in the present disclosure shows a relative inhibition rate of 63.95% of viral RNA load in the tracheal tissue of mice. In some embodiments, the siRNA provided in the present disclosure shows a relative inhibition rate of 91.27% to 94.68% of viral RNA load in lung tissues of mice.
  • the siRNA, composition containing the siRNA, or siRNA conjugate provided in the present disclosure does not show significant off-target effects.
  • Off-target effects can be, for example, suppression of normal expression of genes other than target genes. It is considered that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the target gene effect, the off-target effect is not significant.
  • the siRNA provided in the present disclosure and the siRNA containing the siRNA
  • the composition or siRNA conjugate still shows high viral RNA inhibitory activity.
  • siRNA, pharmaceutical composition and siRNA conjugate provided in the present disclosure can inhibit the expression of SARS-COV-2 virus gene, and effectively treat and/or prevent related disease symptoms caused by SARS-COV-2 virus gene expression , Has a good application prospect.
  • the reagents and media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)) Method to proceed.
  • LipofectamineTM2000 (Invitrogen) is used as a transfection reagent, and the specific operation refers to the instructions provided by the manufacturer.
  • each solution is prepared by using physiological saline as the solvent.
  • the experimental data analysis uses Graphpad prism 8.0 statistical analysis software.
  • siCOVIDa1-M1S, siCOVIDb1-M1S, siCOVIDc1-M1S, siCOVIDd1-M1S, siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDg1-M1S, siCOVIDh1-M1S, siCOVIDe1, siCOVIDf1, siCOVIDe1U-M1S, siCOVIDh1U-M1S, siCOVID siCOVIDh1G-M1S, siCOVIDe1-M1S1 and siCOVIDh1-M1S1 are shown in Table 2.
  • the sense and antisense strands corresponding to the siRNAs numbered NC and NC2 in Table 2 were synthesized by solid-phase synthesis. Use DEPC water to dissolve the obtained equimolar sense strand and antisense strand, and then anneal to obtain reference siRNA, numbered NC and NC2.
  • capital letters C, G, U, A represent the base composition of nucleotides
  • capital letter R represents a degenerate base
  • R is a nucleotide base A or G
  • Y is a base complementary to R
  • lowercase The letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide
  • the lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide
  • the lowercase letter s indicates that the two nucleotides on the left and right sides of the letter s are connected by phosphorothioate groups.
  • nucleoside monomers are sequentially connected or fluorescently labeled according to the base sequence of each primer sequence. ⁇ nucleoside monomers.
  • siRNA, target sequence, primer sequence or probe sequence of the present disclosure are prepared, they are respectively freeze-dried and stored as solid powder for later use.
  • the cells were cultured in H-DMEM complete medium (Hyclone) supplemented with 20% fetal bovine serum (FBS, Hyclone) and 0.2% by volume penicillin double antibody (Penicillin-Streptomycin, Gibco, Invitrogen).
  • H-DMEM complete medium Hyclone
  • FBS fetal bovine serum
  • penicillin double antibody Penicillin-Streptomycin, Gibco, Invitrogen.
  • Culture HEK293A cells purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • modified siRNA with a DNA seeded arm is a powerful tool for mammalian gene silence with significantly reduced off-target, effect.
  • the method described in 2136-2151 construct the detection plasmid, and the siRNA to be evaluated (siCOVIDa1-M1S, siCOVIDb1-M1S, siCOVIDc1-M1S, siCOVIDd1-M1S, siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDg1-M1S and siCOVIDh1-M1S )
  • siCOVIDa1-M1S siCOVIDb1-M1S
  • siCOVIDc1-M1S siCOVIDd1-M1S
  • siCOVIDe1-M1S siCOVIDf1-M1S
  • siCOVIDg1-M1S and siCOVIDh1-M1S Were co-transfected into HEK293A cells, and the inhibitor
  • the psiCHECKTM-2 (PromegaTM) plasmid is used to construct a detection plasmid, which contains a target sequence, that is, the siRNA target sequence.
  • the target sequence is as follows:
  • the target sequence of siCOVIDa1-M1S is:
  • the target sequence of siCOVIDb1-M1S is:
  • the target sequence of siCOVIDc1-M1S is:
  • the target sequence of siCOVIDd1-M1S is:
  • the target sequence of siCOVIDe1-M1S is:
  • the target sequence of siCOVIDf1-M1S is:
  • the target sequence of siCOVIDg1-M1S is:
  • the target sequence of siCOVIDh1-M1S is:
  • the target sequence was cloned into the Xho I/Not I site of the psiCHECKTM-2 plasmid.
  • HEK293A cells Inoculate HEK293A cells in a 96-well plate at 8 ⁇ 103 cells/well. When the cell growth density reaches 70-80% after 16 hours, exhaust the H-DMEM complete medium in the culture wells, and add 80 ⁇ l Opti-MEM medium to each well. (GIBCO company) Continue to train for 1.5h.
  • siRNA working solutions For each siRNA, use DEPC water to dilute the above detection plasmid into 200ng/ ⁇ l detection plasmid working solution. For each siRNA, use siRNA and DEPC water to prepare siRNA working solutions with concentrations of 1 nM, 0.1 nM, and 0.01 nM (based on the amount of siRNA).
  • Each 1A1 solution contains 1 ⁇ l of siRNA working solution with a concentration of 0.1 ⁇ M, 0.05 ⁇ l of detection plasmid working solution (containing 10ng of detection plasmid) and 10 ⁇ l of Opti-MEM medium.
  • Each 1A2 solution contains 1 ⁇ l of siRNA working solution with a concentration of 0.01 ⁇ M, 0.05 ⁇ l of detection plasmid working solution (containing 10ng of detection plasmid) and 10 ⁇ l of Opti-MEM medium.
  • Each 1A3 solution contains 1 ⁇ l of siRNA working solution with a concentration of 0.001 ⁇ M, 0.05 ⁇ l of detection plasmid working solution (containing 10ng of detection plasmid) and 10 ⁇ l of Opti-MEM medium.
  • each 1B solution contains 0.2 ⁇ l Lipofectamine TM 2000 and 10 ⁇ l Opti-MEM medium.
  • each 1C solution contains 0.05 ⁇ l of detection plasmid working solution (containing 10ng of detection plasmid) and 10 ⁇ l of Opti-MEM medium.
  • siRNA For each siRNA, incubate one part of 1B solution, one part of 1A1 solution, one part of 1A2 solution, and one part of 1A3 solution at room temperature for 20 minutes to obtain transfection complexes 1X1, 1X2, or 1X3, respectively. Mix one part of 1B solution with one part of 1C solution, and incubate at room temperature for 20 minutes to obtain the transfection complex 1X4.
  • siRNA For each siRNA, add the transfection complex 1X1 to the three culture wells, mix them evenly, and add 20 ⁇ l/well to obtain a co-transfection mixture with a final siRNA concentration of about 1 nM, which is recorded as test group 1.
  • siRNA concentration For each siRNA, add the transfection complex 1X2 to the other three culture wells, mix them evenly, and add 20 ⁇ l/well to obtain a co-transfection mixture with a final siRNA concentration of about 0.1nM, which is recorded as test group 2. .
  • siRNA For each siRNA, add the transfection complex 1X3 to the other three culture wells, mix them evenly, and add 20 ⁇ l/well to obtain a co-transfection mixture with a final siRNA concentration of about 0.01nM, which is recorded as test group 3. .
  • siRNA For each siRNA, add the transfection complex 1X4 to the other three culture wells to obtain a co-transfection mixture without siRNA. The added amount is 20 ⁇ l/well, which is recorded as the control group.
  • each well was supplemented with 100 ⁇ l of H-DMEM complete medium supplemented with 20% FBS. Place the 96-well plate in a CO2 incubator and continue to incubate for 24 hours.
  • the luminescence ratio Ratio (test) or Ratio (control) of each test group or control group is the average value of the ratio of the three culture wells;
  • the luminescence ratio of the test group is normalized to obtain the ratio R of Ratio (test)/Ratio (control), which represents the expression level of the Renilla reporter gene, that is, the residual activity.
  • Inhibition rate of siRNA (1-R) ⁇ 100%.
  • the inhibitory activity of the reference siRNA NC in the psiCHECK system was also investigated according to the method of experimental examples 1-8. The only difference is that the tested siRNA is the reference siRNA NC.
  • preparation examples 1-8 (siCOVIDa1-M1S, siCOVIDb1-M1S, siCOVIDc1-M1S, siCOVIDd1-M1S, siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDg1-M1S, siCOVIDh1-M1S) all have a concentration of 1nM-0.1nM Good in vitro inhibitory activity. And the inhibitory activity is concentration-dependent. In particular, at a siRNA concentration of 1 nM, the target sequence inhibition rate is at least 96.23% and up to 99.00%, showing an excellent effect of inhibiting SARS-COV-2 virus gene expression.
  • siCOVIDa1-M1S, siCOVIDb1-M1S, siCOVIDc1-M1S, siCOVIDd1-M1S, siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDg1-M1S and siCOVIDh1-M1S in the in vitro psiCHECK system The only difference is that the final concentration of siRNA is 1nM, 0.3nM and 0.1nM; when constructing the detection plasmid, for each siRNA, the target sequence is the following sequence:
  • the target sequence of siCOVIDa1-M1S is:
  • the target sequence of siCOVIDb1-M1S is:
  • the target sequence of siCOVIDc1-M1S is:
  • the target sequence of siCOVIDd1-M1S is:
  • siCOVIDe1-M1S and siCOVIDf1-M1S are:
  • the target sequence of siCOVIDg1-M1S is:
  • the target sequence of siCOVIDh1-M1S is:
  • the inhibitory activity of the reference siRNA NC in the psiCHECK system was also investigated according to the method of Experimental Example 9. The only difference is that the tested siRNA is the reference siRNA NC.
  • the siRNAs of the present disclosure all showed a good effect of inhibiting the gene expression of the target sequence.
  • the inhibitory activity is concentration-dependent.
  • the RNA expression inhibition rate of the target sequence is at least 91.53%, and the highest is 96.99%, showing an excellent inhibitory effect on SARS-COV-2 viral RNA.
  • siCOVIDe1-M1S the inhibitory activity of siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDe1 and siCOVIDf1 in the in vitro psiCHECK system were tested respectively.
  • the only difference is that when constructing the detection plasmid, for siCOVIDe1, the target The sequence is the target sequence of siCOVIDe1-M1S; for siCOVIDf1, the target sequence is the target sequence of siCOVIDf1-M1S; the experimental results are shown in Figure 3.
  • siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDe1 and siCOVIDf1 showed good SARS-COV-2 virus inhibitory activity in vitro.
  • the target sequence expression inhibition rate of siCOVIDe1-M1S The target sequence expression inhibition rate of siCOVIDf1-M1S was 97.75%, 97.15%, the target sequence expression inhibition rate of siCOVIDe1 was 92.54%, and the target sequence expression inhibition rate of siCOVIDf1 was 92.46%, showing excellent inhibition of SARS-COV -2 The effect of viral gene expression.
  • the Vero cells were respectively infected with virus strains that were successively diluted 10 times, cultured at 37°C for 1 hour, and in DMEM supplemented with 2% FBS and 1% low melting point agarose (Promega) Continue to culture the cells for 2 days, fix the cells with 4% formaldehyde aqueous solution and stain the plaque with 0.2% crystal violet aqueous solution for titration to determine the virus titer of the virus solution.
  • the virus diluent used in the following experiments is a virus solution obtained by diluting the virus solution with physiological saline.
  • siRNA of the present disclosure inhibits SARS-COV-2 viral RNA after Vero infects cells
  • the ATCC complete medium is MEM-EBSS medium, containing 2mM L-glutamine-containing MEM basal medium (Hyclone) and Earle's balanced salt solution (EBSS, Giboco).
  • the balanced salt solution contains 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids (NEAA, Corning), 1.0 mM 90% sodium pyruvate and 10% fetal bovine serum (FBS, Hyclone).
  • use 5 ⁇ 105 PFU of SARS-COV-2 virus solution for cell infection specifically: Add 50 ⁇ l of virus dilution containing 5 ⁇ 105PFU to each well, incubate in an incubator containing 5% CO2/95% air at 37°C for 1 h; then discard the supernatant and wash the cells twice with 1 ⁇ PBS solution , Each use 500 ⁇ l/well to obtain virus-infected Vero cells, which are recorded as the blank group with 3 replicate wells in each group.
  • use 5 ⁇ 105 PFU of SARS-COV-2 virus solution for cell infection specifically: Add 50 ⁇ l of virus dilution containing 5 ⁇ 105PFU to each well, incubate at 37°C in an incubator containing 5% CO2/95% air for 1 h; then discard the supernatant and wash the cells twice with 1 ⁇ PBS solution , Each dosage is 500 ⁇ l/well to get virus-infected Vero cells.
  • each siRNA siRNA (siCOVIDb1-M1S, siCOVIDc1-M1S, siCOVIDd1-M1S, siCOVIDe1-M1S, siCOVIDf1-M1S, siCOVIDg1-M1S, siCOVIDh1-M1S and NC2), so that the final concentration of each siRNA is 50nM, and the Vero cells infected with the virus and transfected with each siRNA are obtained, which are respectively marked as the siCOVIDb1-M1S test group and the siCOVIDc1-M1S test siCOVIDd1-M1S test group , SiCOVIDe1-M1S test group, siCOVIDf1-M1S test group, siCOVIDg1-M1S test group, siCOVIDh1-M1S test group and NC2 control group, each with 3 replicate holes.
  • the specific steps are: culture the blank group and each test group for 24 hours and 48 hours, collect the cell culture medium (that is, the supernatant) at 24 hours and 48 hours respectively, and completely lyse the Vero cells that are flat on the cell culture plate .
  • the cDNA was obtained, and the SARS-COV-2 viral RNA load was detected for each test group and the blank group using the probe method qRT-PCR. The results are shown in Figure 4A and Figure 4B.
  • PCR primers used to amplify SARS-COV-2 virus and GAPDH as an internal reference gene are shown in Table 3.
  • the modified siRNA provided by the present disclosure has higher viral RNA inhibitory activity in the Vero cell line infected with SARS-COV-2 virus, and the viral load after transfection of siRNA is significantly lower.
  • the viral RNA load of Vero cells transfected with NC2 was 1.32 ⁇ 107 copies/mL after 24 hours
  • the viral RNA load of siCOVIDb1-M1S was 8.15 ⁇ 106 copies/mL 24 hours after transfection.
  • the viral RNA load 48 hours after transfection of NC2 was 1.23 ⁇ 1010copies/mL, and the viral RNA load 48 hours after transfection with siCOVIDb1-M1S was 1.38 ⁇ 1010copies/mL; transfection with siCOVIDc1-M1S
  • the viral RNA load after 48 hours was 1.47 ⁇ 109 copies/mL, which was only 12.0% of the viral RNA load in Vero cells transfected with NC2 at the same time;
  • the viral RNA load was 7.69 48 hours after transfection with siCOVIDd1-M1S ⁇ 109copies/mL, which is only 62.5% of the viral RNA load in Vero cells transfected with NC2 at the same time; 48 hours after transfection with siCOVIDe1-M1S, the viral RNA load is 7.48 ⁇ 108copies/mL, which is only for NC2 transfection
  • the viral RNA load in Vero cells was 6.1% at the same time; 48 hours after transfection of siCOVIDf1
  • the siRNA of the present disclosure exhibits an excellent effect of inhibiting SARS-COV-2 virus gene expression in the Vero cell line infected with SARS-COV-2 virus.
  • Vero cells were seeded in a 96-well plate at a rate of 8 ⁇ 104 cells/mL, 100 ⁇ L/well, that is, 8 ⁇ 104 cells/well, cultured for 24 hours and then transfected.
  • the siRNAs used are siCOVIDe1-M1S, siCOVIDh1-M1S and NC, respectively.
  • Each 2A1-2A8 solution contains 1 ⁇ l of the above-mentioned 8 concentrations of siRNA working solution, 0.05 ⁇ l of detection plasmid working solution (containing 10ng of detection plasmid) and 10 ⁇ l of Opti-MEM medium .
  • each 2B solution contains 0.2 ⁇ l Lipofectamine TM 2000 and 10 ⁇ l Opti-MEM medium.
  • each siRNA transfection complex 2X1-2X8 In the culture well, add each siRNA transfection complex 2X1-2X8, mix evenly, add 20 ⁇ l/well, and get the final concentration of each siRNA to be about 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125
  • the transfection complexes of nM, 1.563nM and 0.781nM, and each siRNA transfection complex 2X1-2X8 were transfected into 3 culture wells respectively to obtain a co-transfection mixture containing siRNA.
  • the co-transfection mixture containing siRNA was transfected in the culture wells for 4 hours, and each well was supplemented with 100 ⁇ l H-DMEM complete medium containing 20% FBS. Place the 96-well plate in a CO 2 incubator and continue culturing for 72 hours.
  • the siRNAs used are siCOVIDe1-M1S, siCOVIDh1-M1S and NC.
  • OD test group
  • OD blade control group
  • OD control group
  • OD control group
  • the plasmid and the test siRNA were co-transfected into HEK293A cells, and then the expression level of the dual luciferase reporter gene was used to reflect the inhibitory activity of the siRNA.
  • the siRNA to be evaluated are siCOVIDe1-M1S and siCOVIDe1U-M1S (Preparation Example 5 and Preparation Example 11); when constructing the detection plasmid, the target sequence of the siRNA to be tested is TCGAGTAGTCTCTAGTCAGTGTGTTAGC (SEQ ID NO: 1015).
  • Preparation Example 5 and Preparation Example 11 have good in vitro inhibitory activity at a concentration of 1nM-0.1nM, and exhibit a concentration-dependent inhibitory activity.
  • the RNA expression inhibition rate is at least 83.16%, and the highest is 95.72%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.
  • the plasmid and the test siRNA were co-transfected into HEK293A cells, and then the expression level of the dual luciferase reporter gene was used to reflect the inhibitory activity of the siRNA.
  • the siRNA to be evaluated are siCOVIDe1-M1S and siCOVIDe1U-M1S (Preparation Example 5 and Preparation Example 11); when constructing the detection plasmid, the target sequence of the siRNA to be evaluated is TCGAGTAGTTTCTAGTCAGTGTGTTAGC (SEQ ID NO: 1016) .
  • siCOVIDe1U-M1S The inhibitory activity results of different concentrations of siCOVIDe1-M1S and siCOVIDe1U-M1S on the target sequence are shown in Figure 7.
  • siCOVIDe1U-M1S is compared with siCOVIDe1-M1S, the third nucleotide is replaced with uracil from cytosine, which has a mismatch with the target sequence.
  • Preparation Example 5 and Preparation Example 11 have good in vitro inhibitory activity at a concentration of 1nM-0.1nM, and exhibit a concentration-dependent inhibitory activity.
  • the target sequence expression inhibition rate is at least 85.56%, and the highest is 95.47%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.
  • the siRNA when the characteristic sites of the sense strand or the antisense strand of the siRNA provided in the present disclosure undergo base substitution or mutation, the siRNA exhibits the same or similar inhibitory activity as the siRNA corresponding to the non-base substitution or unmutated sequence .
  • the plasmid and the test siRNA were co-transfected into HEK293A cells, and then the expression level of the dual luciferase reporter gene was used to reflect the inhibitory activity of the siRNA.
  • the siRNAs to be evaluated are siCOVIDh1-M1S, siCOVIDh1U-M1S, siCOVIDh1R-M1S, and siCOVIDh1G-M1S (Preparation Example 8, Preparation Examples 12-14); when constructing the detection plasmid, for each of the The target sequence of siRNA is as follows:
  • the target sequence of siCOVIDh1-M1S is:
  • the target sequence of siCOVIDh1U-M1S is:
  • the target sequence of siCOVIDh1R-M1S is:
  • the target sequence of siCOVIDh1G-M1S is:
  • the above four target sequences are a segment of the nucleotide sequence in the SNP mutation sequence of the SARS-CoV-2 virus, which respectively have 1 or 2 nucleotide mutations compared with the corresponding sequence fragment of NC_045512.2.
  • Preparation Example 8 and Preparation Example 12-14 (siCOVIDh1-M1S, siCOVIDh1U-M1S, siCOVIDh1R-M1S, and siCOVIDh1G-M1S) have good in vitro inhibitory activity at a concentration of 1nM-0.1nM, and exhibit a concentration-dependent sexual inhibitory activity.
  • the target sequence expression inhibition rate is at least 80.5% and up to 95.25%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.
  • the siRNA provided in the present disclosure still shows high inhibitory activity for the mutated viral target sequence.
  • the plasmid and the test siRNA were co-transfected into HEK293A cells, and then the expression level of the dual luciferase reporter gene was used to reflect the inhibitory activity of the siRNA.
  • the siRNAs to be evaluated are siCOVIDh1-M1S, siCOVIDh1U-M1S, siCOVIDh1R-M1S, and siCOVIDh1G-M1S (Preparation Example 8, Preparation Examples 12-14); when constructing the detection plasmid, the target sequence of the siRNA to be evaluated Both are TCGAGGTCATTCAATCCAGAAAGC (SEQ ID NO: 1017).
  • Preparation Example 8 and Preparation Example 12-14 (siCOVIDh1-M1S, siCOVIDh1U-M1S, siCOVIDh1R-M1S, and siCOVIDh1G-M1S) have good in vitro inhibitory activity at a concentration of 1nM-0.01nM, and exhibit a concentration-dependent sexual inhibitory activity.
  • the target sequence inhibition rate is at least 69.6% and up to 95.25%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.
  • the siRNA provided in the present disclosure has a base substitution or mismatch at a specific position on the sense strand or antisense strand of the mutant sequence.
  • Completely matched siRNAs have the same or similar inhibitory activity.
  • the plasmid and the test siRNA were co-transfected into HEK293A cells, and then the expression level of the dual luciferase reporter gene was used to reflect the inhibitory activity of the siRNA.
  • the siRNA to be evaluated is siCOVIDh1-M1S.
  • the corresponding four different target sequences are as follows:
  • the target sequence of siCOVIDh1-M1S is:
  • TCGAGGTCATTCAATCCAGAAAGC SEQ ID NO: 1017
  • inhibitory activity results of different concentrations of siCOVIDh1-M1S corresponding to different target sequences are shown in sequence in the histogram in Figure 10.
  • the siRNA to be evaluated that is, Preparation Example 8 has good in vitro inhibitory activity for 4 different target sequences at a concentration of 1 nM-0.01 nM, and exhibits a concentration-dependent inhibitory activity.
  • the target sequence expression inhibition rate was above 95%, and the highest was 97.35%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.
  • the siRNA provided by the present disclosure only completely matches the unmutated sequence, but has at least one base mismatch with the mutated sequence, the siRNA still shows excellent inhibitory activity for the mutated sequence and compares well with the unmutated sequence. The inhibitory activity level of the mutant sequence is close.
  • siRNA of the present disclosure inhibits SARS-COV-2 virus RNA in vivo in humanized ACE2 transgenic mice infected with SARS-COV-2 virus
  • Humanized ACE2 transgenic mice (B6/JGpt-Ace2em1Cin(hACE2)/Gpt) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. First, the humanized ACE2 transgenic mice were randomly divided into groups, with 3 mice in each group, numbered separately, and the PBS control group was added. The mice were anesthetized with 50 mg/kg pentobarbital by intraperitoneal injection, and 50 ⁇ L of virus dilution containing 4 ⁇ 105 pfu SARS-COV-2 was administered by nasal drops to infect the mice.
  • siRNA: liposome A 1:15 (wt/wt)
  • the amine transfection reagent LC8 is a cationic lipid 87 prepared in accordance with Example 12 of Chinese patent CN 103380113B.
  • the composition was prepared according to the method described in Example 9 of CN 103380113B, except that the above-mentioned siRNA and liposome A were used for preparation.
  • mice Two hours after exposure, the three groups of mice were given the test siRNA composition by the nasal drip method, the administration volume was 10 ⁇ L, and the administration dose was 3 mg/kg. Then, at 24h and 48h after exposure, the three groups of mice were repeatedly administered by nasal drops, each with a volume of 10 ⁇ L and a dose of 3mg/kg.
  • mice were sacrificed 6 hours after the last administration, and lung tissues and tracheal tissues in the mice were collected. Save with RNA later (Sigma Aldrich); homogenize lung tissue and tracheal tissue with a tissue homogenizer, and then use Trizol to extract total RNA according to the standard operating procedures for total RNA extraction.
  • Real-time fluorescent quantitative PCR was used to detect the SARS-COV-2 viral RNA load in lung tissues and tracheal tissues, specifically: use the RR064A TAKARA reverse transcription kit according to its instructions to reverse transcription to obtain cDNA, and then use fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.) detects the SARS-COV-2 viral RNA load in lung tissue and trachea tissue by siRNA.
  • Use probe method qRT-PCR to detect the SARS-COV-2 viral RNA load in each test group and the blank group, and calculate the siRNA to SARS-COV-2 viral RNA load, the results are shown in Figure 11A and Figure 11B Show.
  • PCR primers used to amplify SARS-COV-2 virus and GAPDH as an internal reference gene are shown in Table 3.
  • the relative inhibition rate of viral RNA load 1-(viral RNA load of the administration group/viral RNA load of the control group) ⁇ 100%.
  • the mice were transfected with siRNA.
  • the viral RNA load in the body was lower than that of the control group.
  • the viral RNA load was 3.85 ⁇ 109 copies/mL; after administration of the siCOVIDe1-M1S composition, the viral RNA load was 2.23 ⁇ 108 copies/mL, which was only the equivalent of the control group’s viral RNA load 5.80%, the relative inhibition rate of viral RNA load is 94.20%; after administration of the siCOVIDh1-M1S composition, the viral RNA load is 5.50 ⁇ 108 copies/mL, which is only 14.29% of the viral RNA load of the control group, and the viral RNA load is relatively The inhibition rate was 85.71%.
  • the viral RNA load was 5.44 ⁇ 108 copies/mL; after the siCOVIDe1-M1S composition was administered, the viral RNA load was 1.59 ⁇ 106 copies/mL, which was only the amount of viral RNA in the control group. 0.29%, the relative inhibition rate of viral RNA load was 99.71%; after administration of the siCOVIDh1-M1S composition, the viral RNA load was 9.82 ⁇ 106 copies/mL, which was only 1.81% of the viral RNA load of the control group, and the viral RNA load was relatively The inhibition rate was 98.19%. Thus showing an excellent effect of inhibiting the expression of SARS-COV-2 virus RNA.
  • Humanized ACE2 transgenic mice (B6/JGpt-Ace2em1Cin(hACE2)/Gpt) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. First, the humanized ACE2 transgenic mice were randomly divided into groups (all females), 4 mice in each group, numbered separately, and a PBS control group was added. The mice were anesthetized with 50 mg/kg pentobarbital by intraperitoneal injection, and 50 ⁇ L of virus dilution containing 4 ⁇ 105 pfu SARS-COV-2 was administered to each mouse by the nasal drip method to infect the mice.
  • the drug administered to the mice is the siRNA described in the application, namely siCOVIDe1-M1S.
  • the siRNA solution of the corresponding concentration was prepared with normal saline before the experiment.
  • mice Four hours after exposure, the two groups of mice were given the test siRNA and PBS control by inhalation of the throat medicine into the lung trachea.
  • the volume of administration was 50 ⁇ L.
  • the dose of the test siRNA was given as follows: 6mg/kg.
  • mice were sacrificed 48h after exposure, and lung tissues and tracheal tissues were collected from the mice. Save with RNA later (Sigma Aldrich); homogenize lung tissue and tracheal tissue with a tissue homogenizer, and then use Trizol to extract total RNA according to the standard operating procedures for total RNA extraction.
  • Real-time fluorescent quantitative PCR was used to detect the SARS-COV-2 viral RNA load in lung tissues and tracheal tissues, specifically: use the RR064A TAKARA reverse transcription kit according to its instructions to reverse transcription to obtain cDNA, and then use fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.) detects the SARS-COV-2 viral RNA load in lung tissue and trachea tissue by siRNA.
  • Use probe method qRT-PCR to detect SARS-COV-2 viral RNA load in each test group and blank group, and calculate the relative inhibition rate of siRNA to SARS-COV-2 viral RNA load, the result is shown in Figure 12A And shown in Figure 12B.
  • PCR primers used to amplify SARS-COV-2 virus and GAPDH as an internal reference gene are shown in Table 3.
  • the relative inhibition rate of viral RNA load 1-(viral RNA load of the administration group/viral RNA load of the control group) ⁇ 100%.
  • the viral load after siRNA is low.
  • the viral RNA load was 1.29 ⁇ 107 copies/mL after the PBS control was administered; the viral RNA load was 6.51 ⁇ 106 copies/mL after the siCOVIDe1-M1S composition was administered, which was only the viral RNA load of the control group
  • the relative inhibition rate of viral RNA load was 49.5%.
  • the viral RNA load was 2.20 ⁇ 109 copies/mL; after the siCOVIDe1-M1S composition was given, the viral RNA load was 7.93 ⁇ 108 copies/mL, which was only the lower of the viral RNA load of the control group. 36.05%, the relative inhibition rate of viral RNA load was 63.95%. Thus showing an excellent effect of inhibiting the expression of SARS-COV-2 virus RNA.
  • Humanized ACE2 transgenic mice (B6/JGpt-Ace2em1Cin(hACE2)/Gpt) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. First, the humanized ACE2 transgenic mice were randomly divided into groups (all females), 4 mice in each group, numbered separately, and a PBS control group was added. The mice were anesthetized with 50 mg/kg pentobarbital by intraperitoneal injection, and 50 ⁇ L of virus dilution containing 4 ⁇ 105 pfu SARS-COV-2 was administered to each mouse by the nasal drip method to infect the mice.
  • mice Six hours after exposure, the three groups of mice were injected intravenously with the test siRNA composition and PBS control, each with a volume of 50 ⁇ L and a dose of 1 mg/kg.
  • mice were sacrificed on the fourth day after exposure, and the lung tissues of the mice were collected. Save with RNA later (Sigma Aldrich); homogenize lung tissue and tracheal tissue with a tissue homogenizer, and then use Trizol to extract total RNA according to the standard operating procedures for total RNA extraction.
  • Real-time fluorescent quantitative PCR was used to detect the SARS-COV-2 viral RNA load in lung tissues and tracheal tissues, specifically: use the RR064A TAKARA reverse transcription kit according to its instructions to reverse transcription to obtain cDNA, and then use fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.) detects the SARS-COV-2 viral RNA load in lung tissues by siRNA.
  • the probe method qRT-PCR was used to detect the SARS-COV-2 viral RNA load in each test group and the blank group, and the siRNA to SARS-COV-2 viral RNA load was calculated. The results are shown in Figure 13.
  • PCR primers used to amplify SARS-COV-2 virus and GAPDH as an internal reference gene are shown in Table 3.
  • the relative inhibition rate of viral RNA load 1-(viral RNA load of the administration group/viral RNA load of the control group) ⁇ 100%.
  • the modified siRNA provided by the present disclosure administered by intravenous injection has good inhibitory activity in the lung tissue of mice infected with SARS-COV-2 virus, and the viral load after transfection of siRNA is low.
  • the viral RNA load was 2.67 ⁇ 1010copies/mL after the PBS control was administered; the viral RNA load was 1.42 ⁇ 109copies/mL after the siCOVIDe1-M1S composition was administered, which was only the viral RNA load of the control group
  • the relative inhibition rate of viral RNA load was 94.68%.
  • the viral RNA load was 2.33 ⁇ 109 copies/mL, which was only 8.73% of the viral RNA load of the control group.
  • the relative inhibition rate was 91.27%.
  • the inhibitory activity of the tested siRNA in the in vitro psiCHECK system was tested, the difference is only that the tested siRNAs are siCOVIDe1-M1S, siCOVIDe1-M1S1, siCOVIDh1-M1S and siCOVIDh1-M1S1;
  • the target sequence is the following sequence:
  • siCOVIDe1-M1S and siCOVIDe1-M1S1 is:
  • siCOVIDh1-M1S and siCOVIDh1-M1S1 are:
  • the inhibitory activity of the reference siRNA NC in the psiCHECK system was also investigated according to the methods of experimental examples 49-52. The only difference is that the tested siRNA is the reference siRNA NC.
  • the siRNAs of the present disclosure all show a good effect of inhibiting the expression of the target sequence gene. And showed a concentration-dependent inhibitory activity.
  • the target sequence expression inhibition rate is at least 96.42%, and up to 97.57%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.
  • the inhibitory activity of the tested siRNA in the in vitro psiCHECK system was tested, the difference is only that the tested siRNAs are siCOVIDe1-M1S, siCOVIDe1-M1S1, siCOVIDh1-M1S and siCOVIDh1-M1S1;
  • the target sequence is the following sequence:
  • siCOVIDe1-M1S and siCOVIDe1-M1S1 is:
  • siCOVIDh1-M1S and siCOVIDh1-M1S1 are:
  • the inhibitory activity of the reference siRNA NC in the psiCHECK system was also investigated according to the method of experimental examples 53-56. The only difference is that the tested siRNA is the reference siRNA NC.
  • the siRNAs of the present disclosure all show a good effect of inhibiting the expression of the target sequence gene. And the inhibitory activity is concentration-dependent. At a siRNA concentration of 1 nM, the target sequence expression inhibition rate is at least 87.44%, and the highest is 96.43%, showing an excellent effect of inhibiting SARS-COV-2 viral RNA expression.

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Abstract

提供了一种抑制新型冠状病毒基因表达的siRNA及其药物组合物、缀合物、用途和试剂盒。

Description

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新型冠状病毒基因表达的核酸和含有该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
新型冠状病毒病(Corona Virus Disease 2019，下称COVID-19，旧称NCP，即新型冠状病毒肺炎)是指2019被发现的新型冠状病毒感染导致的疾病，为严重急性呼吸综合征冠状病毒2(Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2，下称新型冠状病毒或SARS-COV-2病毒，旧称2019-nCov)感染引起的急性呼吸道传染病。
COVID-19的病程发展较为迅速，患者的主要临床表现为发热、乏力、干咳，鼻塞、流涕等上呼吸道症状少见，会出现缺氧低氧状态。约半数患者多在一周后出现呼吸困难，严重者快速进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍。
SARS-COV-2病毒与SARS，MERS均属于β冠状病毒。SARS-COV-2病毒归类于β属B亚群，是一种单链RNA病毒，这种单链RNA病毒具有多个突变种，例如基因全长为29903bp(NC_045512.2)或全长为29867bp(MT066156.1)或全长为29835bp(MT188341.1)的单链RNA病毒，与2002-2003年出现的SARS病毒(SARS-Cov)具有共同的受体ACE2。
研究表明，SARS-COV-2病毒主要在肺脏扩增和表达，使肺脏不同程度实变，单核和巨噬细胞渗出，II型肺泡上皮细胞增生及脱落，肺组织灶性出血、坏死，肺间质纤维化等；肺支气管粘膜部分上皮脱落，腔内形成黏液(栓)。
此外，SARS-COV-2病毒还在脾脏、心脏、血管、肝脏、胆囊、肾脏、脑组织中扩增和表达。引起这些器官的病变。
因此，急需一种能够抑制SARS-COV-2病毒的表达，或者能够预防或治疗由SARS-COV-2病毒的表达引起的疾病或生理状况，尤其是COVID-19的药物。
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SARS-COV-2病毒基因的表达，实现对由SARS-COV-2病毒的表达引起的疾病或生理状况的治疗或预防，从而完成了本发明。
在一方面，本公开提供了一种siRNA，所述siRNA含有正义链和反义链，所述的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，所述核苷酸序列II至少部分地与第一段核苷酸序列反向互补，所述第一段核苷酸序列为靶RNA中的一段核苷酸序列，所述靶RNA是指SARS-COV-2病毒的RNA。
在一些实施方式中，第一段核苷酸序列为靶RNA保守区序列中的一段核苷酸序列。
在一些实施方式中，本公开提供了一种能够抑制SARS-COV-2病毒基因表达的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，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，所述核苷酸序列I和所述核苷酸序列II选自如下i)-viii)所示序列中的一组：
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，且所述核苷酸序列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'-GCGCUAACAUAGGUUGUAZ 1-3'(SEQ ID NO:1)；
5'-Z 2UACAACCUAUGUUAGCGC-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。
ii)所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GCUUAUGUGUCAACCUAUZ 5-3'(SEQ ID NO:61)；
5'-Z 6AUAGGUUGACACAUAAGC-3'(SEQ ID NO:62)，
其中，Z 5为A，Z 6为U；所述核苷酸序列I中包含位置对应于Z 5的核苷酸Z 7，所述核苷酸序列II中包含位置对应于Z 6的核苷酸Z 8，所述Z 8是所述反义链5'末端的第一个核苷酸。
iii)所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GCGGUUCACUAUAUGUUAZ 9-3'(SEQ ID NO:121)；
5'-Z 10UAACAUAUAGUGAACCGC-3'(SEQ ID NO:122)，
其中，Z 9为A，Z 10为U，所述核苷酸序列I中包含位置对应于Z 9的核苷酸Z11，所述核苷酸序列II中包含位置对应于Z 10的核苷酸Z 12，所述Z 12是所述反义链5'末端的第一个核苷酸。
iv)所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAAUUGCAAUGUCGAUAGZ 13-3'(SEQ ID NO:181)；
5'-Z 14CUAUCGACAUUGCAAUUC-3'(SEQ ID NO:182)，
其中，Z 13为A，Z 14为U，所述核苷酸序列I中包含位置对应于Z 13的核苷酸Z 15，所述核苷酸序列II中包含位置对应于Z 14的核苷酸Z 16，所述Z 16是所述反义链5'末端的第一个核苷酸。
v)所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GUCUCUAGUCAGUGUGUUZ 17-3'(SEQ ID NO:241)；
5'-Z 18AACACACUGACUAGAGAC-3'(SEQ ID NO:242)，
其中，Z 17为A，Z 18为U，所述核苷酸序列I中包含位置对应于Z 17的核苷酸Z 19，所述核苷酸序列II中包含位置对应于Z 18的核苷酸Z 20，所述Z 20是所述反义链5'末端的第一个核苷酸。
vi)所述核苷酸序列I与SEQ ID NO:301所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:302所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GUACUAAGAGGUUUGAUAZ 21-3'(SEQ ID NO:301)；
5'-Z 22UAUCAAACCUCUUAGUAC-3'(SEQ ID NO:302)，
其中，Z 21为A，Z 22为U，所述核苷酸序列I中包含位置对应于Z 21的核苷酸Z 23，所述核苷酸序列II中包含位置对应于Z 22的核苷酸Z 24，所述Z 24是所述反义链5'末端的第一个核苷酸。
vii)所述核苷酸序列I与SEQ ID NO:361所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:362所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAAACAAAGUGUACGUUGZ 25-3'(SEQ ID NO:361)，
5'-Z 26CAACGUACACUUUGUUUC-3'(SEQ ID NO:362)；
其中，Z 25为A，Z 26为U，所述核苷酸序列I中包含位置对应于Z 25的核苷酸Z 27，所述核苷酸序列II中包含位置对应于Z 26的核苷酸Z 28，所述Z 28是所述反义链5'末端的第一个核苷酸。
viii)所述核苷酸序列I与SEQ ID NO:421所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:422所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-CAUUCAAUCCAGAAACUAZ 29-3'(SEQ ID NO:421)，
5'-Z 30UAGUUUCUGGAUUGAAUG-3'(SEQ ID NO:422)；
其中，Z 29为A，Z 30为U，所述核苷酸序列I中包含位置对应于Z 29的核苷酸Z 31，所述核苷酸序列II中包含位置对应于Z 30的核苷酸Z 32，所述Z 32是所述反义链5'末端的第一个核苷酸。
在一些实施方式中，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。
在一些实施方式中，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/或预防由所述SARS-COV-2病毒基因表达引起的相关疾病的药物中的用途。
在一些实施方式中，本公开提供了一种治疗和/或新型冠状病毒引起的疾病的方法，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缀合物给予患有新型冠状病毒引起的疾病的受试者。
在一些实施方式中，本公开提供了一种抑制细胞中SARS-COV-2病毒基因表达的方法，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。
在一些实施方式中，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、专利或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。
附图说明
图1是显示了不同浓度的本公开siRNA和参比siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图2是显示了不同浓度的本公开siRNA和参比siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图3是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图4A是显示了本公开siRNA和参比siRNA在Vero感染细胞中24小时的SARS-COV-2病毒RNA载量的柱状图。
图4B是显示了本公开siRNA和参比siRNA在Vero感染细胞中48小时的SARS-COV-2病毒RNA载量的柱状图。
图5A是显示了本公开siRNA和参比siRNA通过lipo2000转染至Vero细胞后的细胞存活率的浓度-细胞存活率折线图。
图5B是显示了本公开siRNA和参比siRNA自由摄取转染至Vero细胞后的细胞存活率的浓度-细胞存活率折线图。
图6是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图7是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图8是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图9是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图10显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性。
图11A是显示了本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)的SARS-COV-2病毒RNA载量的柱状图。
图11B是显示了本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)的SARS-COV-2病毒RNA载量的柱状图。
图12A是显示了本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)肺部组织的SARS-COV-2病毒RNA载量的柱状图。
图12B是显示了本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)气管组织的SARS-COV-2病毒RNA载量的柱状图。
图13是显示了本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)肺部组织的SARS-COV-2病毒RNA载量的柱状图。
图14是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
图15是显示了不同浓度的本公开siRNA在体外HEK293A细胞中的靶基因抑制活性的柱状图。
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。
在上文及下文中，如无特别说明，COVID-19指发现于2019年的新型冠状病毒病，或称为新型冠状肺炎。SARS-COV-2病毒指引起COVID-19的新型冠状病毒及其变体中的一种。所述变体指SARS-COV-2病毒基因突变后的病毒。
SARS-COV-2病毒基因是指SARS-COV-2病毒及其变体中一种的基因，例如Genbank注册号为NC_045512.2所示的序列。进一步地，若无其它说明，本公开中所使用的术语“靶基因”是指上述SARS-COV-2病毒及其突变体的基因，尤其指Genbank注册号为NC_045512.2所示的序列。术语“靶RNA”是指上述SARS-COV-2病毒基因及其突变体所表达的RNA，尤其指Genbank注册号为NC_045512.2所示的序列的基因对应的RNA。
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，一条链的碱基各自与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U))相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
在上文及下文中，如无特别说明，“基本上反向互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；“实质上反向互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两段核苷酸序列之间不存在碱基错配。
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
在上文及下文中，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。在下文中，有时也将本公开的siRNA缀合物简称为“缀合物”。siRNA缀合物应根据上下文，理解为多个siRNA缀合物的总称或者某个化学式所示的siRNA缀合物。在本公开的上下文中，“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。
如本文所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且该描述包括事件或状况发生的情况和不发生的情况。例如，“任选地取代的烷基”包括下文定义的“烷基”和“取代烷基”。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，C1-C6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，“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任选地被氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。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)。
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本文所使用的，杂芳基可以是单环、双环、三环或四环系统，其中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(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。杂芳基中的杂原子被任选地氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂芳基通过任何环原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1,3-苯并二噁唑基、苯并呋喃基、苯并噁唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1,4]二噁庚英基(benzo[b][1,4]dioxepinyl)、苯并[b][1,4]噁嗪基(benzo[b][1,4]oxazinyl)、1,4-苯并二噁烷基(1,4-benzodioxanyl)、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基(benzodioxolyl)、苯并二噁英基(benzodioxinyl)、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩并[3,2-d]嘧啶基、苯并三唑基、苯并[4,6]咪唑并[1,2-a]吡啶基、咔唑基、噌啉基(cinnolinyl)、环戊烷并[d]嘧啶基、6,7-二氢-5H-环戊烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6-二氢苯并[h]喹唑啉基(5,6-dihydrobenzo[h]quinazolinyl)、5,6-二氢苯并[h]噌啉基(5,6dihydrobenzo[h]cinnolinyl)、6,7-二氢-5H-苯并[6,7]环庚烷并[1,2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃并[3,2-c]吡啶基、5,6,7,8,9,10-六氢环辛烷并[d]嘧啶基、5,6,7,8,9,10- 六氢环辛烷并[d]哒嗪基、5,6,7,8,9,10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基(indazolyl)、吲哚基、异吲哚基、二氢吲哚基、异二氢吲哚基、异喹啉基、吲哚嗪基(indolizinyl)、异噁唑基、5,8-甲醇-5,6,7,8-四氢喹唑啉基(5,8-methano-5,6,7,8-tetrahydroquinazolinyl)、萘啶基(naphthyridinyl)、1,6-萘啶酮基(1,6-naphthyridinonyl)、噁二唑基、2-氧杂吖庚因基(2-oxoazepinyl)、噁唑基、氧杂环丙烷基(oxiranyl)、5,6,6a,7,8,9,10,10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基(phthalazinyl)、蝶啶基(pteridinyl)、嘌呤基、吡咯基、吡唑基、吡唑并[3,4-d]嘧啶基、吡啶基、吡啶并[3,2-d]嘧啶基、吡啶并[3,4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基(quinoxalinyl)、喹啉基、四氢喹啉基、5,6,7,8-四氢喹唑啉基、5,6,7,8-四氢苯并[4,5]噻吩并[2,3-d]嘧啶基、6,7,8,9-四氢-5H-环庚烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6,7,8-四氢吡啶并[4,5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩并[2,3-d]嘧啶基、噻吩并[3,2-d]嘧啶基、噻吩并[2,3-c]吡啶基(thieno[2,3-c]pridinyl)和噻吩基(thiophenyl/thienyl)。
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度对特定的反应条件不敏感，并且可以在分子中的该官能度上添加以及去除，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”、是指获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本文所使用的，“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。
在一方面，本公开提供了第一种至第八种能够抑制SARS-COV-2病毒基因表达的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。
通过本公开所述siRNA的反义链中所述核苷酸序列II与第一段核苷酸序列部分或完全反向互补，实现siRNA的反义链与第一段核苷酸序列的特异性结合，沉默SARS-COV-2病毒基因的表达，并阻断SARS-COV-2病毒的扩增。因此，只要能够实现siRNA的反义链中所述核苷酸序列II与第一段核苷酸序列特异结合，并沉默SARS-COV-2病毒基因的表达，阻断SARS-COV-2病毒的扩增，所述第一段核苷酸序列可以位于靶RNA的任何位置。
在一些实施方式中，第一段核苷酸序列位于靶RNA的保守区中，换句话说，所述第一段核苷酸序列为靶RNA保守区序列中的一段核苷酸序列。这样采用本公开的siRNA可以阻断所有或大多数SARS-COV-2病毒的扩增，而不必考虑SARS-COV-2病毒基因突变造成的靶RNA的改变。所述保守区序列是指SARS-COV-2病毒发生突变时，与核苷酸序列II至少部分地反向互补的一段突变率不超过3个核苷酸的序列。在一些实施方式中，所述保守区序列是指SARS-COV-2病毒发生突变时，与核苷酸序列II至少部分地反向互补的一段突变率不超过1个核苷酸的序列。在一些实施方式中，所述保守区序列是指SARS-COV-2病毒发生突变时，与核苷酸序列II至少部分地反向互补的一段不发生突变的序列。
在一些实施方式中，核苷酸序列II与第一段核苷酸序列基本上反向互补、基本上完全反向互补或完全反向互补。
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、实质上上反向互补或者完全反向互补。
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的长度相同或不同，分别为15-40个核苷酸或者分别为18-30个核苷酸。
下面以列举方式说明适用于本公开的具体的siRNA实例，并不说明本发明仅限于下面列举的siRNA。以下依次对第一种至第八种能够抑制SARS-COV-2病毒基因表达的siRNA进行详细描述。
第一种siRNA
按照本公开，所述siRNA可以是第一种siRNA。
所述第一种siRNA含有正义链和反义链，所述第一种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，且所述核苷酸序列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'-GCGCUAACAUAGGUUGUAZ 1-3'(SEQ ID NO:1)；
5'-Z 2UACAACCUAUGUUAGCGC-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。
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2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2个核苷酸差异。
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RNA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'-GCGCUAACAUAGGUUGUAZ 3-3'(SEQ ID NO:3)；
5'-Z 4UACAACCUAUGUUAGCGC-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；
并且，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RNA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，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A，核苷酸序列IV的碱基为U；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UA，核苷酸序列IV的碱基组成为UA；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CUA，核苷酸序列IV的碱基组成为UAG；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GCUA，核苷酸序列IV的碱基组成为UAGC；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UA，核苷酸序列IV的碱基组成为UA；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。
第二种siRNA
按照本公开，所述siRNA可以是第二种siRNA。
所述第二种siRNA含有正义链和反义链，所述第二种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:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GCUUAUGUGUCAACCUAUZ 5-3'(SEQ ID NO:61)；
5'-Z 6AUAGGUUGACACAUAAGC-3'(SEQ ID NO:62)，
其中，Z 5为A，Z 6为U；所述核苷酸序列I中包含位置对应于Z 5的核苷酸Z 7，所述核苷酸序列II中包含位置对应于Z 6的核苷酸Z 8，所述Z 8是所述反义链5'末端的第一个核苷酸。
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:61所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:61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:62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:62所示的核苷酸序列之间的核苷酸差异包括Z 8位置处的差异，且Z 8选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 8位置处的差异，Z 8选自A、C或G。在一些实施方式中，Z 7是与Z 8互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶RNA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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:63所示的核苷酸序列，核苷酸序列II是SEQ ID NO:64所示的核苷酸序列：
5'-GCUUAUGUGUCAACCUAUZ 7-3'(SEQ ID NO:63)；
5'-Z 8AUAGGUUGACACAUAAGC-3'(SEQ ID NO:64)，
其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 7选自A、U、G或C，并且Z 8是与Z 7互补的核苷酸；在一些实施方式中，Z 7为A，Z 8为U；
并且，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。
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，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RNA中与由SEQ ID NO:61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，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A，核苷酸序列IV的碱基为U；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CA，核苷酸序列IV的碱基组成为UG；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UCA，核苷酸序列IV的碱基组成为UGA；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GUCA，核苷酸序列IV的碱基组成为UGAC；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CA，核苷酸序列IV的碱基组成为UG；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。
第三种siRNA
按照本公开，所述siRNA可以是第三种siRNA。
所述第三种siRNA含有正义链和反义链，所述第三种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:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GCGGUUCACUAUAUGUUAZ 9-3'(SEQ ID NO:121)；
5'-Z 10UAACAUAUAGUGAACCGC-3'(SEQ ID NO:122)，
其中，Z 9为A，Z 10为U；所述核苷酸序列I中包含位置对应于Z 9的核苷酸Z 11，所述核苷酸序列II中包含位置对应于Z 10的核苷酸Z 12，所述Z 12是所述反义链5'末端的第一个核苷酸。
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:121所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:121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:122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:122所示的核苷酸序列之间的核苷酸差异包括Z 12位置处的差异，且Z 12选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 12位置处的差异，Z 12选自A、C或G。在一些实施方式中，Z 11是与Z 12互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶RNA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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:123所示的核苷酸序列，核苷酸序列II是SEQ ID NO:124所示的核苷酸序列：
5'-GCGGUUCACUAUAUGUUA Z 11-3'(SEQ ID NO:123)；
5'-Z 12UAACAUAUAGUGAACCGC-3'(SEQ ID NO:124)，
其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 11选自A、U、G或C，并且Z 12是与Z 11互补的核苷酸；在一些实施方式中，Z 11为U，Z 12为A；
并且，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。
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，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RNA中与由SEQ ID NO:121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，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G，核苷酸序列IV的碱基为C；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UG， 核苷酸序列IV的碱基组成为CA；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GUG，核苷酸序列IV的碱基组成为CUC；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UGUG，核苷酸序列IV的碱基组成为CACA；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UG，核苷酸序列IV的碱基组成为CA；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。
第四种siRNA
按照本公开，所述siRNA可以是第四种siRNA。
所述第四种siRNA含有正义链和反义链，所述第四种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:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAAUUGCAAUGUCGAUAG Z 13-3'(SEQ ID NO:181)；
5'-Z 14CUAUCGACAUUGCAAUUC-3'(SEQ ID NO:182)，
其中，Z 13为A，Z 14为U；所述核苷酸序列I中包含位置对应于Z 13的核苷酸Z 15，所述核苷酸序列II中包含位置对应于Z 14的核苷酸Z 16，所述Z 16是所述反义链5'末端的第一个核苷酸。
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:181所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:181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:182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:182所示的核苷酸序列之间的核苷酸差异包括Z 16位置处的差异，且Z 16选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 16位置处的差异，Z 16选自A、C或G。在一些实施方式中，Z 15是与Z 16互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶RNA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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:183所示的核苷酸序列，核苷酸序列II是SEQ ID NO:184所示的核苷酸序列：
5'-GAAUUGCAAUGUCGAUAG Z 15-3'(SEQ ID NO:183)；
5'-Z 16CUAUCGACAUUGCAAUUC-3'(SEQ ID NO:184)，
其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 15选自A、U、G或C，并且Z 16是与Z 15互补的核苷酸；在一些实施方式中，Z 15为A，Z 16为U；
并且，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。
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，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RNA中与由SEQ ID NO:181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，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G，核苷酸序列IV的碱基为C；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UG，核苷酸序列IV的碱基组成为CA；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UUG，核苷酸序列IV的碱基组成为CAA；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UUUG，核苷酸序列IV的碱基组成为CAAA；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UG，核苷酸序列IV的碱基组成为CA；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。
第五种siRNA
按照本公开，所述siRNA可以是第五种siRNA。
所述第五种siRNA含有正义链和反义链，所述第五种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:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GUCUCUAGUCAGUGUGUUZ 17-3'(SEQ ID NO:241)；
5'-Z 18AACACACUGACUAGAGAC-3'(SEQ ID NO:242)，
其中，Z 17为A，Z 18为U；所述核苷酸序列I中包含位置对应于Z 17的核苷酸Z 18，所述核苷酸序列II中包含位置对应于Z 14的核苷酸Z 19，所述Z 20是所述反义链5'末端的第一个核苷酸。
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:241所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I具有SEQ ID NO:713所示的核苷酸序列，所述核苷酸序列II是与所述核苷酸序列I实质上反向互补或者完全反向互补的核苷酸序列。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:241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:242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:242所示的核苷酸序列之间的核苷酸差异包括Z 20位置处的差异，且Z 20选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 20位置处的差异，Z 20选自A、C或G。在一些实施方式中，Z 19是与Z 20互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶RNA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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:243所示的核苷酸序列，核苷酸序列II是SEQ ID NO:244所示的核苷酸序列：
5'-GUCUCUAGUCAGUGUGUUZ 19-3'(SEQ ID NO:243)；
5'-Z 20AACACACUGACUAGAGAC-3'(SEQ ID NO:244)，
其中，所述Z 20是反义链5'末端的第一个核苷酸，Z 19选自A、U、G或C，并且Z 20是与Z 19互补的核苷酸；在一些实施方式中，Z 19为A，Z 20为U；
并且，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。
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，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RNA中与由SEQ ID NO:241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，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A，核苷酸序列IV的碱基为U；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UA， 核苷酸序列IV的碱基组成为UA；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CUA，核苷酸序列IV的碱基组成为UAG；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ACUA，核苷酸序列IV的碱基组成为UAGU；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UA，核苷酸序列IV的碱基组成为UA；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。
本公开所述的第五种能够抑制SARS-COV-2病毒基因表达的siRNA与北京新发地G614D突变毒株中的病毒RNA序列的对应片段完全匹配。在一些实施方式中，本公开提供的第五种siRNA的正义链或反义链特点位点进行碱基替代或突变后，该siRNA、含该siRNA的组合物或siRNA缀合物显示出与无碱基替代或未突变siRNA相同或相近的抑制活性。在一些实施方式中，本公开提供的第五种siRNA对突变株SARS-CoV-2病毒RNA显示出与对未突变SARS-CoV-2病毒RNA相同或相近的抑制活性。
第六种siRNA
按照本公开，所述siRNA可以是第六种siRNA。
所述第六种siRNA含有正义链和反义链，所述第六种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:301所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:302所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GUACUAAGAGGUUUGAUA Z 21-3'(SEQ ID NO:301)；
5'-Z 22UAUCAAACCUCUUAGUAC-3'(SEQ ID NO:302)，
其中，Z 21为A，Z 22为U；所述核苷酸序列I中包含位置对应于Z 21的核苷酸Z 22，所述核苷酸序列II中包含位置对应于Z 22的核苷酸Z 23，所述Z 24是所述反义链5'末端的第一个核苷酸。
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:301所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:302所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:301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:302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:302所示的核苷酸序列之间的核苷酸差异包括Z 24位置处的差异，且Z 24选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 24位置处的差异，Z 24选自A、C或G。在一些实施方式中，Z 23是与Z 24互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶RNA抑制能力，而这些包含核苷酸差异的siRNA缀合物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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:303所示的核苷酸序列，核苷酸序列II是SEQ ID NO:304所示的核苷酸序列：
5'-GUACUAAGAGGUUUGAUAZ 23-3'(SEQ ID NO:303)；
5'-Z 24UAUCAAACCUCUUAGUAC-3'(SEQ ID NO:304)，
其中，所述Z 24是反义链5'末端的第一个核苷酸，Z 23选自A、U、G或C，并且Z 24是与Z 23互补的核苷酸；在一些实施方式中，Z 23为A，Z 24为U；
并且，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。
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，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RNA中与由SEQ ID NO:301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，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G，核苷酸序列IV的碱基为C；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UG，核苷酸序列IV的碱基组成为CA；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AUG，核苷酸序列IV的碱基组成为CAU；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AAUG，核苷酸序列IV的碱基组成为CAUU；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UG，核苷酸序列IV的碱基组成为CA；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。
第七种siRNA
按照本公开，所述siRNA可以是第七种siRNA。
所述第七种siRNA含有正义链和反义链，所述第七种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:361所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列Ⅱ与SEQ ID NO:362所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-GAAACAAAGUGUACGUUGZ 25-3'(SEQ ID NO:361)；
5'-Z 26CAACGUACACUUUGUUUC-3'(SEQ ID NO:362)，
其中，Z 25为A，Z 26为U；所述核苷酸序列I中包含位置对应于Z 25的核苷酸Z27，所述核苷酸序列II中包含位置对应于Z 26的核苷酸Z 28，所述Z 28是所述反义链5'末端的第一个核苷酸。
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:361所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:362所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:361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:362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:362所示的核苷酸序列之间的核苷酸差异包括Z 28位置处的差异，且Z 28选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 28位置处的差异，Z 28选自A、C或G。在一些实施方式中，Z 27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RNA抑制能力，而这些包含核苷酸差异的siRNA缀合物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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:363所示的核苷酸序列，核苷酸序列II是SEQ ID NO:364所示的核苷酸序列：
5'-GAAACAAAGUGUACGUUGZ 27-3'(SEQ ID NO:363)；
5'-Z 28CAACGUACACUUUGUUUC-3'(SEQ ID NO:364)，
其中，所述Z 28是反义链5'末端的第一个核苷酸，Z 27选自A、U、G或C，并且Z 28是与Z 27互补的核苷酸；在一些实施方式中，Z 27为A，Z 28为U。
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RNA中与由SEQ  ID NO:361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，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A，核苷酸序列IV的碱基为U；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CA，核苷酸序列IV的碱基组成为UG；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UCA，核苷酸序列IV的碱基组成为UGA；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CUCA，核苷酸序列IV的碱基组成为UGAG；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CA，核苷酸序列IV的碱基组成为UG；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。
第八种siRNA
按照本公开，所述siRNA可以是第八种siRNA。
所述第八种siRNA含有正义链和反义链，所述第八种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:421所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列Ⅱ与SEQ ID NO:422所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-CAUUCAAUCCAGAAACUA Z 29-3'(SEQ ID NO:421)；
5'-Z 30UAGUUUCUGGAUUGAAUG-3'(SEQ ID NO:422)，
其中，Z 29为A，Z 30为U；所述核苷酸序列I中包含位置对应于Z 29的核苷酸Z 31，所述核苷酸序列II中包含位置对应于Z 30的核苷酸Z 32，所述Z 8是所述反义链5'末端的第一个核苷酸。
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:421所示的核苷酸序列之间不多于2个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:422所示的核苷酸序列之间不多于2个核苷酸差异。
在一些实施方式中，所述核苷酸序列I具有SEQ ID NO:787、861或935所示的核苷酸序列，所述核苷酸序列II是与所述核苷酸序列I实质上反向互补或者完全反向互补的核苷酸序列。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:421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:422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:422所示的核苷酸序列之间的核苷酸差异包括Z 32位置处的差异，且Z 32选自A、C或G。在一些实施方式中，所述核苷酸差异为Z 32位置处的差异，Z 32选自A、C或G。在一些实施方式中，Z 31是与Z 32互补的核苷酸。具有上述核苷酸差异的siRNA具有较高的靶RNA抑制能力，而这些包含核苷酸差异的siRNA缀合物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。
在一些实施方式中，核苷酸序列I是SEQ ID NO:423所示的核苷酸序列，核苷酸序列II是SEQ ID NO:424所示的核苷酸序列：
5'-CAUUCAAUCCAGAAACUAZ 31-3'(SEQ ID NO:423)；
5'-Z 32UAGUUUCUGGAUUGAAUG-3'(SEQ ID NO:424)，
其中，所述Z 32是反义链5'末端的第一个核苷酸，Z 31选自A、U、G或C，并且Z 32是与Z 31互补的核苷酸；在一些实施方式中，Z 31为A，Z 32为U。
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，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RNA中与由SEQ ID NO:421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，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GU，核苷酸序列IV的碱基组成为AC；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GGU，核苷酸序列IV的碱基组成为ACC；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GGGU，核苷酸序列IV的碱基组成为AAAC；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GU，核苷酸序列IV的碱基组成为AC；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。
本公开所述的第八种能够抑制SARS-COV-2病毒基因表达的siRNA与北京新发地G614D突变毒株中的病毒RNA序列的对应片段完全匹配。并且，在一些实施方式中，本公开提供的第八种siRNA的正义链或反义链特点位点进行碱基替代或突变后，该siRNA、含该siRNA的组合物或siRNA缀合物显示出与无碱基替代或未突变siRNA相同或相近的抑制活性。在一些实施方式中，本公开提供的第八种siRNA对突变株SARS-CoV-2病毒RNA显示出与对未突变SARS-CoV-2病毒RNA相同或相近的抑制活性。
以下，对核苷酸序列V、核酸序列、siRNA中的核苷酸修饰以及修饰序列的描述适用于上述第一种siRNA至第八种siRNA中的任意一种。即如果没有特指，下面对siRNA的描述应视为是对第一种siRNA、第二种siRNA、第三种siRNA、第四种siRNA、第五种siRNA、第六种siRNA、第七种siRNA和第八种siRNA逐一进行了描述。例如，如不特别指明具体的siRNA，“所述siRNA还含有核苷酸序列V”的意思是“第一种siRNA、第二种siRNA、第三种siRNA、第四种siRNA、第五种siRNA、第六种siRNA、第七种siRNA或第八种siRNA还含有核苷酸序列V”。
在一些实施方式中，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。在一些实施方式中，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RNA的亲和力，核苷酸序列V与靶RNA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RNA沉默活性。
靶RNA的相应位置的核苷酸是指与靶RNA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RNA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，对于所述第一种siRNA，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'-GCGCUAACAUAGGUUGUAZ 3-3'(SEQ ID NO:5)，
5'-Z 4UACAACCUAUGUUAGCGCUA-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'-UAGCGCUAACAUAGGUUGUAZ 3-3'(SEQ ID NO:7)，
5'-Z 4UACAACCUAUGUUAGCGCUAGC-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 4选自A、U、G或C，并且Z 3是与Z 4互补的核苷酸。
在一些实施方式中，对于所述第二种siRNA，所述siRNA的正义链含有如SEQ ID NO:65所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:66所示的核苷酸序列：
5'-GCUUAUGUGUCAACCUAUZ 7-3'(SEQ ID NO:65)，
5'-Z 8AUAGGUUGACACAUAAGCUG-3'(SEQ ID NO:66)；
或者，所述siRNA的正义链含有如SEQ ID NO:67所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:68所示的核苷酸序列：
5'-UAGCUUAUGUGUCAACCUAUZ 7-3'(SEQ ID NO:67)，
5'-Z 8AUAGGUUGACACAUAAGCUGAC-3'(SEQ ID NO:68)；
其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 8选自A、U、G或C，并且Z 7是与Z 8互补的核苷酸。
在一些实施方式中，对于所述第三种siRNA，所述siRNA的正义链含有如SEQ ID NO:125所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:126所示的核苷酸序列：
5'-GCGGUUCACUAUAUGUUA Z 11-3'(SEQ ID NO:125)，
5'-Z 12UAACAUAUAGUGAACCGCCA-3'(SEQ ID NO:126)；
或者，所述siRNA的正义链含有如SEQ ID NO:127所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:128所示的核苷酸序列：
5'-UGGCGGUUCACUAUAUGUUAZ 11-3'(SEQ ID NO:127)，
5'-Z 12UAACAUAUAGUGAACCGCCACA-3'(SEQ ID NO:128)；
其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 12选自A、U、G或C，并且Z 11是与Z 12互补的核苷酸。
在一些实施方式中，对于所述第四种siRNA，所述siRNA的正义链含有如SEQ ID NO:185所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:186所示的核苷酸序列：
5'-GAAUUGCAAUGUCGAUAGZ 15-3'(SEQ ID NO:185)，
5'-Z 16CUAUCGACAUUGCAAUUCCA-3'(SEQ ID NO:186)；
或者，所述siRNA的正义链含有如SEQ ID NO:187所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:188所示的核苷酸序列：
5'-UGGAAUUGCAAUGUCGAUAGZ 15-3'(SEQ ID NO:187)，
5'-Z 16CUAUCGACAUUGCAAUUCCAAA-3'(SEQ ID NO:188)；
其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 16选自A、U、G或C，并且Z 15是与Z 16互补的核苷酸。
在一些实施方式中，对于所述第五种siRNA，所述siRNA的正义链含有如SEQ ID NO:245所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:246所示的核苷酸序列：
5'-GUCUCUAGUCAGUGUGUUZ 19-3'(SEQ ID NO:245)，
5'-Z 20AACACACUGACUAGAGACUA-3'(SEQ ID NO:246)；
或者，所述siRNA的正义链含有如SEQ ID NO:247所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:248所示的核苷酸序列：
5'-UAGUCUCUAGUCAGUGUGUUZ Z 19-3'(SEQ ID NO:247)，
5'-Z 20AACACACUGACUAGAGACUAGU-3'(SEQ ID NO:248)；
其中，所述Z 20是反义链5'末端的第一个核苷酸，Z 20选自A、U、G或C，并且Z 19是与Z 20互补的核苷酸。
在一些实施方式中，对于所述第六种siRNA，所述siRNA的正义链含有如SEQ ID NO:305所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:306所示的核苷酸序列：
5'-GUACUAAGAGGUUUGAUAZ 23-3'(SEQ ID NO:305)，
5'-Z 24UAUCAAACCUCUUAGUACCA-3'(SEQ ID NO:306)；
或者，所述siRNA的正义链含有如SEQ ID NO:307所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:308所示的核苷酸序列：
5'-UGGUACUAAGAGGUUUGAUAZ 23-3'(SEQ ID NO:307)，
5'-Z 24UAUCAAACCUCUUAGUACCAUU-3'(SEQ ID NO:308)；
其中，所述Z 24是反义链5'末端的第一个核苷酸，Z 24选自A、U、G或C，并且Z 23是与Z 24互补的核苷酸。
在一些实施方式中，对于所述第七种siRNA，所述siRNA的正义链含有如SEQ ID NO:365所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:366所示的核苷酸序列：
5'-GAAACAAAGUGUACGUUGZ 27-3'(SEQ ID NO:365)，
5'-Z 28CAACGUACACUUUGUUUCUG-3'(SEQ ID NO:366)；
或者，所述siRNA的正义链含有如SEQ ID NO:367所示的核苷酸序列，所述反义链含有如SEQ ID NO:368所示的核苷酸序列：
5'-CAGAAACAAAGUGUACGUUGZ 27-3'(SEQ ID NO:367)，
5'-Z 28CAACGUACACUUUGUUUCUGAG-3'(SEQ ID NO:368)；
其中，所述Z 28是反义链5'末端的第一个核苷酸，Z 28选自A、U、G或C，并且Z 27是与Z 28互补的核苷酸。
在一些实施方式中，对于所述第八种siRNA，所述siRNA的正义链含有如SEQ ID NO:425所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:426所示的核苷酸序列：
5'-CAUUCAAUCCAGAAACUA Z 31-3'(SEQ ID NO:425)，
5'-Z 32UAGUUUCUGGAUUGAAUGAC-3'(SEQ ID NO:426)；
或者，所述siRNA的正义链含有如SEQ ID NO:427所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:428所示的核苷酸序列：
5'-GUCAUUCAAUCCAGAAACUAZ 31-3'(SEQ ID NO:427)，
5'-Z 32UAGUUUCUGGAUUGAAUGACCA-3'(SEQ ID NO:428)；
其中，所述Z 32是反义链5'末端的第一个核苷酸，Z 32选自A、U、G或C，并且Z 31是与Z 32互补的核苷酸。
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且具有2个核苷酸差异。在一些实施方式中，所述核苷酸序列I具有所述核苷酸序列I具有SEQ ID NO:713、787、861或935所示的核苷酸序列，所述核苷酸序列II是与所述核苷酸序列I完全反向互补的核苷酸序列。
在一些实施方式中，本公开所述siRNA为表1a-表1h中列出的siCOVIDa1、siCOVIDa2、siCOVIDb1、siCOVIDb2、siCOVIDc1、siCOVIDc2、siCOVIDd1、siCOVIDd2、siCOVIDe1、siCOVIDe2、siCOVIDe1U、siCOVIDe2U、siCOVIDf1、siCOVIDf2、siCOVIDg1、siCOVIDg2、siCOVIDh1和siCOVIDh2、siCOVIDh1U、siCOVIDh2U、siCOVIDh1R、siCOVIDh2R、siCOVIDh1G和siCOVIDh2G中的一种。
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缀合物抑制SARS-COV-2病毒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，或者核苷酸上的碱基是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'-NH2)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 PCTCN2021086016-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 PCTCN2021086016-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 PCTCN2021086016-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 PCTCN2021086016-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1a-表1h中列出的siCOVIDa1-M1、siCOVIDa1-M2、siCOVIDa1-M3、siCOVIDa2-M1、siCOVIDa2-M2、siCOVIDa2-M3、siCOVIDb1-M1、siCOVIDb1-M2、siCOVIDb1-M3、siCOVIDb2-M1、siCOVIDb2-M2、siCOVIDb2-M3、siCOVIDc1-M1、siCOVIDc1-M2、siCOVIDc1-M3、siCOVIDc2-M1、siCOVIDc2-M2、siCOVIDc2-M3、siCOVIDd1-M1、siCOVIDd1-M2、siCOVIDd1-M3、siCOVIDd2-M1、siCOVIDd2-M2、siCOVIDd2-M3、siCOVIDe1-M1、siCOVIDe1-M2、siCOVIDe1-M3、siCOVIDe2-M1、siCOVIDe2-M2、siCOVIDe2-M3、siCOVIDe1U-M1、siCOVIDe1U-M2、siCOVIDe1U-M3、siCOVIDe2U-M1、siCOVIDe2U-M2、siCOVIDe2U-M3、siCOVIDf1-M1、siCOVIDf1-M2、siCOVIDf1-M3、siCOVIDf2-M1、siCOVIDf2-M2、siCOVIDf2-M3、siCOVIDg1-M1、siCOVIDg1-M2、siCOVIDg1-M3、siCOVIDg2-M1、siCOVIDg2-M2、siCOVIDg2-M3、siCOVIDh1-M1、siCOVIDh1-M2、siCOVIDh1-M3、siCOVIDh2-M1、siCOVIDh2-M2、siCOVIDh2-M3、siCOVIDh1U-M1、siCOVIDh1U-M2、siCOVIDh1U-M3、siCOVIDh2U-M1、siCOVIDh2U-M2、siCOVIDh2U-M3、siCOVIDh1R-M1、siCOVIDh1R-M2、siCOVIDh1R-M3、siCOVIDh2R-M1、siCOVIDh2R-M2、siCOVIDh2R-M3、siCOVIDh1G-M1、siCOVIDh1G-M2、siCOVIDh1G-M3、siCOVIDh2G-M1、 siCOVIDh2G-M2、siCOVIDh2G-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RNA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基：
Figure PCTCN2021086016-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1a-表1h中列出的siCOVIDa1-M1S、siCOVIDa1-M2S、siCOVIDa1-M3S、siCOVIDa2-M1S、siCOVIDa2-M2S、siCOVIDa2-M3S、siCOVIDa1-M1S1、siCOVIDa1-M2S1、siCOVIDa1-M3S1、siCOVIDa2-M1S1、siCOVIDa2-M2S1、siCOVIDa2-M3S1、siCOVIDb1-M1S、siCOVIDb1-M2S、siCOVIDb1-M3S、siCOVIDb2-M1S、siCOVIDb2-M2S、siCOVIDb2-M3S、siCOVIDb1-M1S1、siCOVIDb1-M2S1、siCOVIDb1-M3S1、siCOVIDb2-M1S1、siCOVIDb2-M2S1、siCOVIDb2-M3S1、siCOVIDc1-M1S、siCOVIDc1-M2S、siCOVIDc1-M3S、siCOVIDc2-M1S、siCOVIDc2-M2S、siCOVIDc2-M3S、siCOVIDc1-M1S1、siCOVIDc1-M2S1、siCOVIDc1-M3S1、siCOVIDc2-M1S1、siCOVIDc2-M2S1、siCOVIDc2-M3S1、siCOVIDd1-M1S、siCOVIDd1-M2S、siCOVIDd1-M3S、siCOVIDd2-M1S、siCOVIDd2-M2S、siCOVIDd2-M3S、siCOVIDd1-M1S1、siCOVIDd1-M2S1、siCOVIDd1-M3S1、siCOVIDd2-M1S1、siCOVIDd2-M2S1、siCOVIDd2-M3S1、siCOVIDe1-M1S、siCOVIDe1-M2S、siCOVIDe1-M3S、siCOVIDe2-M1S、siCOVIDe2-M2S、siCOVIDe2-M3S、siCOVIDe1-M1S1、siCOVIDe1-M2S1、siCOVIDe1-M3S1、siCOVIDe2-M1S1、siCOVIDe2-M2S1、siCOVIDe2-M3S1、siCOVIDe1U-M1S、siCOVIDe1U-M2S、siCOVIDe1U-M3S、siCOVIDe2U-M1S、siCOVIDe2U-M2S、siCOVIDe2U-M3S、siCOVIDe1U-M1S1、siCOVIDe1U-M2S1、siCOVIDe1U-M3S1、siCOVIDe2U-M1S1、siCOVIDe2U-M2S1、siCOVIDe2U-M3S1、siCOVIDf1-M1S、siCOVIDf1-M2S、siCOVIDf1-M3S、siCOVIDf2-M1S、siCOVIDf2-M2S、siCOVIDf2-M3S、siCOVIDf1-M1S1、siCOVIDf1-M2S1、siCOVIDf1-M3S1、siCOVIDf2-M1S1、siCOVIDf2-M2S1、siCOVIDf2-M3S1、siCOVIDg1-M1S、siCOVIDg1-M2S、siCOVIDg1-M3S、siCOVIDg2-M1S、siCOVIDg2-M2S、siCOVIDg2-M3S、siCOVIDg1-M1S1、siCOVIDg1-M2S1、siCOVIDg1-M3S1、siCOVIDg2-M1S1、siCOVIDg2-M2S1、siCOVIDg2-M3S1、siCOVIDh1-M1S、siCOVIDh1-M2S、siCOVIDh1-M3S、siCOVIDh2-M1S、siCOVIDh2-M2S、siCOVIDh2-M3S、siCOVIDh1-M1S1、siCOVIDh1-M2S1、siCOVIDh1-M3S1、siCOVIDh2-M1S1、 siCOVIDh2-M2S1、siCOVIDh2-M3S1、siCOVIDh1U-M1S、siCOVIDh1U-M2S、siCOVIDh1U-M3S、siCOVIDh2U-M1S、siCOVIDh2U-M2S、siCOVIDh2U-M3S、siCOVIDh1U-M1S1、siCOVIDh1U-M2S1、siCOVIDh1U-M3S1、siCOVIDh2U-M1S1、siCOVIDh2U-M2S1、siCOVIDh2U-M3S1、siCOVIDh1R-M1S、siCOVIDh1R-M2S、siCOVIDh1R-M3S、siCOVIDh2R-M1S、siCOVIDh2R-M2S、siCOVIDh2R-M3S、siCOVIDh1R-M1S1、siCOVIDh1R-M2S1、siCOVIDh1R-M3S1、siCOVIDh2R-M1S1、siCOVIDh2R-M2S1、siCOVIDh2R-M3S1、siCOVIDh1G-M1S、siCOVIDh1G-M2S、siCOVIDh1G-M3S、siCOVIDh2G-M1S、siCOVIDh2G-M2S、siCOVIDh2G-M3S、siCOVIDh1G-M1S1、siCOVIDh1G-M2S1、siCOVIDh1G-M3S1、siCOVIDh2G-M1S1、siCOVIDh2G-M2S1、siCOVIDh2G-M3S1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 PCTCN2021086016-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 PCTCN2021086016-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1a-表1h中列出的siCOVIDa1-M1P1、siCOVIDa1-M2P1、siCOVIDa1-M3P1、siCOVIDa2-M1P1、siCOVIDa2-M2P1、siCOVIDa2-M3P1、siCOVIDa1-M1SP1、siCOVIDa1-M2SP1、siCOVIDa1-M3SP1、siCOVIDa2-M1SP1、siCOVIDa2-M2SP1、siCOVIDa2-M3SP1、siCOVIDa1-M1S1P1、siCOVIDa1-M2S1P1、siCOVIDa1-M3S1P1、siCOVIDa2-M1S1P1、siCOVIDa2-M2S1P1、siCOVIDa2-M3S1P1、siCOVIDb1-M1P1、siCOVIDb1-M2P1、siCOVIDb1-M3P1、siCOVIDb2-M1P1、siCOVIDb2-M2P1、siCOVIDb2-M3P1、siCOVIDb1-M1SP1、siCOVIDb1-M2SP1、siCOVIDb1-M3SP1、siCOVIDb2-M1SP1、siCOVIDb2-M2SP1、siCOVIDb2-M3SP1、siCOVIDb1-M1S1P1、siCOVIDb1-M2S1P1、siCOVIDb1-M3S1P1、siCOVIDb2-M1S1P1、siCOVIDb2-M2S1P1、siCOVIDb2-M3S1P1、siCOVIDc1-M1P1、siCOVIDc1-M2P1、siCOVIDc1-M3P1、siCOVIDc2-M1P1、siCOVIDc2-M2P1、siCOVIDc2-M3P1、siCOVIDc1-M1SP1、siCOVIDc1-M2SP1、siCOVIDc1-M3SP1、siCOVIDc2-M1SP1、siCOVIDc2-M2SP1、siCOVIDc2-M3SP1、siCOVIDc1-M1S1P1、siCOVIDc1-M2S1P1、siCOVIDc1-M3S1P1、siCOVIDc2-M1S1P1、siCOVIDc2-M2S1P1、siCOVIDc2-M3S1P1、siCOVIDd1-M1P1、siCOVIDd1-M2P1、siCOVIDd1-M3P1、siCOVIDd2-M1P1、siCOVIDd2-M2P1、siCOVIDd2-M3P1、siCOVIDd1-M1SP1、siCOVIDd1-M2SP1、siCOVIDd1-M3SP1、siCOVIDd2-M1SP1、siCOVIDd2-M2SP1、siCOVIDd2-M3SP1、siCOVIDd1-M1S1P1、siCOVIDd1-M2S1P1、siCOVIDd1-M3S1P1、siCOVIDd2-M1S1P1、siCOVIDd2-M2S1P1、siCOVIDd2-M3S1P1、siCOVIDe1-M1P1、siCOVIDe1-M2P1、siCOVIDe1-M3P1、siCOVIDe2-M1P1、siCOVIDe2-M2P1、siCOVIDe2-M3P1、siCOVIDe1-M1SP1、siCOVIDe1-M2SP1、siCOVIDe1-M3SP1、siCOVIDe2-M1SP1、siCOVIDe2-M2SP1、siCOVIDe2-M3SP1、siCOVIDe1-M1S1P1、siCOVIDe1-M2S1P1、 siCOVIDe1-M3S1P1、siCOVIDe2-M1S1P1、siCOVIDe2-M2S1P1、siCOVIDe2-M3S1P1、siCOVIDe1U-M1P1、siCOVIDe1U-M2P1、siCOVIDe1U-M3P1、siCOVIDe2U-M1P1、siCOVIDe2U-M2P1、siCOVIDe2U-M3P1、siCOVIDe1U-M1SP1、siCOVIDe1U-M2SP1、siCOVIDe1U-M3SP1、siCOVIDe2U-M1SP1、siCOVIDe2U-M2SP1、siCOVIDe2U-M3SP1、siCOVIDe1U-M1S1P1、siCOVIDe1U-M2S1P1、siCOVIDe1U-M3S1P1、siCOVIDe2U-M1S1P1、siCOVIDe2U-M2S1P1、siCOVIDe2U-M3S1P1、siCOVIDf1-M1P1、siCOVIDf1-M2P1、siCOVIDf1-M3P1、siCOVIDf2-M1P1、siCOVIDf2-M2P1、siCOVIDf2-M3P1、siCOVIDf1-M1SP1、siCOVIDf1-M2SP1、siCOVIDf1-M3SP1、siCOVIDf2-M1SP1、siCOVIDf2-M2SP1、siCOVIDf2-M3SP1、siCOVIDf1-M1S1P1、siCOVIDf1-M2S1P1、siCOVIDf1-M3S1P1、siCOVIDf2-M1S1P1、siCOVIDf2-M2S1P1、siCOVIDf2-M3S1P1、siCOVIDg1-M1P1、siCOVIDg1-M2P1、siCOVIDg1-M3P1、siCOVIDg2-M1P1、siCOVIDg2-M2P1、siCOVIDg2-M3P1、siCOVIDg1-M1SP1、siCOVIDg1-M2SP1、siCOVIDg1-M3SP1、siCOVIDg2-M1SP1、siCOVIDg2-M2SP1、siCOVIDg2-M3SP1、siCOVIDg1-M1S1P1、siCOVIDg1-M2S1P1、siCOVIDg1-M3S1P1、siCOVIDg2-M1S1P1、siCOVIDg2-M2S1P1、siCOVIDg2-M3S1P1、siCOVIDh1-M1P1、siCOVIDh1-M2P1、siCOVIDh1-M3P1、siCOVIDh2-M1P1、siCOVIDh2-M2P1、siCOVIDh2-M3P1、siCOVIDh1-M1SP1、siCOVIDh1-M2SP1、siCOVIDh1-M3SP1、siCOVIDh2-M1SP1、siCOVIDh2-M2SP1、siCOVIDh2-M3SP1、siCOVIDh1-M1S1P1、siCOVIDh1-M2S1P1、siCOVIDh1-M3S1P1、siCOVIDh2-M1S1P1、siCOVIDh2-M2S1P1、siCOVIDh2-M3S1P1、siCOVIDh1U-M1P1、siCOVIDh1U-M2P1、siCOVIDh1U-M3P1、siCOVIDh2U-M1P1、siCOVIDh2U-M2P1、siCOVIDh2U-M3P1、siCOVIDh1U-M1SP1、siCOVIDh1U-M2SP1、siCOVIDh1U-M3SP1、siCOVIDh2U-M1SP1、siCOVIDh2U-M2SP1、siCOVIDh2U-M3SP1、siCOVIDh1U-M1S1P1、siCOVIDh1U-M2S1P1、siCOVIDh1U-M3S1P1、siCOVIDh2U-M1S1P1、siCOVIDh2U-M2S1P1、siCOVIDh2U-M3S1P1、siCOVIDh1R-M1P1、siCOVIDh1R-M2P1、siCOVIDh1R-M3P1、siCOVIDh2R-M1P1、siCOVIDh2R-M2P1、siCOVIDh2R-M3P1、siCOVIDh1R-M1SP1、siCOVIDh1R-M2SP1、siCOVIDh1R-M3SP1、siCOVIDh2R-M1SP1、siCOVIDh2R-M2SP1、siCOVIDh2R-M3SP1、siCOVIDh1R-M1S1P1、siCOVIDh1R-M2S1P1、siCOVIDh1R-M3S1P1、siCOVIDh2R-M1S1P1、siCOVIDh2R-M2S1P1、siCOVIDh2R-M3S1P1、siCOVIDh1G-M1P1、siCOVIDh1G-M2P1、siCOVIDh1G-M3P1、siCOVIDh2G-M1P1、siCOVIDh2G-M2P1、siCOVIDh2G-M3P1、siCOVIDh1G-M1SP1、siCOVIDh1G-M2SP1、siCOVIDh1G-M3SP1、siCOVIDh2G-M1SP1、siCOVIDh2G-M2SP1、siCOVIDh2G-M3SP1、siCOVIDh1G-M1S1P1、siCOVIDh1G-M2S1P1、siCOVIDh1G-M3S1P1、siCOVIDh2G-M1S1P1、siCOVIDh2G-M2S1P1、siCOVIDh2G-M3S1P1中的任意一种。
本公开的发明人意外发现，本公开提供的上述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RNA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。
所述药物组合物中，对siRNA和药学上可接受的载体的含量可以是常规的含量，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)，在一些的实施方式中，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部、或通过喷雾经肺部给药到其它脏器组织(如肝脏)、或通过口咽吸入、或鼻腔给药等方式递送所述药物组合物。在一些实施方式中，所述药物组合物用于喷雾给药。
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 PCTCN2021086016-appb-000008
其中：
每个X101和X102各自独立地是O、S、N-A或C-A，其中A是氢或C1-C20烃链；
每个Y101和Z101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；
每个R101、R102、R103、R104、R105、R106和R107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R102是氢；
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如式(202)或式(203)所示的结构：
Figure PCTCN2021086016-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，R103是多胺。在其它实施方式中，R103是缩酮。在一些实施方式中，在式(201)中的R101和R102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R103可以是下述式(204)-式(213)中的任一个：
Figure PCTCN2021086016-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R103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 PCTCN2021086016-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与一种或多种聚乙二醇部分共价地缀合的脂质。在一些实施方式中，所述聚乙二醇化脂质为一种或多种聚乙二醇部分共价地缀合的脂质。在一些实施方式中，本公开的转染复合物中使用的聚乙二醇化脂质为1,2-二棕榈酰基-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，所述药物组合物中还包含阳离子脂质。在一些实施方式中，所述阳离子脂质为1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)。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、所述聚乙二醇化脂质和所述阳离子脂质四者之间的摩尔比为(19.7-80)：(19.7-80)：(0.3-50)：(50-150)，例如可以为(50-70)：(20-40)：(3-20)：(80-120)。
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、聚乙二醇化脂质和/或阳离子脂质)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、聚乙二醇化脂质和阳离子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。
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和任选的接头(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.
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，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。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；靶向肝细胞表面的受体的配体，如亲脂分子，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；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；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、转铁蛋白等适配体；抗体；量子点。
在一些实施方式中，至少一个靶向基团是能够与新型冠状病毒在细胞表面的受体结合的配体。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，至少一个靶向基团是靶向肺细胞表面的受体的配体，在一些实施方式中，至少一个靶向基团是能够靶向ACE2或integrinαvβ6的配体。在一些实施方式中，至少一个靶向基团是多肽，例如透膜肽；或者小分子配体，例如WO2019010274A1表6中列举的靶向基团，或WO2019089765A1中描述的各种靶向基团(如[0094]段中描述的编号为Structure 1-Structure 37的配体)，以引用的方式将其全部公开内容并入本文。
本公开的发明人意外发现，本公开的siRNA缀合物表现出较高的SARS-COV-2病毒RNA沉默活性。在一些实施方式中，本公开的siRNA可以为表1a-1h中示出的siRNA中的一种。采用这些siRNA，本公开的siRNA缀合物表现出更高的SARS-COV-2病毒RNA沉默活性。
表1a 本公开的第一种siRNA序列
Figure PCTCN2021086016-appb-000012
Figure PCTCN2021086016-appb-000013
Figure PCTCN2021086016-appb-000014
表1b 本公开的第二种siRNA序列
Figure PCTCN2021086016-appb-000015
Figure PCTCN2021086016-appb-000016
Figure PCTCN2021086016-appb-000017
Figure PCTCN2021086016-appb-000018
表1c 本公开的第三种siRNA序列
Figure PCTCN2021086016-appb-000019
Figure PCTCN2021086016-appb-000020
Figure PCTCN2021086016-appb-000021
Figure PCTCN2021086016-appb-000022
表1d 本公开的第四种siRNA序列
Figure PCTCN2021086016-appb-000023
Figure PCTCN2021086016-appb-000024
Figure PCTCN2021086016-appb-000025
表1e 本公开的第五种siRNA序列
Figure PCTCN2021086016-appb-000026
Figure PCTCN2021086016-appb-000027
Figure PCTCN2021086016-appb-000028
Figure PCTCN2021086016-appb-000029
Figure PCTCN2021086016-appb-000030
Figure PCTCN2021086016-appb-000031
表1f 本公开的第六种siRNA序列
Figure PCTCN2021086016-appb-000032
Figure PCTCN2021086016-appb-000033
Figure PCTCN2021086016-appb-000034
Figure PCTCN2021086016-appb-000035
表1g 本公开的第七种siRNA序列
Figure PCTCN2021086016-appb-000036
Figure PCTCN2021086016-appb-000037
Figure PCTCN2021086016-appb-000038
表1h 本公开的第八种siRNA序列
Figure PCTCN2021086016-appb-000039
Figure PCTCN2021086016-appb-000040
Figure PCTCN2021086016-appb-000041
Figure PCTCN2021086016-appb-000042
Figure PCTCN2021086016-appb-000043
Figure PCTCN2021086016-appb-000044
Figure PCTCN2021086016-appb-000045
Figure PCTCN2021086016-appb-000046
Figure PCTCN2021086016-appb-000047
Figure PCTCN2021086016-appb-000048
其中，大写字母C、G、U、A表示核苷酸的碱基组成；大写字母R表示简并碱基，具体地为随机的核苷酸碱基A或G，Y是与R互补的碱基；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NH4+，有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方式中，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。在一种方式中，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。
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。
本公开的siRNA及含该siRNA的药物组合物及缀合物的应用
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/或预防新型冠状病毒引起的疾病的药物中的用途。在一些实施方式中，新型冠状病毒引起的疾病是COVID-19。
在一些实施方式中，本公开提供了一种预防和/或新型冠状病毒引起的疾病的方法，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/或治疗新型冠状病毒所引起的疾病的目的。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/或治疗新型冠状病毒引起的疾病，或用于制备用于预防和/或治疗新型冠状病毒引起的疾病的药物。
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/或治疗新型冠状病毒引起的疾病的手段，在一些实施方式中采用能够将药物递送至肺部的给药方式。
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LD50(使50％的群体死亡的致死剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指能引起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。在一些实施方式中，根据给药方式的不同对所述siRNA、药物组合物或siRNA缀合物所制成的制剂在给药过程中的剂量进行调整。
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30-45g的ob/ob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。
在一些实施方式中，本公开提供了一种抑制肺细胞中SARS-COV-2病毒基因表达的方法，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缀合物与所述肺细胞接触，将本公开的siRNA和/或药物组合物和/或siRNA缀合物导入所述肺细胞，通过RNA干扰的机制达到抑制肺细胞中SARS-COV-2病毒基因表达的目的。
采用本公开提供的方法抑制SARS-COV-2病毒基因在细胞中表达，所提供的修饰的siRNA、药物组合物和/或siRNA缀合物中的siRNA用量一般是这样的量：其足以减少靶基因的表达，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修饰的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修饰的siRNA。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修饰的siRNA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修饰的siRNA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，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述修饰的siRNA、药物组合物和/或siRNA缀合物和/或缀合物，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，所述修饰的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/或缀合物和任选的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。
有益效果
本公开提供的siRNA、含该siRNA的组合物和siRNA缀合物具有良好的稳定性，较高的基因抑制活性，很低的细胞毒性，和/或能显著治疗或缓解由新型冠状病毒引起的疾病的症状。
在一些实施方式中，本公开提供的siRNA、含该siRNA的组合物(以下，有时也简称为siRNA组合物)或siRNA缀合物在体外细胞实验中显示出优异的靶基因抑制活性。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在细胞中显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的靶基因表达抑制率。在一些实施方式中，在1nM的siRNA浓度下，本公开提供的siRNA在HEK293A细胞中显示出96.23％-99.00％的靶RNA表达量抑制率。在一些实施方式中，在1nM的siRNA浓度下，本公开提供的siRNA在HEK293A细胞中显示出91.53％-96.99％的靶RNA表达量抑制率。在一些实施方式中，在1nM的siRNA浓度下，本公开提供的siRNA在HEK293A细胞中显示出92.54％-97.75％的靶RNA表达量抑制率。在一些实施方式中，在终浓度50nM的siRNA转染浓度下，本公开提供的siRNA在感染了SARS-COV-2病毒的Vero细胞系中，转染后48h时显示出至少37.5％、甚至高达97.9％的SARS-COV-2病毒RNA抑制率。
在一些实施方式中，本公开提供的siRNA、含该siRNA的组合物或siRNA缀合物可在体内具有更高的稳定性和/或更高的活性。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的靶基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的SARS-COV-2病毒基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的肺内SARS-COV-2病毒基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的动物模型中肺内SARS-COV-2病毒基因表达抑制率。在一些实施方式中，本公开提供的在一些实施方式中siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的动物模型中气管组织内SARS-COV-2病毒基因表达抑制率。在一些实施方式中，本公开提供的siRNA、siRNA组合物或siRNA缀合物在体内显示出至少20％，30％，40％，50％，60％，70％，80％，90％或95％的人类受试者中肺内SARS-COV-2病毒基因表达抑制率。在一些实施方式中，本公开提供的siRNA在小鼠体内肺部组织显示出85.71％-94.20％的病毒RNA载量相对抑制率。在一些实施方式中，本公开提供的siRNA在小鼠体内气管组织显示出98.19％-99.71％的病毒RNA载量相对抑制率。在一些实施方式中，本公开提供的siRNA在小鼠体内肺部组织显示出49.5％的病毒RNA载量相对抑制率。在一些实施方式中，本公开提供的siRNA在小鼠体内气管组织显示出63.95％的病毒RNA载量相对抑制率。在一些实施方式中，本公开提供的siRNA在小鼠体内肺部组织显示出91.27％-94.68％的病毒RNA载量相对抑制率。
在一些实施方式中，本公开提供的siRNA、含该siRNA的组合物或siRNA缀合物未显示出显著的脱靶效应。脱靶效应可以是例如抑制非靶基因的基因正常表达。据认为，如果脱靶基因表达的结合/抑制与在靶基因效果相比低于50％、40％、30％、20％或10％时，该脱靶效应就是不显著的。
在一些实施方式中，对于突变株SARS-CoV-2病毒，即使是其病毒RNA序列在特定位点存在特定的1个或2个碱基替代或突变，本公开提供的siRNA、含该siRNA的组合物或siRNA缀合物仍显示出较高的病毒RNA抑制活性。
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SARS-COV-2病毒基因的表达，有效治疗和/或预防由SARS-COV-2病毒基因表达引起的相关疾病症状，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。
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。
本公开合成的针对SARS-COV-2病毒基因的siRNA或作为阴性对照的siRNA转染细胞时，使用LipofectamineTM2000(Invitrogen)作为转染试剂，具体操作参照制造商提供的说明书。
若无其它说明，以下提供的试剂比例均按体积比(v/v)计算。若无其它说明，各溶液均为使用生理盐水作为溶剂配制而成。
实验数据分析采用Graphpad prism 8.0统计分析软件。
制备例1-16
本公开提供的siRNA的合成
通过常规固相合成方法分别合成表2中所列的siRNA序列的正义链或反义链，使用DEPC水，分别溶解等摩尔表2中相互互补的正义链和反义链，随后退火得到本公开提供的siCOVIDa1-M1S、siCOVIDb1-M1S、siCOVIDc1-M1S、siCOVIDd1-M1S、siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDg1-M1S、siCOVIDh1-M1S、siCOVIDe1、siCOVIDf1、siCOVIDe1U-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S、siCOVIDh1G-M1S、siCOVIDe1-M1S1和siCOVIDh1-M1S1，如表2所示。
对比制备例1-2
参比siRNA的合成
通过固相合成方法分别合成了表2中siRNA编号为NC和NC2的siRNA对应的正义链和反义链。使用DEPC水，分别溶解得到的等摩尔的正义链和反义链，随后退火得到参比siRNA，编号为NC和NC2。
表2 siRNA序列
Figure PCTCN2021086016-appb-000049
Figure PCTCN2021086016-appb-000050
Figure PCTCN2021086016-appb-000051
其中，大写字母C、G、U、A表示核苷酸的碱基组成，大写字母R表示简并碱基，R为核苷酸碱基A或G，Y是与R互补的碱基；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基连接。
上述序列的制备过程中，当目标序列中包含未修饰的核苷酸时，在切割与脱保护条件中，在氨水处理后，相对于单链核酸的量，用0.4ml/μmol N-甲基吡咯烷酮溶解产品，随后加入0.3ml/μmol三乙胺和0.6ml/μmol三乙胺三氢氟酸盐，以脱除核糖上的2'-TBDMS保护。制备完成后，使用超纯水(电阻率18.2MΩ*cm(25℃))将siRNA稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)对上述siRNA的分子量进行检测，实测值与理论值一致，确认所获得的siRNA分别具有对应于表2中所示的各个siRNA的序列。
含有简并碱基的核苷酸序列的制备与上述相同，区别仅在于：在正义链的连接过程中，连接至上文所述简并碱基位置处时，按照A:G＝1:1的比例投入原料核苷单体A或G，连接简并碱基R，其中R为随机的A或G；与之相对应地，在反义链的连接过程中，连接至上文所述简并碱基位置处时，按照U:C＝1:1的比例投入原料核苷单体U或C，连接简并碱基Y，其中Y为随机的U或C。
另外，以下各实验例中使用的目标序列、引物序列与探针序列的制备与上述相同，区别仅在于，按照各引物序列的碱基顺序，依次连接对应的核苷单体或带有荧光标记的核苷单体。
在上述本公开的siRNA、目标序列、引物序列或探针序列制备完成后，分别冻干为固体粉末保存备用。
实验例1-8
本公开的siRNA体外(in vitro)的抑制活性
用加入20％的胎牛血清(FBS，Hyclone公司)及0.2体积％的青链霉素双抗(Penicillin-Streptomycin，Gibco，Invitrogen公司)的H-DMEM完全培养基(Hyclone公司)培养细胞，于37℃在含5％CO2/95％空气的培养箱中培养HEK293A细胞(购自南京科佰生物科技有限公司)。
根据Kumico Ui-Tei et.al.,Functional dissection of siRNA sequence by systematic DNA substitution:modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.Nucleic Acids Research,2008.36(7),2136-2151描述的方法，构建检测质粒，与待评价的siRNA(siCOVIDa1-M1S、siCOVIDb1-M1S、siCOVIDc1-M1S、siCOVIDd1-M1S、siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDg1-M1S和siCOVIDh1-M1S)分别共转染至HEK293A细胞中，通过双萤光素酶报告基因的表达水平，来反映siRNA的抑制活性。具体步骤如下：
[1]构建检测质粒
采用psiCHECKTM-2(PromegaTM)质粒构建检测质粒，该质粒含有一个目标序列，即siRNA靶序列。对于每一待评价的siRNA，目标序列分别如下所示：
siCOVIDa1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000052
siCOVIDb1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000053
siCOVIDc1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000054
siCOVIDd1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000055
siCOVIDe1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000056
siCOVIDf1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000057
siCOVIDg1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000058
siCOVIDh1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000059
将目标序列克隆到psiCHECKTM-2质粒的Xho I/Not I位点。
[2]转染
将HEK293A细胞以8×103细胞/孔接种于96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80μl Opti-MEM培养基(GIBCO公司)继续培养1.5h。
对于每一siRNA，用DEPC化水将上述检测质粒稀释成200ng/μl的检测质粒工作液。对于每一siRNA，用siRNA和DEPC化水配制成浓度分别为1nM、0.1nM和0.01nM(以siRNA的量计)的siRNA工作液。
配制1A1溶液，每份1A1溶液含有浓度为0.1μM的siRNA工作液1μl、检测质粒工作液0.05μl(含检测质粒10ng)和10μl的Opti-MEM培养基。
配制1A2溶液，每份1A2溶液含有浓度为0.01μM的siRNA工作液1μl、检测质粒工作液0.05μl(含检测质粒10ng)和10μl的Opti-MEM培养基。
配制1A3溶液，每份1A3溶液含有浓度为0.001μM的siRNA工作液1μl、检测质粒工作液0.05μl(含检测质粒10ng)和10μl的Opti-MEM培养基。
配制1B溶液，每份1B溶液含有0.2μl Lipofectamine TM 2000和10μl Opti-MEM培养基。
配制1C溶液，每份1C溶液含有检测质粒工作液0.05μl(含检测质粒10ng)和10μl的Opti-MEM培养基。
对于每一个siRNA，分别将一份1B溶液与一份1A1溶液、一份1A2溶液、一份1A3溶液，室温下孵育20min，分别得到转染复合物1X1、1X2或1X3。将一份1B溶液与一份1C溶液混合，室温下孵育20min得到转染复合物1X4。
对于每一个siRNA，在三个培养孔中，分别加入转染复合物1X1，均匀混合，加入量为20μl/孔，得到siRNA终浓度约为1nM的共转染混合物，记为测试组1。
对于每一个siRNA，在另外三个培养孔中，分别加入转染复合物1X2，均匀混合，加入量为20μl/孔，得到siRNA终浓度约为0.1nM的共转染混合物，记为测试组2。
对于每一个siRNA，在另外三个培养孔中，分别加入转染复合物1X3，均匀混合，加入量为20μl/孔，得到siRNA终浓度约为0.01nM的共转染混合物，记为测试组3。
对于每一个siRNA，在另外三个培养孔中，分别加入转染复合物1X4，得到不含siRNA的共转染混合物，加入量为20μl/孔，记为对照组。
将含siRNA的共转染混合物和不含siRNA的转染混合物在培养孔中共转染4h后，每孔补加100μl添加20％FBS的H-DMEM完全培养基。将96孔板置于CO2培养箱继续培养24h。
[3]检测
吸去培养孔中的培养基，每孔加入150μl的
Figure PCTCN2021086016-appb-000060
Luciferase试剂与H-DMEM混合溶液(体积比1:1)，充分混匀，室温孵育10min后，转移120μl混合液到96孔酶标板上，使用Synergy II多功 能酶标仪(BioTek公司)读取Firefly化学发光值(Fir)；再向每孔加入60μl
Figure PCTCN2021086016-appb-000061
Stop&
Figure PCTCN2021086016-appb-000062
试剂，充分混匀，室温孵育10min后，按照读取Fir的排布方式，使用酶标仪读取Renilla的化学发光值(Ren)。
计算每孔发光比值Ratio＝Ren/Fir，各测试组或对照组的发光比值Ratio(测试)或Ratio(对照)为三个培养孔Ratio的平均值；以对照组的发光比值为基准，对各测试组的发光比值进行归一化，获得Ratio(测试)/Ratio(对照)的比值R，以此表示Renilla报告基因的表达水平，即残留活性。siRNA的抑制率＝(1-R)×100％。
不同浓度的siCOVIDa1-M1S、siCOVIDb1-M1S、siCOVIDc1-M1S、siCOVIDd1-M1S、siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDg1-M1S、siCOVIDh1-M1S对目标序列的抑制活性结果如图1所示。
对比实验例1
参比siRNA NC体外(in vitro)的抑制活性。
按照实验例1-8的方法同时考察了参比siRNA NC在psiCHECK系统中的抑制活性，区别仅在于，所测试的siRNA为参比siRNA NC。
结果如图1所示。
结果表明，制备例1-8(siCOVIDa1-M1S、siCOVIDb1-M1S、siCOVIDc1-M1S、siCOVIDd1-M1S、siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDg1-M1S、siCOVIDh1-M1S)在1nM-0.1nM浓度时都具有较好的体外抑制活性。且抑制活性呈现出浓度依赖性。特别是，在1nM的siRNA浓度下，目标序列抑制率至少为96.23％，最高可达99.00％，显示出优异的抑制SARS-COV-2病毒基因表达的效果。
实验例9-16
本公开的siRNA体外(in vitro)的抑制活性
进一步地，使用与实验例1相同的方法，分别测试siCOVIDa1-M1S、siCOVIDb1-M1S、siCOVIDc1-M1S、siCOVIDd1-M1S、siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDg1-M1S和siCOVIDh1-M1S在体外psiCHECK系统中的抑制活性，区别仅在于，siRNA的终浓度依次为1nM、0.3nM和0.1nM；在构建检测质粒时，对于每一siRNA而言，目标序列为以下的序列：
siCOVIDa1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000063
siCOVIDb1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000064
siCOVIDc1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000065
Figure PCTCN2021086016-appb-000066
siCOVIDd1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000067
siCOVIDe1-M1S和siCOVIDf1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000068
siCOVIDg1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000069
siCOVIDh1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000070
上述各目标序列均是NC_045512.2中的序列片段。
实验结果参见图2所示。
对比实验例2
参比siRNA NC体外(in vitro)的抑制活性。
按照实验例9的方法同时考察了参比siRNA NC在psiCHECK系统中的抑制活性，区别仅在于，所测试的siRNA为参比siRNA NC。
结果如图2所示。
由图2结果可知，在参比siRNA显示对目标序列无抑制活性的同时，本公开的siRNA均显示出良好的抑制目标序列基因表达的效果。且抑制活性呈现出浓度依赖性。在1nM的siRNA浓度下，目标序列的RNA表达量抑制率至少为91.53％，最高可达96.99％，显示出优异的抑制SARS-COV-2病毒RNA的效果。
实验例17-20
本公开的siRNA体外(in vitro)的抑制活性
进一步地，使用与实验例1相同的方法，分别测试siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDe1和siCOVIDf1在体外psiCHECK系统中的抑制活性，区别仅在于，在构建检测质粒时，对于siCOVIDe1而言，目标序列为siCOVIDe1-M1S的目标序列；对于siCOVIDf1而言，目标序列为siCOVIDf1-M1S的目标序列；实验结果参见图3所示。
由图3结果可见，siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDe1和siCOVIDf1在体外显示出较好的SARS-COV-2病毒抑制活性，在1nM的siRNA浓度下，siCOVIDe1-M1S的目标序列表达量抑制率为97.75％，siCOVIDf1-M1S的目标序列表达量抑制率为97.15％，siCOVIDe1的目标序列表达量抑制率为92.54％，siCOVIDf1的目标序列表达量抑制率为92.46％，显示出优异的抑制SARS-COV-2病毒基因表达的效果。
以下，全部涉及传染性SARS-CoV-2的实验均在军事科学医学研究院的生物安全3级(BSL3)密闭实验室中进行。
在37℃下，在添加有2％FBS(PAN Biotech)、1％青链霉素双抗(GIBCO)以及1％HEPES缓冲液(GIBCO)的DMEM培养基(GIBCO)中培养Vero细胞，用于SARS-CoV-2病毒扩增和滴定。SARS-CoV-2病毒株BetaCoV/wuhan/AMMS01/2020由军事科学医学研究院秦成峰实验室分离获得，使用上述Vero细胞对病毒进行扩增，得到病毒液，并通过标准噬斑形成测定法来测定病毒液的病毒滴度。具体地，在12孔板中，分别使用依次10倍稀释的病毒株感染Vero细胞，在37℃下培养1小时，并在添加2％FBS和1％的低熔点琼脂糖(Promega)的DMEM中继续培养细胞2天，用4％甲醛水溶液固定细胞并使用0.2％结晶紫水溶液染色噬菌斑进行滴定，确定病毒液的病毒滴度。
下述实验中所用的病毒稀释液为用生理盐水稀释病毒液所得的病毒溶液。
实验例21-27
本公开的siRNA在Vero感染细胞后对SARS-COV-2病毒RNA的抑制
用ATCC完全培养基，于37℃在含5％CO2/95％空气的培养箱中培养Vero细胞(购自南京科佰生物科技有限公司)。其中，ATCC完全培养基为MEM-EBSS培养基，含有含2mM L-谷氨酰胺的MEM基础培养基(Hyclone公司)和Earle's平衡盐溶液(EBSS，Giboco公司)。该平衡盐溶液含1.5g/L碳酸氢钠，0.1mM非必须氨基酸(NEAA，Corning公司)，1.0mM 90％丙酮酸钠和10％胎牛血清(FBS，Hyclone公司)。
将Vero细胞以8×104细胞/孔接种于24孔板中，16-24h后细胞生长密度达到70-80％时，使用5×105PFU的SARS-COV-2病毒液进行细胞感染，具体地为向每孔中加入50μl含5×105PFU的病毒稀释液，于37℃在含5％CO2/95％空气的培养箱中培养1h；随后弃掉上层清液，使用1×PBS溶液洗涤细胞2次，每次用量500μl/孔，得到感染了病毒的Vero细胞，记为空白组，每组3个复孔。
将Vero细胞以8×104细胞/孔接种于24孔板中，16-24h后细胞生长密度达到70-80％时，使用5×105PFU的SARS-COV-2病毒液进行细胞感染，具体地为向每孔中加入50μl含5×105PFU的病毒稀释液，于37℃在含5％CO2/95％空气的培养箱中培养1h；随后弃掉上层清液，使用1×PBS溶液洗涤细胞2次，每次用量500μl/孔，得到感染了病毒的Vero细胞。根据Lipofectamine TM 2000(Invitrogen公司)的使用说明，分别在感染了病毒的Vero细胞中转染各siRNA(siCOVIDb1-M1S、siCOVIDc1-M1S、siCOVIDd1-M1S、siCOVIDe1-M1S、siCOVIDf1-M1S、siCOVIDg1-M1S、siCOVIDh1-M1S和NC2)， 使得每个siRNA的终浓度均为50nM，得到感染了病毒和转染了各siRNA的Vero细胞，分别记为siCOVIDb1-M1S测试组、siCOVIDc1-M1S测试siCOVIDd1-M1S测试组、siCOVIDe1-M1S测试组、siCOVIDf1-M1S测试组、siCOVIDg1-M1S测试组、siCOVIDh1-M1S测试组和NC2对照组，每组3个复孔。
于37℃在含5％CO 2/95％空气的培养箱中培养24h和48h后，分别提取上清液，通过实时荧光定量PCR(Quantitative Real-Time PCR)分别检测上述各测试组和对照组SARS-COV-2病毒RNA载量。
具体步骤为：培养空白组和各测试组24小时和48小时，分别在24小时和48小时时收集细胞培养基(即上清液)，并使平铺在细胞培养板中的Vero细胞完全裂解。使用Thermo PureLink RNA Mini Kit根据产品说明书的操作方法提取上清液及完全裂解的Vero细胞中的总RNA；分别取1μg总RNA，使用RR064A TAKARA反转录试剂盒按其说明书的操作方法反转录得到cDNA，使用探针法qRT-PCR对各测试组和空白组进行SARS-COV-2病毒RNA载量的检测，结果如图4A和图4B所示。
其中，用于扩增SARS-COV-2病毒和作为内参基因的GAPDH的PCR引物如表3所示。
表3 引物信息
Figure PCTCN2021086016-appb-000071
对比实验例3
参比siRNA NC2在Vero感染细胞中对SARS-COV-2病毒RNA表达的抑制活性
按照实验例21-27的方法同时考察了对比制备例2制备得到的参比siRNA NC2在Vero感染细胞中对SARS-COV-2病毒RNA表达的抑制活性，区别仅在于，所测试的siRNA为参比siRNA NC2。结果如图4A和图4B所示。
由图4A和图4B可见，本公开提供的修饰的siRNA在感染了SARS-COV-2病毒的Vero细胞系中有较高的病毒RNA抑制活性，转染siRNA后病毒的载量显著更低。根据图4A的结果可知，转染NC2的Vero细胞在24小时后的病毒RNA载量为1.32×107copies/mL，转染siCOVIDb1-M1S的24小时后病毒RNA载量为8.15×106copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的61.7％；转染siCOVIDc1-M1S的24小时后病毒RNA载量为8.39×106copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的63.6％；转染siCOVIDd1-M1S的24小时后病毒RNA载 量为4.13×106copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的31.3％；转染siCOVIDe1-M1S的24小时后病毒RNA载量为2.42×106copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的18.3％；转染siCOVIDf1-M1S的24小时后病毒RNA载量为3.82×106copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的28.9％；转染siCOVIDg1-M1S的24小时后病毒RNA载量为5.83×106copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的44.2％；转染siCOVIDh1-M1S的24小时后病毒RNA载量为3.79×105copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的2.87％。
根据图4B的结果可知，转染NC2的48小时后病毒RNA载量为1.23×1010copies/mL，转染siCOVIDb1-M1S的48小时后病毒RNA载量为1.38×1010copies/mL；转染siCOVIDc1-M1S的48小时后病毒RNA载量为1.47×109copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的12.0％；转染siCOVIDd1-M1S的48小时后病毒RNA载量为7.69×109copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的62.5％；转染siCOVIDe1-M1S的48小时后病毒RNA载量为7.48×108copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的6.1％；转染siCOVIDf1-M1S的48小时后病毒RNA载量为1.77×109copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的14.4％；转染siCOVIDg1-M1S的48小时后病毒RNA载量为5.49×109copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的44.6％；转染siCOVIDh1-M1S的48小时后病毒RNA载量为2.57×108copies/mL，仅为转染NC2的Vero细胞中相同时间下病毒RNA载量的2.1％。从而，本公开的siRNA在感染了SARS-COV-2病毒的Vero细胞系中显示出优异的抑制SARS-COV-2病毒基因表达的效果。
实验例28-29
本公开的siRNA对Vero细胞活性的影响
用加入10％的胎牛血清(FBS，Hyclone公司)及0.2体积％的青链霉素双抗(Penicillin-Streptomycin，Gibco，Invitrogen公司)的H-DMEM完全培养基(Hyclone公司)于37℃在含5％CO2/95％空气的培养箱中培养Vero细胞(购自南京科佰生物科技有限公司)。
将Vero细胞以8×104细胞/mL接种于96孔板中，100μL/孔，即8×104细胞/孔，培养24h后进行转染。
[1]转染
通过自由摄取和lipo2000转染这两种方式分别进行实验：
(1)通过Lipofectamine TM 2000转染
用DEPC化水将上述检测质粒稀释成266ng/mL，即浓度为20μM的检测质粒工作液；用DEPC化水将下面的siRNA中的每一个siRNA分别配制成100nM、50nM、25nM、12.5nM、6.25nM、3.125nM、1.563nM、0.781nM共8种不同浓度的siRNA工作液，所用siRNA分别为siCOVIDe1-M1S、siCOVIDh1-M1S和NC。
对于每一个siRNA，分别配制2A1-2A8溶液，每份2A1-2A8溶液依次含有上述8个浓度的siRNA工作液1μl、检测质粒工作液0.05μl(含检测质粒10ng)和10μl的Opti-MEM培养基。
配制2B溶液，每份2B溶液含有0.2μl Lipofectamine TM 2000和10μl Opti-MEM培养基。
分别将一份2B溶液与得到的一份每个siRNA的2A1-2A8的溶液混合，分别室温下孵育20min，得到每个siRNA的转染复合物2X1-2X8。
在培养孔中，分别加入每一个siRNA转染复合物2X1-2X8，均匀混合，加入量为20μl/孔，得到每个siRNA终浓度分别约为100nM、50nM、25nM、12.5nM、6.25nM、3.125nM、1.563nM和0.781nM的转染复合物，每个siRNA的转染复合物2X1-2X8分别转染3个培养孔，得到含siRNA的共转染混合物。
分别将含siRNA的共转染混合物在培养孔中转染4h后，每孔补加100μl含20％FBS的H-DMEM完全培养基。将96孔板置于CO 2培养箱继续培养72h。
(2)自由摄取
用DEPC化水将上述检测质粒稀释成13.3ng/ml，即浓度为1μM的检测质粒工作液；用DEPC化水将下面的siRNA中的每一个siRNA分别配制成3000nM、1000nM、300nM、100nM、30nM、10nM、3nM和1nM共8种不同浓度的siRNA工作液，所用siRNA分别为siCOVIDe1-M1S、siCOVIDh1-M1S和NC。
使用97μL含10％FBS-DMEM对培养孔中细胞进行换液，分别向培养孔中加入8种不同浓度的siRNA工作液3μL，每种浓度的siRNA对应3个培养孔，于37℃在含5％CO 2/95％空气的培养箱中进行培养，72h后检测。
[2]检测
在Lipofectamine TM 2000转染和自由摄取72h后，使用CCK8试剂盒(购自北京拜尔迪，DE5001-3000T)提供的试剂，按试剂盒说明书方法进行操作，对96孔板中每孔更换100μL含20％FBS的H-DMEM完全培养基，再向每孔加入10μL的CCK8试剂盒中试剂，将96孔板在于37℃在含5％CO 2/95％空气的培养箱内孵育4小时，检测OD值。再向无细胞培养孔加入100μL新鲜培养基再加入10μL的CCK8试剂盒中试剂作为空白对照组。
对各测试组和对照组的OD450吸光度进行检测后，计算细胞活性(相对值)，计算方法如下：
细胞活性＝[OD(测试组)-OD(空白对照组)]/[OD(对照组)-OD(空白对照组)]×100％
其中，OD(测试组)代表含有细胞、CCK8试剂和siRNA的培养孔中的OD450吸光度；OD(空白对照组)代表仅含有CCK8试剂的培养孔中的OD450吸光度；OD(对照组)代表含有细胞、CCK8试剂但不含有siRNA的培养孔中的OD450吸光度。不同浓度的siCOVIDe1-M1S、siCOVIDh1-M1S对细胞的活性影响结果如图5A和图5B所示。
由图5A可见，在Lipo2000转染时，不同浓度下的siCOVIDe1-M1S和siCOVIDh1-M1S对Vero细胞均无明显毒性作用，细胞活性均大于80％。由图5B可见，在自由摄取时，不同浓度下的siCOVIDe1-M1S和siCOVIDh1-M1S对Vero细胞均无明显毒性作用，细胞活性均大于80％。
实验例30-31
本公开的siRNA体外(in vitro)的抑制活性
按照实验例1-8的方法，通过构建检测质粒后，将质粒与待测siRNA分别共转染至HEK293A细胞中，然后通过双萤光素酶报告基因的表达水平，来反映siRNA的抑制活性，区别仅在于：待评价的siRNA为siCOVIDe1-M1S和siCOVIDe1U-M1S(制备例5和制备例11)；在构建检测质粒时，对于待测siRNA的目标序列均为TCGAGTAGTCTCTAGTCAGTGTGTTAGC(SEQ ID NO:1015)。
不同浓度的siCOVIDe1-M1S、siCOVIDe1U-M1S对目标序列的抑制活性结果如图6示。
结果表明，制备例5和制备例11(siCOVIDe1-M1S、siCOVIDe1U-M1S)在1nM-0.1nM浓度时都具有较好的体外抑制活性，且呈现出浓度依赖性抑制活性。特别是，在1nM的siRNA浓度下，RNA表达量抑制率至少为83.16％，最高可达95.72％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。
实验例32-33
本公开的siRNA体外(in vitro)的抑制活性
按照实验例1-8的方法，通过构建检测质粒后，将质粒与待测siRNA分别共转染至HEK293A细胞中，然后通过双萤光素酶报告基因的表达水平，来反映siRNA的抑制活性，区别仅在于：待评价的siRNA为siCOVIDe1-M1S和siCOVIDe1U-M1S(制备例5和制备例11)；在构建检测质粒时，对于待评价的siRNA的目标序列均为TCGAGTAGTTTCTAGTCAGTGTGTTAGC(SEQ ID NO:1016)。
不同浓度的siCOVIDe1-M1S、siCOVIDe1U-M1S对目标序列的抑制活性结果如图7所示。其中，siCOVIDe1U-M1S与siCOVIDe1-M1S相比，第3位核苷酸由胞嘧啶替换为尿嘧啶，与目标序列具有1位错配。
结果表明，制备例5和制备例11(siCOVIDe1-M1S、siCOVIDe1U-M1S)在1nM-0.1nM浓度时都具有较好的体外抑制活性，且呈现出浓度依赖性抑制活性。特别是，在1nM的siRNA浓度下，目标序列表达量抑制率至少为85.56％，最高可达95.47％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。尤其，显示出当本公开提供的siRNA的正义链或反义链特点位点进行碱基替代或突变后，该siRNA显示出与无碱基替代或未突变序列对应的siRNA相同或相近的抑制活性。
实验例34-37
本公开的siRNA对病毒突变序列的体外(in vitro)抑制活性
按照实验例1-8的方法，通过构建检测质粒后，将质粒与待测siRNA分别共转染至HEK293A细胞中，然后通过双萤光素酶报告基因的表达水平，来反映siRNA的抑制活性，区别仅在于：待评价 的siRNA为siCOVIDh1-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S和siCOVIDh1G-M1S(制备例8、制备例12-14)；在构建检测质粒时，对于对于所述每一待评价的siRNA，目标序列如下所示：
siCOVIDh1-M1S的目标序列为：
Figure PCTCN2021086016-appb-000072
siCOVIDh1U-M1S的目标序列为：
Figure PCTCN2021086016-appb-000073
siCOVIDh1R-M1S的目标序列为：
Figure PCTCN2021086016-appb-000074
siCOVIDh1G-M1S的目标序列为：
Figure PCTCN2021086016-appb-000075
上述4条目标序列为SARS-CoV-2病毒的SNP突变序列中的一段核苷酸序列，与NC_045512.2的对应序列片段相比分别具有1个或2个核苷酸突变。
不同浓度的siCOVIDh1-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S和siCOVIDh1G-M1S对目标序列的抑制活性结果如图8所示。
结果表明，制备例8和制备例12-14(siCOVIDh1-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S和siCOVIDh1G-M1S)在1nM-0.1nM浓度时都具有较好的体外抑制活性，且呈现出浓度依赖性抑制活性。特别是，在1nM的siRNA浓度下，目标序列表达量抑制率至少在80.25％以上，最高可达95.25％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。尤其是，对于发生了突变的病毒目标序列，本公开提供的siRNA仍显示出较高的的抑制活性。
实验例38-41
本公开的siRNA体外(in vitro)的抑制活性
按照实验例1-8的方法，通过构建检测质粒后，将质粒与待测siRNA分别共转染至HEK293A细胞中，然后通过双萤光素酶报告基因的表达水平，来反映siRNA的抑制活性，区别仅在于：待评价的siRNA为siCOVIDh1-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S和siCOVIDh1G-M1S(制备例8、制备例12-14)；在构建检测质粒时，对于待评价的siRNA的目标序列均为TCGAGGTCATTCAATCCAGAAAGC(SEQ ID NO:1017)。
不同浓度的siCOVIDh1-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S和siCOVIDh1G-M1S对目标序列的抑制活性结果如图9所示。
结果表明，制备例8和制备例12-14(siCOVIDh1-M1S、siCOVIDh1U-M1S、siCOVIDh1R-M1S和siCOVIDh1G-M1S)在1nM-0.01nM浓度时都具有较好的体外抑制活性，且呈现出浓度依赖性抑制活性。特别是，在1nM的siRNA浓度下，目标序列抑制率至少在69.6％以上，最高可达95.25％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。尤其是，和与目标序列完全匹配的siRNA相比，本公开提供的siRNA的即使是与突变序列的正义链或反义链特定位点存在碱基替代或错配，该siRNA仍显示出与前述完全匹配的siRNA相同或相近的抑制活性。
实验例42
本公开的siRNA对病毒突变序列的体外(in vitro)抑制活性
按照实验例1-8的方法，通过构建检测质粒后，将质粒与待测siRNA分别共转染至HEK293A细胞中，然后通过双萤光素酶报告基因的表达水平，来反映siRNA的抑制活性，区别仅在于：待评价的siRNA为siCOVIDh1-M1S，对于所述该待评价的siRNA，对应4条不同的目标序列依次如下所示：
siCOVIDh1-M1S的目标序列为：
(1)TCGAGGTCATTCAATCCAGAAAGC(SEQ ID NO:1017)；
(2)TCGAGGTCATTTAATCCAGAAAGC(SEQ ID NO:1018)；
(3)TCGAGGTCATTCAATCCRGAAAGC(SEQ ID NO:1019)；
(4)TCGAGGTCATTCAATCCGGAAAGC(SEQ ID NO:1020)。
不同浓度的siCOVIDh1-M1S对应不同目标序列的抑制活性结果如图10中柱状图依次所示。
结果表明，待评价的siRNA，即制备例8对4条不同目标序列在1nM-0.01nM浓度时都具有较好的体外抑制活性，且呈现出浓度依赖性抑制活性。特别是，在1nM的siRNA浓度下，目标序列表达量抑制率均在95％以上，最高可达97.35％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。 尤其，显示出当本公开提供的siRNA尽管仅与未突变序列完全匹配，而与突变序列存在至少一个碱基错配，然而对于突变序列，该siRNA仍显示出优异的抑制活性，且与对未突变序列的抑制活性水平接近。
实验例43-44
本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)对SARS-COV-2病毒RNA的抑制效果
人源化ACE2转基因小鼠(B6/JGpt-Ace2em1Cin(hACE2)/Gpt)，购自江苏集萃药康生物科技股份有限公司。首先，将人源化ACE2转基因小鼠随机分组，每组3只小鼠，分别进行编号，并增加PBS对照组。通过腹腔注射向小鼠体内注射入50mg/kg戊巴比妥进行麻醉，通过滴鼻法施与50μL含有4×105pfu SARS-COV-2的病毒稀释液，使小鼠染毒。
染毒后，向每只小鼠给予本申请中所述的siRNA和脂质体A形成的组合物(siRNA:脂质体A＝1:15(wt/wt))，具体的组成为，用生理盐水将各siRNA分别配制成0.6mg/mL siRNA溶液(siCOVIDe1-M1S或siCOVIDh1-M1S)，脂质体A由LC8:胆固醇:C16-PEG＝59:29:12(mol)组成；其中，含胺转染试剂LC8是按照中国专利CN 103380113B中实施例12制备的阳离子脂质87。所述组合物按照CN 103380113B中实施例9所述的方法制备获得，区别仅在于使用上述的siRNA和脂质体A进行制备。
染毒后2h后，通过滴鼻法向三组小鼠分别给予待测siRNA组合物，给药体积均为10μL，给药剂量为3mg/kg。然后，分别在染毒后24h和48h，重复向三组小鼠分别通过滴鼻法给药，给药体积均为10μL，给药剂量为3mg/kg。
在最后一次给药的6h后处死小鼠，收集小鼠体内肺部组织和气管组织。用RNA later(Sigma Aldrich公司)保存；用组织匀浆仪匀浆肺部组织和气管组织，再用Trizol根据总RNA提取的标准操作步骤提取得到总RNA。
采用实时荧光定量PCR检测肺部组织和气管组织中SARS-COV-2病毒RNA载量，具体地：使用RR064A TAKARA反转录试剂盒按其说明书的操作方法反转录得到cDNA，接着用荧光定量PCR试剂盒(北京康为世纪生物科技有限公司)检测siRNA对肺部组织和气管组织中的SARS-COV-2病毒RNA载量。使用探针法qRT-PCR对各测试组和空白组进行SARS-COV-2病毒RNA载量的检测，并计算得到siRNA对SARS-COV-2病毒RNA载量，结果如图11A和图11B所示。
其中，用于扩增SARS-COV-2病毒和作为内参基因的GAPDH的PCR引物如表3所示。
病毒RNA载量相对抑制率＝1-(给药组病毒RNA载量/对照组病毒RNA载量)×100％。
结果表明，通过滴鼻法方式给予的本公开提供的修饰的siRNA在感染了SARS-COV-2病毒的小鼠的肺部组织和气管组织中都具有良好的抑制活性，转染siRNA后小鼠体内病毒RNA载量与对照组相比更低。在肺部组织中，给予对照组后，病毒RNA载量为3.85×109copies/mL；给予siCOVIDe1-M1S组合物后，病毒RNA载量为2.23×108copies/mL，仅为对照组病毒RNA载量的5.80％，病毒RNA载量相对抑制率为94.20％；给予siCOVIDh1-M1S组合物后，病毒RNA载量为5.50×108copies/mL，仅为对照组病毒RNA载量的14.29％，病毒RNA载量相对抑制率为85.71％。在气管组织中，给予PBS对照物后，病毒RNA载量为5.44×108copies/mL；给予siCOVIDe1-M1S组合物后，病毒RNA载量为1.59×106copies/mL，仅为对照组病毒RNA载量的0.29％，病毒RNA载量相对抑制率为99.71％；给予siCOVIDh1-M1S组合物后，病毒RNA载量为9.82×106copies/mL，仅为对照组病毒RNA载量的1.81％，病毒RNA载量相对抑制率为98.19％。从而显示出优异的抑制SARS-COV-2病毒RNA表达的效果。
实验例45-46
本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)的SARS-COV-2病毒RNA载量
人源化ACE2转基因小鼠(B6/JGpt-Ace2em1Cin(hACE2)/Gpt)，购自江苏集萃药康生物科技股份有限公司。首先，将人源化ACE2转基因小鼠随机分组(均为雌性)，每组4只小鼠，分别进行编号，并增加PBS对照组。通过腹腔注射向小鼠体内注射入50mg/kg戊巴比妥进行麻醉，通过滴鼻法向每只小鼠给予50μL含有4×105pfu SARS-COV-2的病毒稀释液使小鼠染毒。
小鼠给药的药品为本申请中所述的siRNA，即siCOVIDe1-M1S，根据小鼠体重和给药剂量，在 实验前以生理盐水配制为相应浓度的siRNA溶液。
染毒后4h后，通过喉部给予雾化药品吸入肺部气管的方式向两组小鼠分别给予待测siRNA和PBS对照，给药体积均为50μL，给予的待测siRNA的给药剂量为6mg/kg。
在染毒后48h后处死小鼠，收集小鼠体内肺部组织和气管组织。用RNA later(Sigma Aldrich公司)保存；用组织匀浆仪匀浆肺部组织和气管组织，再用Trizol根据总RNA提取的标准操作步骤提取得到总RNA。
采用实时荧光定量PCR检测肺部组织和气管组织中SARS-COV-2病毒RNA载量，具体地：使用RR064A TAKARA反转录试剂盒按其说明书的操作方法反转录得到cDNA，接着用荧光定量PCR试剂盒(北京康为世纪生物科技有限公司)检测siRNA对肺部组织和气管组织中的SARS-COV-2病毒RNA载量。使用探针法qRT-PCR对各测试组和空白组进行SARS-COV-2病毒RNA载量的检测，并计算得到siRNA对SARS-COV-2病毒RNA载量的相对抑制率，结果如图12A和图12B所示。
其中，用于扩增SARS-COV-2病毒和作为内参基因的GAPDH的PCR引物如表3所示。
病毒RNA载量相对抑制率＝1-(给药组病毒RNA载量/对照组病毒RNA载量)×100％。
结果表明，通过雾化药品吸入肺部气管的方式给予本公开提供的修饰的siRNA在感染了SARS-COV-2病毒的小鼠的肺部组织和气管组织中都具有良好的抑制活性，转染siRNA后病毒的载量低。在肺部组织中，给予PBS对照物后，病毒RNA载量为1.29×107copies/mL；给予siCOVIDe1-M1S组合物后，病毒RNA载量为6.51×106copies/mL，仅为对照组病毒RNA载量的50.5％，病毒RNA载量相对抑制率为49.5％。在气管组织中，给予PBS对照物后，病毒RNA载量为2.20×109copies/mL；给予siCOVIDe1-M1S组合物后，病毒RNA载量为7.93×108copies/mL，仅为对照组病毒RNA载量的36.05％，病毒RNA载量相对抑制率为63.95％。从而显示出优异的抑制SARS-COV-2病毒RNA表达的效果。
实验例47-48
本公开的siRNA在感染SARS-COV-2病毒的人源化ACE2转基因小鼠体内(in vivo)的SARS-COV-2病毒RNA载量
人源化ACE2转基因小鼠(B6/JGpt-Ace2em1Cin(hACE2)/Gpt)，购自江苏集萃药康生物科技股份有限公司。首先，将人源化ACE2转基因小鼠随机分组(均为雌性)，每组4只小鼠，分别进行编号，并增加PBS对照组。通过腹腔注射向小鼠体内注射入50mg/kg戊巴比妥进行麻醉，通过滴鼻法向每只小鼠给予50μL含有4×105pfu SARS-COV-2的病毒稀释液使小鼠染毒。
小鼠给药的药品为本申请中所述的siRNA和脂质体B的组合物(siRNA:脂质＝1:15(wt/wt))，其具体的组成为0.6mg/mL siRNA(siCOVIDe1-M1S或siCOVIDh1-M1S)和脂质体B，具体组成为LC8:胆固醇:C16-PEG:DOTAP＝59:29:12:100(mol)，其中，含胺转染试剂LC8是按照中国专利CN103380113B中实施例12制备的阳离子脂质87，DOTAP购自上海毕得医药科技股份有限公司。所述组合物按照CN 103380113B中实施例9所述的方法制备获得，区别仅在于使用上述的siRNA和脂质体B进行制备。
染毒后6h后，通过静脉注射方式向三组小鼠分别给予待测siRNA组合物和PBS对照，给药体积均为50μL，给药剂量为1mg/kg。
在染毒后第四天处死小鼠，收集小鼠体内肺部组织。用RNA later(Sigma Aldrich公司)保存；用组织匀浆仪匀浆肺部组织和气管组织，再用Trizol根据总RNA提取的标准操作步骤提取得到总RNA。
采用实时荧光定量PCR检测肺部组织和气管组织中SARS-COV-2病毒RNA载量，具体地：使用RR064A TAKARA反转录试剂盒按其说明书的操作方法反转录得到cDNA，接着用荧光定量PCR试剂盒(北京康为世纪生物科技有限公司)检测siRNA对肺部组织中的SARS-COV-2病毒RNA载量。使用探针法qRT-PCR对各测试组和空白组进行SARS-COV-2病毒RNA载量的检测，并计算得到siRNA对SARS-COV-2病毒RNA载量，结果如图13所示。
其中，用于扩增SARS-COV-2病毒和作为内参基因的GAPDH的PCR引物如表3所示。
病毒RNA载量相对抑制率＝1-(给药组病毒RNA载量/对照组病毒RNA载量)×100％。
结果表明，通过静脉注射方式给予的本公开提供的修饰的siRNA在感染了SARS-COV-2病毒的小鼠的肺部组织中具有好的抑制活性，转染siRNA后病毒的载量低。在肺部组织中，给予PBS对照物后，病毒RNA载量为2.67×1010copies/mL；给予siCOVIDe1-M1S组合物后，病毒RNA载量为 1.42×109copies/mL，仅为对照组病毒RNA载量的5.32％，病毒RNA载量相对抑制率为94.68％；给予siCOVIDh1-M1S组合物后，病毒RNA载量为2.33×109copies/mL，仅为对照组病毒RNA载量的8.73％，病毒RNA载量相对抑制率为91.27％。从而显示出优异的抑制SARS-COV-2病毒RNA表达的效果。
实验例49-52
本公开的siRNA体外(in vitro)的抑制活性
进一步地，使用与实验例1相同的方法，分别测试待测siRNA在体外psiCHECK系统中的抑制活性，区别仅在于，待测siRNA为siCOVIDe1-M1S、siCOVIDe1-M1S1、siCOVIDh1-M1S和siCOVIDh1-M1S1；在构建检测质粒时，对于每一siRNA而言，目标序列为以下的序列：
siCOVIDe1-M1S和siCOVIDe1-M1S1的目标序列为：
Figure PCTCN2021086016-appb-000076
siCOVIDh1-M1S和siCOVIDh1-M1S1的目标序列为：
Figure PCTCN2021086016-appb-000077
上述各目标序列均是NC_045512.2中的序列片段。
实验结果参见图14所示。
对比实验例4
参比siRNA NC体外(in vitro)的抑制活性。
按照实验例49-52的方法同时考察了参比siRNA NC在psiCHECK系统中的抑制活性，区别仅在于，所测试的siRNA为参比siRNA NC。
结果如图14所示。
由图14结果可知，本公开的siRNA均显示出良好的抑制目标序列基因表达的效果。且呈现出浓度依赖性抑制活性。在1nM的siRNA浓度下，目标序列表达量抑制率至少为96.42％，最高可达97.57％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。
实验例53-56
本公开的siRNA体外(in vitro)的抑制活性
进一步地，使用与实验例1相同的方法，分别测试待测siRNA在体外psiCHECK系统中的抑制活性，区别仅在于，待测siRNA为siCOVIDe1-M1S、siCOVIDe1-M1S1、siCOVIDh1-M1S和siCOVIDh1-M1S1；在构建检测质粒时，对于每一siRNA而言，目标序列为以下的序列：
siCOVIDe1-M1S和siCOVIDe1-M1S1的目标序列为：
Figure PCTCN2021086016-appb-000078
siCOVIDh1-M1S和siCOVIDh1-M1S1的目标序列为：
Figure PCTCN2021086016-appb-000079
上述各目标序列均是NC_045512.2中的序列片段。
实验结果参见图15所示。
对比实验例5
参比siRNA NC体外(in vitro)的抑制活性。
按照实验例53-56的方法同时考察了参比siRNA NC在psiCHECK系统中的抑制活性，区别仅在于，所测试的siRNA为参比siRNA NC。
结果如图15所示。
由图15结果可知，本公开的siRNA均显示出良好的抑制目标序列基因表达的效果。且抑制活性呈现出浓度依赖性。在1nM的siRNA浓度下，目标序列表达量抑制率至少为87.44％，最高可达96.43％，显示出优异的抑制SARS-COV-2病毒RNA表达的效果。
以上详细描述了本发明的一些实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
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，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (48)

  1. 一种siRNA，其特征在于，所述siRNA含有正义链和反义链，所述的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，所述核苷酸序列II至少部分地与第一段核苷酸序列反向互补，所述第一段核苷酸序列为靶RNA中的一段核苷酸序列，所述靶RNA是指SARS-COV-2病毒的RNA。
  2. 根据权利要求1所述的siRNA，其中，第一段核苷酸序列为靶RNA保守区序列中的一段核苷酸序列。
  3. 根据权利要求1所述的siRNA，其中，所述核苷酸序列II与所述第一段核苷酸序列基本上反向互补、基本上完全反向互补或完全反向互补。
  4. 根据权利要求1所述的siRNA，其中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或者完全反向互补。
  5. 根据权利要求1所述的siRNA，其中，所述核苷酸序列I和所述核苷酸序列II的长度相同或不同，分别为15-40个核苷酸。
  6. 根据权利要求5所述的siRNA，其中，所述核苷酸序列I和所述核苷酸序列II的长度相同或不同，分别为18-30个核苷酸。
  7. 根据权利要求1-6中任意一项所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列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'-GCGCUAACAUAGGUUGUAZ 1-3'(SEQ ID NO:1)；
    5'-Z 2UACAACCUAUGUUAGCGC-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；或者，
    所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GCUUAUGUGUCAACCUAUZ 5-3'(SEQ ID NO:61)；
    5'-Z 6AUAGGUUGACACAUAAGC-3'(SEQ ID NO:62)，
    其中，Z 5为A，Z 6为U；
    所述核苷酸序列I中包含位置对应于Z 5的核苷酸Z 7，所述核苷酸序列II中包含位置对应于Z 6的核苷酸Z 8，所述Z 8是所述反义链5'末端的第一个核苷酸；或者，
    所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GCGGUUCACUAUAUGUUAZ 9-3'(SEQ ID NO:121)；
    5'-Z 10UAACAUAUAGUGAACCGC-3'(SEQ ID NO:122)，
    其中，Z 9为A，Z 10为U；
    所述核苷酸序列I中包含位置对应于Z 9的核苷酸Z 11，所述核苷酸序列II中包含位置对应于Z 10的核苷酸Z 12，所述Z 12是所述反义链5'末端的第一个核苷酸；或者，
    所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GAAUUGCAAUGUCGAUAGZ 13-3'(SEQ ID NO:181)；
    5'-Z 14CUAUCGACAUUGCAAUUC-3'(SEQ ID NO:182)，
    其中，Z 13为A，Z 14为U；
    所述核苷酸序列I中包含位置对应于Z 13的核苷酸Z 15，所述核苷酸序列II中包含位置对应于Z 14的核苷酸Z 16，所述Z 16是所述反义链5'末端的第一个核苷酸；或者，
    所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GUCUCUAGUCAGUGUGUUZ 17-3'(SEQ ID NO:241)；
    5'-Z 18AACACACUGACUAGAGAC-3'(SEQ ID NO:242)，
    其中，Z 17为A，Z 18为U；
    所述核苷酸序列I中包含位置对应于Z 17的核苷酸Z 19，所述核苷酸序列II中包含位置对应于Z 18的核苷酸Z 20，所述Z 20是所述反义链5'末端的第一个核苷酸；或者，
    所述核苷酸序列I与SEQ ID NO:301所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:302所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GUACUAAGAGGUUUGAUAZ 21-3'(SEQ ID NO:301)；
    5'-Z 22UAUCAAACCUCUUAGUAC-3'(SEQ ID NO:302)，
    其中，Z 21为A，Z 22为U；
    所述核苷酸序列I中包含位置对应于Z 21的核苷酸Z 23，所述核苷酸序列II中包含位置对应于Z 22的核苷酸Z 24，所述Z 24是所述反义链5'末端的第一个核苷酸；或者，
    所述核苷酸序列I与SEQ ID NO:361所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:362所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-GAAACAAAGUGUACGUUGZ 25-3'(SEQ ID NO:361)；
    5'-Z 26CAACGUACACUUUGUUUC-3'(SEQ ID NO:362)，
    其中，Z 25为A，Z 26为U；
    所述核苷酸序列I中包含位置对应于Z 25的核苷酸Z 27，所述核苷酸序列II中包含位置对应于Z 26的核苷酸Z 28，所述Z 28是所述反义链5'末端的第一个核苷酸；或者，
    所述核苷酸序列I与SEQ ID NO:421所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:422所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-CAUUCAAUCCAGAAACUAZ 29-3'(SEQ ID NO:421)；
    5'-Z 30UAGUUUCUGGAUUGAAUG-3'(SEQ ID NO:422)，
    其中，Z 29为A，Z 30为U；
    所述核苷酸序列I中包含位置对应于Z 29的核苷酸Z 31，所述核苷酸序列II中包含位置对应于Z 30的核苷酸Z 32，所述Z 32是所述反义链5'末端的第一个核苷酸。
  8. 根据权利要求7所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于2个核苷酸差异；或者，
    所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于2个核苷酸差异；或者，
    所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于2个核苷酸差异；或者，
    所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于2个核苷酸差异；或者，
    所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于2个核苷酸 差异；或者，
    所述核苷酸序列I与SEQ ID NO:301所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:302所示的核苷酸序列长度相等，且不多于2个核苷酸差异；或者，
    所述核苷酸序列I与SEQ ID NO:361所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:362所示的核苷酸序列长度相等，且不多于2个核苷酸差异；或者，
    所述核苷酸序列I与SEQ ID NO:421所示的核苷酸序列长度相等，且不多于2个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:422所示的核苷酸序列长度相等，且不多于2个核苷酸差异。
  9. 根据权利要求7或8所述的siRNA，其中，所述核苷酸序列I具有SEQ ID NO:713、787、861或935所示的核苷酸序列，所述核苷酸序列II是与所述核苷酸序列I实质上反向互补或者完全反向互补的核苷酸序列。
  10. 根据权利要求7或8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；
    或者，所述核苷酸序列I与SEQ ID NO:61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:62所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:121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:122所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:181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:182所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:241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:242所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:301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:302所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:361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:62所示的核苷酸序列之间不多于1个核苷酸差异；
    或者，所述核苷酸序列I与SEQ ID NO:421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:422所示的核苷酸序列之间不多于1个核苷酸差异。
  11. 根据权利要求7、8或10中任意一项所述的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；
    或者，所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列之间的核苷酸差异包括Z 8位置处的差异，且Z 8选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列之间的核苷酸差异包括Z 12位置处的差异，且Z 12选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列之间的核苷酸差异包括Z 16位置处的差异，且Z 16选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列之间的核苷酸差异包括Z 20位置处的差异，且Z 20选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:302所示的核苷酸序列之间的核苷酸差异包括Z 24位置处的差异，且Z 24选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:362所示的核苷酸序列之间的核苷酸差异包括Z 28位置处的差异，且Z 28选自A、C或G；
    或者，所述核苷酸序列II与SEQ ID NO:422所示的核苷酸序列之间的核苷酸差异包括Z 32位置处的差异，且Z 32选自A、C或G。
  12. 根据权利要求7-8和10-11中任一项所述的siRNA，其中Z 3是与Z 4互补的核苷酸；或者，Z 7是与Z 8互补的核苷酸；或者，Z 11是与Z 12互补的核苷酸；或者，Z 15是与Z 16互补的核苷酸；或者，Z 19是与Z 20互补的核苷酸；或者，Z 23是与Z 24互补的核苷酸；或者，Z 27是与Z 28互补的核苷酸；或者，Z 31是与Z 32互补的核苷酸。
  13. 根据权利要求1-12中任一项所述的siRNA，其中，所述正义链和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'-GCGCUAACAUAGGUUGUAZ 3-3'(SEQ ID NO:3)；
    5'-Z 4UACAACCUAUGUUAGCGC-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；
    或者，所述核苷酸序列I是SEQ ID NO:63所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:64所示的核苷酸序列：
    5'-GCUUAUGUGUCAACCUAUZ 7-3'(SEQ ID NO:63)；
    5'-Z 8AUAGGUUGACACAUAAGC-3'(SEQ ID NO:64)，
    其中，Z 7选自A、U、G或C，Z 8是与Z 7互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:123所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:124所示的核苷酸序列：
    5'-GCGGUUCACUAUAUGUUAZ 11-3'(SEQ ID NO:123)；
    5'-Z 12UAACAUAUAGUGAACCGC-3'(SEQ ID NO:124)，
    其中，Z 11选自A、U、G或C，Z 12是与Z 11互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:183所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:184所示的核苷酸序列：
    5'-GAAUUGCAAUGUCGAUAG Z 15-3'(SEQ ID NO:183)；
    5'-Z 16CUAUCGACAUUGCAAUUC-3'(SEQ ID NO:184)，
    其中，Z 15选自A、U、G或C，Z 16是与Z 15互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:243所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:244所示的核苷酸序列：
    5'-GUCUCUAGUCAGUGUGUUZ 19-3'(SEQ ID NO:243)；
    5'-Z 20AACACACUGACUAGAGAC-3'(SEQ ID NO:244)，
    其中，Z 19选自A、U、G或C，Z 20是与Z 19互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:303所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:304所示的核苷酸序列：
    5'-GUACUAAGAGGUUUGAUAZ 23-3'(SEQ ID NO:303)；
    5'-Z 24UAUCAAACCUCUUAGUAC-3'(SEQ ID NO:304)，
    其中，Z 23选自A、U、G或C，Z 24是与Z 23互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:363所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:364所示的核苷酸序列：
    5'-GAAACAAAGUGUACGUUGZ 27-3'(SEQ ID NO:363)；
    5'-Z 28CAACGUACACUUUGUUUC-3'(SEQ ID NO:364)，
    其中，Z 27选自A、U、G或C，Z 28是与Z 27互补的核苷酸；
    或者，所述核苷酸序列I是SEQ ID NO:423所示的核苷酸序列，所述核苷酸序列II是SEQ ID NO:424所示的核苷酸序列：
    5'-CAUUCAAUCCAGAAACUAZ 31-3'(SEQ ID NO:423)；
    5'-Z 32UAGUUUCUGGAUUGAAUG-3'(SEQ ID NO:424)，
    其中，Z 31选自A、U、G或C，Z 32是与Z 31互补的核苷酸。
  14. 根据权利要求13所述的siRNA，其中，Z 3为A，Z 4为U；或者Z 7为A，Z 8为U；或者Z 11为A，Z 12为U；或者Z 15为A，Z 16为U；或者Z 19为A，Z 20为U；或者Z 23为A， Z 24为U；或者Z 27为A，Z 28为U；或者Z 31为A，Z 32为U。
  15. 根据权利要求1-14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。
  16. 根据权利要求15所述的siRNA，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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A；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UA；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CUA；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GCUA；
    或者，所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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A；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CA；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UCA；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GUCA；
    或者，所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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G；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UG；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GUG；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UGUG；
    或者，所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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G；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UG；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UUG；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UUUG；
    或者，所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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A；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UA；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CUA；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ACUA；
    或者，所述核苷酸序列I与SEQ ID NO:301所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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G；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UG；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AUG；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AAUG；
    或者，所述核苷酸序列I与SEQ ID NO:361所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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A；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CA；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UCA；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CUCA；
    或者，所述核苷酸序列I与SEQ ID NO:421所示的核苷酸序列长度相等，且不多于3个核苷酸差异，并且，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GU；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GGU；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UGGU。
  17. 根据权利要求1-16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。
  18. 根据权利要求17所述的siRNA，其中，所述核苷酸序列V的长度为2个核苷酸。
  19. 根据权利要求17或18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RNA相应位置的核苷酸互补。
  20. 根据权利要求1-19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'-GCGCUAACAUAGGUUGUAZ 3-3'(SEQ ID NO:5)；
    5'-Z 4UACAACCUAUGUUAGCGCUA-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'-UAGCGCUAACAUAGGUUGUAZ 3-3'(SEQ ID NO:7)；
    5'-Z 4UACAACCUAUGUUAGCGCUAGC-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；
    或者，所述siRNA的正义链含有如SEQ ID NO:65所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:66所示的核苷酸序列：
    5'-GCUUAUGUGUCAACCUAUZ 7-3'(SEQ ID NO:65)；
    5'-Z 8AUAGGUUGACACAUAAGCUG-3'(SEQ ID NO:66)，
    或者，所述siRNA的正义链含有如SEQ ID NO:67所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:68所示的核苷酸序列：
    5'-UAGCUUAUGUGUCAACCUAUZ 7-3'(SEQ ID NO:67)；
    5'-Z 8AUAGGUUGACACAUAAGCUGAC-3'(SEQ ID NO:68)，
    其中，所述Z 8是反义链5'末端的第一个核苷酸，Z 7选自A、U、G或C，并且Z 8是与Z 7互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:125所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:126所示的核苷酸序列：
    5'-GCGGUUCACUAUAUGUUAZ 11-3'(SEQ ID NO:125)；
    5'-Z 12UAACAUAUAGUGAACCGCCA-3'(SEQ ID NO:126)，
    或者，所述siRNA的正义链含有如SEQ ID NO:127所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:128所示的核苷酸序列：
    5'-UGGCGGUUCACUAUAUGUUAZ 11-3'(SEQ ID NO:127)；
    5'-Z 12UAACAUAUAGUGAACCGCCACA-3'(SEQ ID NO:128)，
    其中，所述Z 12是反义链5'末端的第一个核苷酸，Z 11选自A、U、G或C，并且Z 12是与Z 11互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:185所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:186所示的核苷酸序列：
    5'-GAAUUGCAAUGUCGAUAGZ 15-3'(SEQ ID NO:185)；
    5'-Z 16CUAUCGACAUUGCAAUUCCA-3'(SEQ ID NO:186)，
    或者，所述siRNA的正义链含有如SEQ ID NO:187所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:188所示的核苷酸序列：
    5'-UGGAAUUGCAAUGUCGAUAGZ 15-3'(SEQ ID NO:187)；
    5'-Z 16CUAUCGACAUUGCAAUUCCAAA-3'(SEQ ID NO:188)，
    其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 15选自A、U、G或C，并且Z 16是与Z 15互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:245所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:246所示的核苷酸序列：
    5'-GUCUCUAGUCAGUGUGUUZ 19-3'(SEQ ID NO:245)；
    5'-Z 20AACACACUGACUAGAGACUA-3'(SEQ ID NO:246)，
    或者，所述siRNA的正义链含有如SEQ ID NO:247所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:248所示的核苷酸序列：
    5'-UAGUCUCUAGUCAGUGUGUUZZ 19-3'(SEQ ID NO:247)；
    5'-Z 20AACACACUGACUAGAGACUAGU-3'(SEQ ID NO:248)，
    其中，所述Z 16是反义链5'末端的第一个核苷酸，Z 19选自A、U、G或C，并且Z 20是与Z 19互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:305所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:306所示的核苷酸序列：
    5'-GUACUAAGAGGUUUGAUAZ 23-3'(SEQ ID NO:305)；
    5'-Z 24UAUCAAACCUCUUAGUACCA-3'(SEQ ID NO:306)，
    或者，所述siRNA的正义链含有如SEQ ID NO:307所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:308所示的核苷酸序列：
    5'-UGGUACUAAGAGGUUUGAUAZ 23-3'(SEQ ID NO:307)；
    5'-Z 24UAUCAAACCUCUUAGUACCAUU-3'(SEQ ID NO:308)，
    其中，所述Z 24是反义链5'末端的第一个核苷酸，Z 23选自A、U、G或C，并且Z 24是与Z 23互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:365所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:366所示的核苷酸序列：
    5'-GAAACAAAGUGUACGUUGZ 27-3'(SEQ ID NO:365)；
    5'-Z 28CAACGUACACUUUGUUUCUG-3'(SEQ ID NO:366)；
    或者，所述siRNA的正义链含有如SEQ ID NO:367所示的核苷酸序列，所述反义链含有如SEQ ID NO:368所示的核苷酸序列：
    5'-CAGAAACAAAGUGUACGUUGZ 27-3'(SEQ ID NO:367)；
    5'-Z 28CAACGUACACUUUGUUUCUGAG-3'(SEQ ID NO:368)；
    其中，所述Z 28是反义链5'末端的第一个核苷酸，Z 27选自A、U、G或C，并且Z 28是与Z 28互补的核苷酸；
    或者，所述siRNA的正义链含有如SEQ ID NO:425所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:426所示的核苷酸序列：
    5'-CAUUCAAUCCAGAAACUAZ 31-3'(SEQ ID NO:425)；
    5'-Z 32UAGUUUCUGGAUUGAAUGAC-3'(SEQ ID NO:426)，
    或者，所述siRNA的正义链含有如SEQ ID NO:427所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:428所示的核苷酸序列：
    5'-GUCAUUCAAUCCAGAAACUAZ 31-3'(SEQ ID NO:427)；
    5'-Z 32UAGUUUCUGGAUUGAAUGACCA-3'(SEQ ID NO:428)，
    其中，所述Z 32是反义链5'末端的第一个核苷酸，Z 31选自A、U、G或C，并且Z 32是与Z 31互补的核苷酸。
  21. 根据权利要求1-20中任一项所述的siRNA，其中，所述siRNA为siCOVIDa1、siCOVIDa2、siCOVIDb1、siCOVIDb2、siCOVIDc1、siCOVIDc2、siCOVIDd1、siCOVIDd2、siCOVIDe1、siCOVIDe2、siCOVIDe1U、siCOVIDe2U、siCOVIDf1、siCOVIDf2、siCOVIDg1、siCOVIDg2、siCOVIDh1、siCOVIDh2、siCOVIDh1U、siCOVIDh2U、siCOVIDh1R、siCOVIDh2R、siCOVIDh1G和siCOVIDh2G中的一种。
  22. 根据权利要求1-21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。
  23. 根据权利要求1-22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。
  24. 根据权利要求23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。
  25. 根据权利要求24所述的siRNA，其中，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。
  26. 根据权利要求23-25中任一项所述的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。
  27. 根据权利要求26所述的siRNA，其中，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；核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。
  28. 根据权利要求23-27中任意一项所述的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。
  29. 根据权利要求25所述的siRNA，其中，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。
  30. 根据权利要求1-29中任一项所述的siRNA，其中，所述siRNA为siCOVIDa1-M1、siCOVIDa1-M2、siCOVIDa1-M3、siCOVIDa2-M1、siCOVIDa2-M2、siCOVIDa2-M3、siCOVIDb1-M1、 siCOVIDb1-M2、siCOVIDb1-M3、siCOVIDb2-M1、siCOVIDb2-M2、siCOVIDb2-M3、siCOVIDc1-M1、siCOVIDc1-M2、siCOVIDc1-M3、siCOVIDc2-M1、siCOVIDc2-M2、siCOVIDc2-M3、siCOVIDd1-M1、siCOVIDd1-M2、siCOVIDd1-M3、siCOVIDd2-M1、siCOVIDd2-M2、siCOVIDd2-M3、siCOVIDe1-M1、siCOVIDe1-M2、siCOVIDe1-M3、siCOVIDe2-M1、siCOVIDe2-M2、siCOVIDe2-M3、siCOVIDe1U-M1、siCOVIDe1U-M2、siCOVIDe1U-M3、siCOVIDe2U-M1、siCOVIDe2U-M2、siCOVIDe2U-M3、siCOVIDf1-M1、siCOVIDf1-M2、siCOVIDf1-M3、siCOVIDf2-M1、siCOVIDf2-M2、siCOVIDf2-M3、siCOVIDg1-M1、siCOVIDg1-M2、siCOVIDg1-M3、siCOVIDg2-M1、siCOVIDg2-M2、siCOVIDg2-M3、siCOVIDh1-M1、siCOVIDh1-M2、siCOVIDh1-M3、siCOVIDh2-M1、siCOVIDh2-M2、siCOVIDh2-M3、siCOVIDh1U-M1、siCOVIDh1U-M2、siCOVIDh1U-M3、siCOVIDh2U-M1、siCOVIDh2U-M2、siCOVIDh2U-M3、siCOVIDh1R-M1、siCOVIDh1R-M2、siCOVIDh1R-M3、siCOVIDh2R-M1、siCOVIDh2R-M2、siCOVIDh2R-M3、siCOVIDh1G-M1、siCOVIDh1G-M2、siCOVIDh1G-M3、siCOVIDh2G-M1、siCOVIDh2G-M2、siCOVIDh2G-M3中的任意一种。
  31. 根据权利要求22所述的siRNA，其中，所述具有修饰基团的磷酸酯基为磷酸酯基的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。
  32. 根据权利要求22或31所述的siRNA，其中，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基：
    Figure PCTCN2021086016-appb-100001
  33. 根据权利要求31或32所述的siRNA，其中，所述siRNA中，硫代磷酸酯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。
  34. 根据权利要求1-33中任一项所述的siRNA，其中，所述siRNA为siCOVIDa1-M1S、siCOVIDa1-M2S、siCOVIDa1-M3S、siCOVIDa2-M1S、siCOVIDa2-M2S、siCOVIDa2-M3S、siCOVIDa1-M1S1、siCOVIDa1-M2S1、siCOVIDa1-M3S1、siCOVIDa2-M1S1、siCOVIDa2-M2S1、siCOVIDa2-M3S1、siCOVIDb1-M1S、siCOVIDb1-M2S、siCOVIDb1-M3S、siCOVIDb2-M1S、siCOVIDb2-M2S、siCOVIDb2-M3S、siCOVIDb1-M1S1、siCOVIDb1-M2S1、siCOVIDb1-M3S1、siCOVIDb2-M1S1、siCOVIDb2-M2S1、siCOVIDb2-M3S1、siCOVIDc1-M1S、siCOVIDc1-M2S、siCOVIDc1-M3S、siCOVIDc2-M1S、siCOVIDc2-M2S、siCOVIDc2-M3S、siCOVIDc1-M1S1、siCOVIDc1-M2S1、siCOVIDc1-M3S1、siCOVIDc2-M1S1、siCOVIDc2-M2S1、siCOVIDc2-M3S1、siCOVIDd1-M1S、siCOVIDd1-M2S、siCOVIDd1-M3S、siCOVIDd2-M1S、siCOVIDd2-M2S、siCOVIDd2-M3S、siCOVIDd1-M1S1、siCOVIDd1-M2S1、siCOVIDd1-M3S1、siCOVIDd2-M1S1、siCOVIDd2-M2S1、siCOVIDd2-M3S1、siCOVIDe1-M1S、siCOVIDe1-M2S、siCOVIDe1-M3S、siCOVIDe2-M1S、siCOVIDe2-M2S、siCOVIDe2-M3S、siCOVIDe1-M1S1、siCOVIDe1-M2S1、siCOVIDe1-M3S1、siCOVIDe2-M1S1、siCOVIDe2-M2S1、siCOVIDe2-M3S1、siCOVIDe1U-M1S、 siCOVIDe1U-M2S、siCOVIDe1U-M3S、siCOVIDe2U-M1S、siCOVIDe2U-M2S、siCOVIDe2U-M3S、siCOVIDe1U-M1S1、siCOVIDe1U-M2S1、siCOVIDe1U-M3S1、siCOVIDe2U-M1S1、siCOVIDe2U-M2S1、siCOVIDe2U-M3S1、siCOVIDf1-M1S、siCOVIDf1-M2S、siCOVIDf1-M3S、siCOVIDf2-M1S、siCOVIDf2-M2S、siCOVIDf2-M3S、siCOVIDf1-M1S1、siCOVIDf1-M2S1、siCOVIDf1-M3S1、siCOVIDf2-M1S1、siCOVIDf2-M2S1、siCOVIDf2-M3S1、siCOVIDg1-M1S、siCOVIDg1-M2S、siCOVIDg1-M3S、siCOVIDg2-M1S、siCOVIDg2-M2S、siCOVIDg2-M3S、siCOVIDg1-M1S1、siCOVIDg1-M2S1、siCOVIDg1-M3S1、siCOVIDg2-M1S1、siCOVIDg2-M2S1、siCOVIDg2-M3S1、siCOVIDh1-M1S、siCOVIDh1-M2S、siCOVIDh1-M3S、siCOVIDh2-M1S、siCOVIDh2-M2S、siCOVIDh2-M3S、siCOVIDh1-M1S1、siCOVIDh1-M2S1、siCOVIDh1-M3S1、siCOVIDh2-M1S1、siCOVIDh2-M2S1、siCOVIDh2-M3S1、siCOVIDh1U-M1S、siCOVIDh1U-M2S、siCOVIDh1U-M3S、siCOVIDh2U-M1S、siCOVIDh2U-M2S、siCOVIDh2U-M3S、siCOVIDh1U-M1S1、siCOVIDh1U-M2S1、siCOVIDh1U-M3S1、siCOVIDh2U-M1S1、siCOVIDh2U-M2S1、siCOVIDh2U-M3S1、siCOVIDh1R-M1S、siCOVIDh1R-M2S、siCOVIDh1R-M3S、siCOVIDh2R-M1S、siCOVIDh2R-M2S、siCOVIDh2R-M3S、siCOVIDh1R-M1S1、siCOVIDh1R-M2S1、siCOVIDh1R-M3S1、siCOVIDh2R-M1S1、siCOVIDh2R-M2S1、siCOVIDh2R-M3S1、siCOVIDh1G-M1S、siCOVIDh1G-M2S、siCOVIDh1G-M3S、siCOVIDh2G-M1S、siCOVIDh2G-M2S、siCOVIDh2G-M3S、siCOVIDh1G-M1S1、siCOVIDh1G-M2S1、siCOVIDh1G-M3S1、siCOVIDh2G-M1S1、siCOVIDh2G-M2S1、siCOVIDh2G-M3S1中的任意一种。
  35. 根据权利要求1-34中任一项所述的siRNA，其中，所述siRNA为siCOVIDa1-M1P1、siCOVIDa1-M2P1、siCOVIDa1-M3P1、siCOVIDa2-M1P1、siCOVIDa2-M2P1、siCOVIDa2-M3P1、siCOVIDa1-M1SP1、siCOVIDa1-M2SP1、siCOVIDa1-M3SP1、siCOVIDa2-M1SP1、siCOVIDa2-M2SP1、siCOVIDa2-M3SP1、siCOVIDa1-M1S1P1、siCOVIDa1-M2S1P1、siCOVIDa1-M3S1P1、siCOVIDa2-M1S1P1、siCOVIDa2-M2S1P1、siCOVIDa2-M3S1P1、siCOVIDb1-M1P1、siCOVIDb1-M2P1、siCOVIDb1-M3P1、siCOVIDb2-M1P1、siCOVIDb2-M2P1、siCOVIDb2-M3P1、siCOVIDb1-M1SP1、siCOVIDb1-M2SP1、siCOVIDb1-M3SP1、siCOVIDb2-M1SP1、siCOVIDb2-M2SP1、siCOVIDb2-M3SP1、siCOVIDb1-M1S1P1、siCOVIDb1-M2S1P1、siCOVIDb1-M3S1P1、siCOVIDb2-M1S1P1、siCOVIDb2-M2S1P1、siCOVIDb2-M3S1P1、siCOVIDc1-M1P1、siCOVIDc1-M2P1、siCOVIDc1-M3P1、siCOVIDc2-M1P1、siCOVIDc2-M2P1、siCOVIDc2-M3P1、siCOVIDc1-M1SP1、siCOVIDc1-M2SP1、siCOVIDc1-M3SP1、siCOVIDc2-M1SP1、siCOVIDc2-M2SP1、siCOVIDc2-M3SP1、siCOVIDc1-M1S1P1、siCOVIDc1-M2S1P1、siCOVIDc1-M3S1P1、siCOVIDc2-M1S1P1、siCOVIDc2-M2S1P1、siCOVIDc2-M3S1P1、siCOVIDd1-M1P1、siCOVIDd1-M2P1、siCOVIDd1-M3P1、siCOVIDd2-M1P1、siCOVIDd2-M2P1、siCOVIDd2-M3P1、siCOVIDd1-M1SP1、siCOVIDd1-M2SP1、siCOVIDd1-M3SP1、siCOVIDd2-M1SP1、siCOVIDd2-M2SP1、siCOVIDd2-M3SP1、siCOVIDd1-M1S1P1、siCOVIDd1-M2S1P1、siCOVIDd1-M3S1P1、siCOVIDd2-M1S1P1、siCOVIDd2-M2S1P1、siCOVIDd2-M3S1P1、siCOVIDe1-M1P1、siCOVIDe1-M2P1、siCOVIDe1-M3P1、siCOVIDe2-M1P1、siCOVIDe2-M2P1、siCOVIDe2-M3P1、siCOVIDe1-M1SP1、siCOVIDe1-M2SP1、siCOVIDe1-M3SP1、siCOVIDe2-M1SP1、siCOVIDe2-M2SP1、siCOVIDe2-M3SP1、siCOVIDe1-M1S1P1、siCOVIDe1-M2S1P1、siCOVIDe1-M3S1P1、siCOVIDe2-M1S1P1、siCOVIDe2-M2S1P1、siCOVIDe2-M3S1P1、siCOVIDe1U-M1P1、siCOVIDe1U-M2P1、siCOVIDe1U-M3P1、siCOVIDe2U-M1P1、siCOVIDe2U-M2P1、siCOVIDe2U-M3P1、siCOVIDe1U-M1SP1、siCOVIDe1U-M2SP1、siCOVIDe1U-M3SP1、siCOVIDe2U-M1SP1、siCOVIDe2U-M2SP1、siCOVIDe2U-M3SP1、siCOVIDe1U-M1S1P1、siCOVIDe1U-M2S1P1、siCOVIDe1U-M3S1P1、siCOVIDe2U-M1S1P1、siCOVIDe2U-M2S1P1、siCOVIDe2U-M3S1P1、siCOVIDf1-M1P1、siCOVIDf1-M2P1、siCOVIDf1-M3P1、siCOVIDf2-M1P1、siCOVIDf2-M2P1、siCOVIDf2-M3P1、siCOVIDf1-M1SP1、siCOVIDf1-M2SP1、siCOVIDf1-M3SP1、siCOVIDf2-M1SP1、siCOVIDf2-M2SP1、siCOVIDf2-M3SP1、siCOVIDf1-M1S1P1、siCOVIDf1-M2S1P1、siCOVIDf1-M3S1P1、siCOVIDf2-M1S1P1、siCOVIDf2-M2S1P1、 siCOVIDf2-M3S1P1、siCOVIDg1-M1P1、siCOVIDg1-M2P1、siCOVIDg1-M3P1、siCOVIDg2-M1P1、siCOVIDg2-M2P1、siCOVIDg2-M3P1、siCOVIDg1-M1SP1、siCOVIDg1-M2SP1、siCOVIDg1-M3SP1、siCOVIDg2-M1SP1、siCOVIDg2-M2SP1、siCOVIDg2-M3SP1、siCOVIDg1-M1S1P1、siCOVIDg1-M2S1P1、siCOVIDg1-M3S1P1、siCOVIDg2-M1S1P1、siCOVIDg2-M2S1P1、siCOVIDg2-M3S1P1、siCOVIDh1-M1P1、siCOVIDh1-M2P1、siCOVIDh1-M3P1、siCOVIDh2-M1P1、siCOVIDh2-M2P1、siCOVIDh2-M3P1、siCOVIDh1-M1SP1、siCOVIDh1-M2SP1、siCOVIDh1-M3SP1、siCOVIDh2-M1SP1、siCOVIDh2-M2SP1、siCOVIDh2-M3SP1、siCOVIDh1-M1S1P1、siCOVIDh1-M2S1P1、siCOVIDh1-M3S1P1、siCOVIDh2-M1S1P1、siCOVIDh2-M2S1P1、siCOVIDh2-M3S1P1、siCOVIDh1U-M1P1、siCOVIDh1U-M2P1、siCOVIDh1U-M3P1、siCOVIDh2U-M1P1、siCOVIDh2U-M2P1、siCOVIDh2U-M3P1、siCOVIDh1U-M1SP1、siCOVIDh1U-M2SP1、siCOVIDh1U-M3SP1、siCOVIDh2U-M1SP1、siCOVIDh2U-M2SP1、siCOVIDh2U-M3SP1、siCOVIDh1U-M1S1P1、siCOVIDh1U-M2S1P1、siCOVIDh1U-M3S1P1、siCOVIDh2U-M1S1P1、siCOVIDh2U-M2S1P1、siCOVIDh2U-M3S1P1、siCOVIDh1R-M1P1、siCOVIDh1R-M2P1、siCOVIDh1R-M3P1、siCOVIDh2R-M1P1、siCOVIDh2R-M2P1、siCOVIDh2R-M3P1、siCOVIDh1R-M1SP1、siCOVIDh1R-M2SP1、siCOVIDh1R-M3SP1、siCOVIDh2R-M1SP1、siCOVIDh2R-M2SP1、siCOVIDh2R-M3SP1、siCOVIDh1R-M1S1P1、siCOVIDh1R-M2S1P1、siCOVIDh1R-M3S1P1、siCOVIDh2R-M1S1P1、siCOVIDh2R-M2S1P1、siCOVIDh2R-M3S1P1、siCOVIDh1G-M1P1、siCOVIDh1G-M2P1、siCOVIDh1G-M3P1、siCOVIDh2G-M1P1、siCOVIDh2G-M2P1、siCOVIDh2G-M3P1、siCOVIDh1G-M1SP1、siCOVIDh1G-M2SP1、siCOVIDh1G-M3SP1、siCOVIDh2G-M1SP1、siCOVIDh2G-M2SP1、siCOVIDh2G-M3SP1、siCOVIDh1G-M1S1P1、siCOVIDh1G-M2S1P1、siCOVIDh1G-M3S1P1、siCOVIDh2G-M1S1P1、siCOVIDh2G-M2S1P1、siCOVIDh2G-M3S1P1中的任意一种。
  36. 一种药物组合物，其特征在于，该药物组合物含有权利要求1-33中任意一项所述的siRNA和药学上可接受的载体。
  37. 根据权利要求36所述的药物组合物，其中，所述siRNA与药学上可接受的载体的重量比为1:(1-500)。
  38. 根据权利要求37所述的药物组合物，其中，所述siRNA与药学上可接受的载体的重量比为1:(1-50)。
  39. 一种siRNA缀合物，其特征在于，所述siRNA缀合物含有权利要求1-35中任意一项所述的siRNA以及缀合连接至该siRNA的缀合基团。
  40. 根据权利要求39所述的siRNA缀合物，其中，所述缀合基团包含药学上可接受的靶向基团和接头，并且，所述siRNA、所述接头和所述靶向基团依次共价或非共价连接。
  41. 根据权利要求40所述的siRNA缀合物，其中，所述靶向基团选自能够和细胞表面受体结合的配体。
  42. 根据权利要求40所述的siRNA缀合物，其中，所述靶向基团选自于靶向ACE2或integrinαvβ6的基团。
  43. 根据权利要求42所述的siRNA缀合物，其中，每个所述靶向基团独立地为多肽或小分子配体。
  44. 权利要求1-35中任意一项所述的siRNA和/或权利要求36-38中任意一项所述的药物组合物和/或权利要求39-43中任意一项所述的siRNA缀合物在制备用于治疗和/或预防新型冠状病毒引起的疾病的药物中的用途。
  45. 根据权利要求44所述的用途，其中，所述新型冠状病毒所引起的疾病为COVID-19。
  46. 一种治疗和/或预防新冠状病毒所引起的疾病的方法，其中，所述方法包括将有效量的权利要求1-35中任意一项所述的siRNA和/或权利要求36-38中任意一项所述的药物组合物和/或权利要求39-43中任意一项所述的siRNA缀合物给予患有新型冠状病毒所引起的疾病的受试者。
  47. 一种抑制细胞中SARS-COV-2病毒基因表达的方法，该方法包括将有效量的权利要求1-35中任意一项所述的siRNA和/或权利要求36-38中任意一项所述的药物组合物和/或权利要求39-43中任意一项所述的siRNA缀合物与所述细胞接触。
  48. 一种试剂盒，其中，该试剂盒含有权利要求1-35中任意一项所述的siRNA和/或权利要求36-38中任意一项所述的药物组合物和/或权利要求39-43中任意一项所述的siRNA缀合物。
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